20strains date-
SAT-20中文资料
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20±0.5 30±0.5 40±0.6
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CAT-9 CAT-10 n CAT-10-75 CAT-12 CAT-15
NEW
n CAT-15-75 CAT-20
n CAT-20-75 CAT-25 CAT-30 CAT-40
FREQUENCY RANGE, MHz
fL-fU
DC-1500 DC-1500 DC-1500 DC-500 DC-1500
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* Attenuation Flatness over DC-2000 MHz: CAT-25, NAT-25, 0.75 dB typ.; NAT-1-2W, 0.20 dB typ.; NAT-2-2W, 0.40 dB typ.; NAT-3-2W, 0.50 dB typ. NAT-4-2W, 0.8 dB typ.; NAT-5-2W, 0.9 dB typ.; NAT-6-2W, 1.0 dB typ.
酵母表达载体pPICZ手册
pPICZ A, B, and CPichia expression vectors for selection onZeocin™ and purification of recombinant proteins Catalog no. V190-20Rev. Date: 7 July 2010Manual part no. 25-0148MAN00000034User ManualiiTable of ContentsKit Contents and Storage (iv)Accessory Products (v)Introduction (1)Overview (1)Methods (3)Cloning into pPICZ A, B, and C (3)Pichia Transformation (9)Expression in Pichia (13)Purification (15)Appendix (17)Recipes (17)Zeocin™ (19)Map and Features of pPICZ A, B, and C (21)Lithium Chloride Transformation Method (23)Construction of In Vitro Multimers (24)Technical Support (32)Purchaser Notification (33)References (34)iiiKit Contents and StorageContents The following components are included with Catalog no. V190–20. Note that thepPICZ expression vectors are supplied in suspension.Component QuantityCompositionpPICZ A Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ B Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ C Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0GS115/pPICZ/lacZ Positive1 stab --Control strainShipping/Storage The components included with Catalog no. V190–20 are shipped on wet ice.Upon receipt, store as directed below.For long-term storage of your positive control stab strain, we recommendpreparing a glycerol stock immediately upon receipt and storing at –80°C.Component ShippingStorage pPICZ A Expression Vector Wet ice Store at –20°CpPICZ B Expression Vector Wet ice Store at –20°CpPICZ C Expression Vector Wet ice Store at –20°CGS115/pPICZ/lacZ positive control strain Wet ice Store at 4°CivAccessory ProductsAdditional ProductsThe products listed in this section are intended for use with the pPICZ vectors.For more information, visit our web site at or contactTechnical Support (page 32).Product QuantityCatalogno. X-33 Pichia strain 1 stab C180-00GS115 Pichia strain 1 stab C181-00KM71H Pichia strain 1 stab C182-00SMD1168H Pichia strain 1 stab C184-00pPICZα A, B, and C 20 μg each V195-20pPIC6α A,B, and C 20 μg each V215-20pPIC6 A, B, and C 20 μg each V210-20pPIC6 Starter Kit 1 kit K210-01Original Pichia Expression Kit 1 kit K1710-01EasySelect™Pichia Expression Kit 1 kit K1740-01Pichia EasyComp™ Transformation Kit 1 kit K1730-01Pichia Protocols 1 book G100-01PureLink™ Gel Extraction Kit 50 preps250 prepsK2100–12K2100–25S.N.A.P ™ Gel Purification Kit 25 preps K1999–25PureLink™ Quick Plasmid Miniprep Kit 50 preps250 prepsK2100–10K2100–11PureLink™ HiPure Plasmid Midiprep Kit 25 preps50 prepsK2100–04K2100–13One Shot® TOP10 (chemically competent E. coli) 10 reactions20 reactionsC4040–10C4040–03One Shot® TOP10 Electrocompetent E. Coli 10 reactions20 reactionsC4040-50C4040-52TOP10 Electrocomp™ Kits 20 reactions C664–55Positope™ Control Protein 5 μg R900-50CIAP (Calf Intestinal Alkaline Phosphatase) 1,000 units 18009–019T4 DNA Ligase 100 units500 units15224–01715224–025Zeocin™ 1g5 gR250-01R250-05β-Gal Assay Kit 1 kit K1455-01β-Gal Staining Kit 1 kit K1465-01E-Gel® Agarose Gels E-Gel® Agarose Gels are bufferless, pre-cast agarose gels designed for fast, convenient electrophoresis of DNA samples. E-Gel® agarose gels are available in different agarose percentage and well format for your convenience.For more details on these products, visit our web site at or contact Technical Support (page 32).Continued on next pagevAccessory Products, ContinuedZeocin™Zeocin™ may be obtained from Invitrogen (see above). For your convenience, the drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquotsat a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for sixmonths if stored at –20°C.Detection of Fusion Protein A number of antibodies are available from Invitrogen to detect expression ofyour fusion protein from the pPICZ vector. Horseradish peroxidase (HRP)-conjugated antibodies allow one-step detection in Western blots usingcolorimetric or chemiluminescent detection methods. The amount of antibodysupplied is sufficient for 25 Western Blots.Antibody Epitope Catalogno.Anti-myc R950–25 Anti-myc-HRPDetects the 10 amino acid epitopederived from c-myc (Evans et al., 1985):EQKLISEEDLR951–25Anti-His(C-term) R930–25Anti-His(C-term)-HRPDetects the C-terminal polyhistidine(6xHis) tag (requires the free carboxylgroup for detection) (Lindner et al., 1997):HHHHHH-COOHR931–25Purification of Fusion Protein The polyhistidine (6xHis) tag allows purification of the recombinant fusionprotein using metal-chelating resins such as ProBond™. Ordering information forProBond™ resin is provided below.Product QuantityCatalogno. ProBond™ Purification System 6 purifications K850–01ProBond ™ Purification System with Anti-myc-HRP Antibody1 Kit K852–01ProBond ™ Purification System with Anti-His(C-term)-HRP Antibody1 Kit K853–01ProBond™ Nickel-Chelating Resin 50 ml150 mlR801–01R801–15Purification Columns 50 each R640–50viIntroductionOverviewIntroduction pPICZ A, B, and C are 3.3 kb expression vectors used to express recombinantproteins in Pichia pastoris. Recombinant proteins are expressed as fusions to aC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.The vector allows high-level, methanol inducible expression of the gene ofinterest in Pichia, and can be used in any Pichia strain including X33, GS115,SMD1168H, and KM71H. pPICZ contains the following elements:•5′ fragment containing the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989;Tschopp et al., 1987a)•Zeocin™ resistance gene for selection in both E. coli and Pichia (Baron et al.,1992; Drocourt et al., 1990)•C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis)tag for detection and purification of a recombinant fusion protein (if desired)•Three reading frames to facilitate in-frame cloning with the C-terminalpeptideReference Sources The pPICZ A, B, and C expression vectors may be used with the Original Pichia Expression Kit, and are included in the EasySelect™Pichia Expression Kit (see page v for ordering information). Additional general information about recombinant protein expression in Pichia pastoris is provided in the manuals for the Original Pichia Expression Kit and the EasySelect™Pichia Expression Kit. For more information about the Original Pichia Expression Kit, the EasySelect™Pichia Expression Kit, or their manuals, visit our web site at or contact Technical Support (page 32).More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference:Higgins, D. R., and Cregg, J. M. (1998) Pichia Protocols. In Methods in Molecular Biology, Vol. 103. (J. M. Walker, ed. Humana Press, Totowa, NJ) (see page v for ordering information).Recommended Pichia Host Strain We recommend using the X-33 Pichia strain as the host for expression of recombinant proteins from pPICZ. Other Pichia strains including GS115, KM71H, and SMD1168H are suitable. The X-33 Pichia strain and other strains are available from Invitrogen (see page v for ordering information). The X-33 Pichia strain has the following genotype and phenotype:Genotype: Wild-typePhenotype: Mut+1Overview, ContinuedExperimental Overview The following table describes the basic steps needed to clone and express your gene of interest in pPICZ.Step Action1 Propagate pPICZ A, B, and C by transformation into a rec A, end A1E. coli strain such as TOP10, DH5 , or JM109.2 Develop a cloning strategy and ligate your gene into one of the pPICZvectors in frame with the C-terminal tag.3 TransformintoE. coli and select transformants on Low Salt LB platescontaining 25 μg/ml Zeocin™.4 Analyze 10–20 transformants by restriction mapping or sequencing toconfirm in-frame fusion of your gene with the C-terminal tag.5 Purify and linearize the recombinant plasmid for transformation intoPichia pastoris.6 TransformyourPichia strain and plate onto YPDS plates containing the appropriate concentration of Zeocin™.7 Select for Zeocin™-resistant transformants.8 Optimize expression of your gene.9 Purify your fusion protein on metal-chelating resin (i.e. ProBond™).Continued on next page2MethodsCloning into pPICZ A, B, and CIntroduction The pPICZ vector is supplied with the multiple cloning site in three readingframes (A, B, and C) to facilitate cloning your gene of interest in frame with theC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.Use the diagrams provided on pages 5–7 to help you design a strategy to cloneyour gene of interest in frame with the C-terminal peptide. Generalconsiderations for cloning and transformation are discussed in this section.General Molecular Biology Techniques For assistance with E. coli transformations, restriction enzyme analysis, DNA biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).E. coli Strain Many E. coli strains are suitable for the propagation of the pPICZ vectorsincluding TOP10, JM109, and DH5 . We recommend that you propagate thepPICZ vectors in E. coli strains that are recombination deficient (rec A) andendonuclease A deficient (end A).For your convenience, TOP10 E. coli are available as chemically competent orelectrocompetent cells from Invitrogen (page v).Transformation Method You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of Plasmids The pPICZ vectors contain the Zeocin™ resistance (Sh ble) gene to allow selection of the plasmid using Zeocin™. To propagate and maintain the pPICZ plasmids, we recommend using the following procedure:e 10 ng of your vector to transform a rec A, end A E. coli strain like TOP10,DH5 , JM109, or equivalent (see above).2.Select transformants on Low Salt LB plates containing 25 μg/ml Zeocin™ (seepage 17 for a recipe).3.Prepare a glycerol stock from each transformant containing plasmid forlong-term storage (see page 8).Continued on next page3Cloning into pPICZ A, B, and C, ContinuedGeneral Considerations The following are some general points to consider when using pPICZ to express your gene of interest in Pichia:•The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae.•Many Saccharomyces genes have proven to be functional in Pichia.•The premature termination of transcripts because of "AT rich regions" has been observed in Pichia and other eukaryotic systems (Henikoff & Cohen, 1984; Irniger et al., 1991; Scorer et al., 1993; Zaret & Sherman, 1984). If you have problems expressing your gene, check for premature termination by northern analysis and check your sequence for AT rich regions. It may be necessary to change the sequence in order to express your gene (Scorer et al., 1993).•The native 5´ end of the AOX1 mRNA is noted in the diagram for each multiple cloning site. This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason.Cloning Considerations For proper initiation of translation, your insert should contain an initiation ATG codon as part of a yeast consensus sequence (Romanos et al., 1992). An example of a yeast consensus sequence is provided below. The ATG initiation codon is shown underlined.(G/A)NNATG GTo express your gene as a recombinant fusion protein, you must clone your gene in frame with the C-terminal peptide containing the c-myc epitope and the polyhistidine tag. The vector is supplied in three reading frames to facilitate cloning. Refer to the diagrams on pages 5–7 to develop a cloning strategy.If you wish to express your protein without the C-terminal peptide, be sure to include a stop codon.Construction of Multimeric Plasmids pPICZ A, B, and C contain unique Bgl II and Bam H I sites to allow construction of plasmids containing multiple copies of your gene. For information on how to construct multimers, refer to pages 24–31.Continued on next page4Multiple CloningSite of pPICZ A Below is the multiple cloning site for pPICZ A. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ A from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATTTGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT 1218Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTTAGCCT TAGACATGAC AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´polyadenylation sitePolyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Apa I 104210981158871931991Continued on next pageMultiple CloningSite of pPICZ B Below is the multiple cloning site for pPICZ B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ B from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1216Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATTATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Xba I 104010961156871931991Continued on next pageMultiple CloningSite of pPICZ C Below is the multiple cloning site for pPICZ C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ C from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1217Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming siteTGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho I Sac II Not I Sna B I 104110971157871931991Continued on next pageE. coli Transformation Transform your ligation mixtures into a competent rec A, end A E. coli strain(e.g. TOP10, DH5, JM109) and select on Low Salt LB agar plates containing25 μg/ml Zeocin™ (see below). Note that there is no blue/white screening for the presence of insert with pPICZ A, B, or C. Once you have obtained Zeocin™-resistant colonies, pick 10 transformants and screen for the presence and orientation of your insert.Important To facilitate selection of Zeocin™-resistant E. coli, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare Low Salt LB broth and plates using the recipe in the Appendix, page 17.Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug.C We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression and cloned in frame with the C-terminal peptide (if desired). Refer to the diagrams on pages 5–7 for the sequences and location of the priming sites.Preparing a Glycerol Stock Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.1.Streak the original colony out on an Low Salt LB plate containing 25 μg/mlZeocin™. Incubate the plate at 37°C overnight.2.Isolate a single colony and inoculate into 1–2 ml of Low Salt LB containing25 μg/ml Zeocin™.3.Grow the culture to mid-log phase (OD600 = 0.5–0.7).4.Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to acryovial.5.Store at –80°C.Plasmid Preparation Once you have cloned and sequenced your insert, generate enough plasmid DNA to transform Pichia (5–10 μg of each plasmid per transformation). We recommend isolating plasmid DNA using the PureLink™ Quick Plasmid Miniprep Kit or the PureLink™ HiPure Plasmid Midiprep Kit (page v), or CsCl gradient centrifugation.Once you have purified plasmid DNA, proceed to Pichia Transformation, next page.Pichia TransformationIntroduction You should now have your gene cloned into one of the pPICZ vectors. Yourconstruct should be correctly fused to the C-terminal peptide (if desired). Thissection provides general guidelines to prepare plasmid DNA, transform yourPichia strain, and select for Zeocin™-resistant clones.Zeocin™ Selection We generally use 100 μg/ml Zeocin™ to select for transformants when using the X-33 Pichia strain. If you are transforming your pPICZ construct into anotherPichia strain, note that selection conditions may vary. We recommendperforming a dose response curve to determine the appropriate concentration ofZeocin™ to use for selection of transformants in your strain.Method of Transformation We do not recommend spheroplasting for transformation of Pichia with plasmids containing the Zeocin™ resistance marker. Spheroplasting involves removal of the cell wall to allow DNA to enter the cell. Cells must first regenerate the cell wall before they are able to express the Zeocin™ resistance gene. For this reason, plating spheroplasts directly onto selective medium containing Zeocin™ does not yield any transformants.We recommend electroporation for transformation of Pichia with pPICZ A, B, or C. Electroporation yields 103 to 104 transformants per μg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, use the LiCl protocol on page 23 or the Pichia EasyComp™Transformation Kit available from Invitrogen (see below).PichiaEasyComp™Transformation Kit If you wish to perform chemical transformation of your Pichia strain with pPICZ A, B, or C, the Pichia EasyComp™ Transformation Kit is available from Invitrogen (see page v for ordering information). The Pichia EasyComp™ Transformation Kit provides reagents to prepare 6 preparations of competent cells. Each preparation will yield enough competent cells for 20 transformations. Competent cells may be used immediately or frozen and stored for future use. For more information, visit our web site at or contact Technical Support (page 32).Important Since pPICZ does not contain the HIS4 gene, integration can only occur at the AOX1 locus. Vector linearized within the 5´ AOX1 region will integrate by gene insertion into the host 5´ AOX1 region. Therefore, the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol (Mut+) or not (Mut S). To generate a Mut+ recombinant strain, you must use a Pichia host that contains the native AOX1 gene (e.g. X-33, GS115, SMD1168H). If you wish to generate a Mut S recombinant strain, then use a Pichia host that has a disrupted AOX1 gene (i.e. KM71H).Continued on next pageHis4 Host Strains Host strains containing the his4 allele (e.g. GS115) and transformed with thepPICZ vectors require histidine when grown in minimal media. Add histidine toa final concentration of 0.004% to ensure growth of your transformants.The pPICZ vectors do not contain a yeast origin of replication. Transformantscan only be isolated if recombination occurs between the plasmid and the Pichiagenome.Materials Needed You will need the following items:Note: Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear tobe somewhat osmotically sensitive.•5–10 μg pure pPICZ containing your insert•YPD Medium•50 ml conical polypropylene tubes• 1 liter cold (4°C) sterile water (place on ice the day of the experiment)•25 ml cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)•30°C incubator•Electroporation device and 0.2 cm cuvettes•YPDS plates containing the appropriate concentration of Zeocin™ (seepage 18 for recipe)Linearizing YourpPICZ ConstructTo promote integration, we recommend that you linearize your pPICZ constructwithin the 5′ AOX1 region. The table below lists unique sites that may be used tolinearize pPICZ prior to transformation. Other restriction sites are possible.Note that for the enzymes listed below, the cleavage site is the same for versionsA, B, and C of pPICZ. Be sure that your insert does not contain the restriction siteyou wish to use to linearize your vector.Enzyme Restriction Site (bp) SupplierSac I 209 ManyPme I 414 New England BiolabsBst X I 707 ManyRestriction Digest 1.Digest ~5–10 μg of plasmid DNA with one of the enzymes listed above.2.Check a small aliquot of your digest by agarose gel electrophoresis forcomplete linearization.3.If the vector is completely linearized, heat inactivate or add EDTA to stopthe reaction, phenol/chloroform extract once, and ethanol precipitate using1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol.4.Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol,air-dry, and resuspend in 10 μl sterile, deionized water. Use immediately orstore at –20°C.Continued on next pagePreparation of Pichia for Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation.1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at30°C overnight.2. Inoculate 500 ml of fresh medium in a 2 liter flask with 0.1–0.5 ml of theovernight culture. Grow overnight again to an OD600 = 1.3–1.5.3. Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pelletwith 500 ml of ice-cold (0–4°C), sterile water.4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml ofice-cold (0–4°C), sterile water.5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold (0–4°C) 1 M sorbitol.6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold(0–4°C) 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day. Do not store cells.Transformation by Electroporation 1.Mix 80 μl of the cells from Step 6 (above) with 5–10 μg of linearized pPICZDNA (in 5–10 μl sterile water) and transfer them to an ice-cold (0–4°C)0.2 cm electroporation cuvette.2.Incubate the cuvette with the cells on ice for 5 minutes.3.Pulse the cells according to the parameters for yeast (Saccharomycescerevisiae) as suggested by the manufacturer of the specific electroporation device being used.4.Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer thecuvette contents to a sterile 15 ml tube.5.Let the tube incubate at 30°C without shaking for 1 to 2 hours.6.Spread 50-200 μl each on separate, labeled YPDS plates containing theappropriate concentration of Zeocin™.7.Incubate plates for 2–3 days at 30°C until colonies form.8.Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD orYPDS plates containing the appropriate concentration of Zeocin™.Continued on next pageGenerally, several hundred Zeocin™-resistant colonies are generated using theprotocol on the previous page. If more colonies are needed, the protocol may bemodified as described below. Note that you will need ~20, 150 mm plates withYPDS agar containing the appropriate concentration of Zeocin™.1. Set up two transformations per construct and follow Steps 1 through 5 ofthe Transformation by Electroporation protocol, page 11.2. After 1 hour in 1 M sorbitol at 30°C (Step 5, previous page), add 1 ml YPDmedium to each tube.3. Shake (~200 rpm) the cultures at 30°C.4. After 1 hour, take one of the tubes and plate out all of the cells by spreading200 μl on 150 mm plates containing the appropriate concentration ofZeocin™.5. Optional: Continue incubating the other culture for three more hours (for atotal of four hours) and then plate out all of the cells by spreading 200 μl on150 mm plates containing the appropriate concentration of Zeocin™.6. Incubate plates for 2–4 days at 30°C until colonies form.Mut Phenotype If you used a Pichia strain containing a native AOX1 gene (e.g. X-33, GS115,SMD1168H) as the host for your pPICZ construct, your Zeocin™-resistanttransformants will be Mut+. If you used a strain containing a deletion in theAOX1 gene (e.g. KM71H), your transformants will be Mut S.If you wish to verify the Mut phenotype of your Zeocin™-resistant transformants,you may refer to the general guidelines provided in the EasySelect™PichiaExpression Kit manual or the Original Pichia Expression Kit manual or topublished reference sources (Higgins & Cregg, 1998).You are now ready to test your transformants for expression of your gene ofinterest. See Expression in Pichia, next page.。
ANSYS中粘弹性材料的参数意义
ANSYS中粘弹性材料的参数意义ANSYS中粘弹性材料的参数意义：我用的材料知道时温等效方程（W.L.F.方程），ANSYS 中的本构模型用MAXWELL模型表示。
１．活化能与理想气体常数的比值（T ool-Narayanaswamy Shift Function）或者时温方程的第一个常数。
２．一个常数当用T ool-Narayanaswamy Shift Function的方程描述，或者是时温方程第２个常数３．定义体积衰减函数的MAXWELL单元数（在时温方程中用不到）４．时温方程的参考温度５．决定１、２、３、４参数的值６－１５定义体积衰减函数的系数，１６－２５定义fictive temperature的松弛时间这２０个数最终用来定义fictive temperature（在理论手册中介绍，不用在时温方程中）２６－３０和３１－３５分别定义了材料在不同物理状态时的热扩散系数３６－４５用来定义fictive temperature的fictive temperature 的一些插值一类的数值，时温方程也用不到４６剪切模量开始松弛的值４７松弛时间无穷大的剪切模量的值４８体积模量开始松弛的值４９松弛时间无穷大的体积模量的值５０描述剪切松弛模量的MAXWELL模型的单元数５１－６０拟合剪切松弛模量的prony级数的系数值６１－７０拟合剪切松弛模量的prony级数的指数系数值（形式参看理论手册）７１描述体积松弛模量的MAXWELL模型的单元数７６－８５拟合体积松弛模量的prony级数的系数值８５－９５拟合体积松弛模量的prony级数的指数系数值（形式参看理论手册）进入ansys非线性粘弹性材料有两项：（1）maxwell(麦克斯韦)模型最多可以输入95个常数（2）prony(普朗尼）模型这个模型下面又有三项：（a)shear Responsea1: 即理论中的C1-Relative modulus: 相对剪切模量t1: 即理论中的C2-Relative time: 相对时间（b）V olumetric Response（容积响应）a1: 即理论中的C1-Relative modulus: 相对弹性模量t1: 即理论中的C2-Relative time: 相对时间（c）Shift function (转换函数)有三项可以选择：（I）William-Landel, ferry: 时温等效方程Tref: 即理论中的C1-Relative temperature: 相对温度（对应《粘弹性理论》中的时温等效方程（WFL方程）应该是玻璃化转变温度）C1,C2: 没有什么好说的了，就是WFL方程的常量，与材料有关；（II）Tool-Narayanaswamy 方程Tref: 即理论中的C1-Relative temperature: 相对温度（应该是玻璃化转变温度）C1: 没有什么好说的了，就是TN常量；（III）用户定义Tref: 即理论中的C1-Relative temperature: 相对温度（应该是玻璃化转变温度）C1: 没有什么好说的了，就是方程的常量；-------------------------------------------------------------------------《粘弹性理论》TB, Lab, MAT, NTEMP, NPTS, TBOPT, EOSOPT如果Lab:MATMaterial reference number (defaults to 1; maximum equals 100,000).NTEMP:Number of temperatures for which data will be provided. Default = 1; Max = 6.NPTS:Number of pairs of Prony series. Default = 1 pair; Max = 6 pairs.TBOPT:Defines the relaxation behavior for viscoelasticity.1--(or SHEAR) relaxation behavior of the shear response.2--(or BULK) relaxation behavior of the volumetric response.如果Lab:SHIFTNTEMP:Allows one temperature for which data will be provided.NPTS:Number of material constants to be entered as determined by the shift function specified by3--for TBOPT = WLF2--TBOPT = TNTBOPT:Defines the shift function1--( or WLF) William-Landel-Ferry shift function. 2--(or TN) Tool-Narayanaswamy shift function. 100--(or USER) User-defined shift function。
USC 20 20-TB 3 机械切割器说明书
2. Make certain the machine is properly grounded. 3. Before operating the machine, remove ties, rings, watches, other jewelry, and roll up
sleeves above the elbows. Remove all loose clothing and confine long hair. Do not wear gloves. 4. Keep the floor around the machine clean. 5. Keep machine guards in place at all times when the machine is in use. 6. Do not over reach. Maintain a balanced stance at all times so that do not fall or lean against blades or other moving parts. 7. Make all machine adjustments or maintenance with the machine unplugged from the power source. 8. Replace warning labels if they become obscured or removed. 9. Make sure the power source switch is in the OFF position before connecting the machine to the power source. 10. Make a habit of checking to see that the keys and adjusting wrenches are removed before turning on the machine. 11. Keep belt guard and blade guards in place and in working order. 12. Failure to comply with all of these warning could lead to serious injury.
ASTM A20 中文版
压力容器用钢板通用要求SA-20/SA-20M(与ASTM标准A20/A20M-04完全等同)1 适用范围1.1 本标准包括一组通用要求，除专用材料标准中另有规定外，这些要求应适用于下列各项ASTM压力容器用轧制钢板标准：1.1.1 本标准还包括一组补充要求，适用于上列若干个标准中本身已指明的场合。
这些补充要求仅当买方在订货单中要求附加试验或检验并作了单独规定时采用。
1.2 附录X1叙述了制造压力容器钢板用卷材的生产及一些特性。
1.3 附录X2提供了关于压力容器用钢板拉伸性能变化性的资料。
1.4 附录X3提供了关于压力容器用钢板夏比V型缺口冲击试验性能变化的材料。
1.5 附录X4提供了包括冷弯时建议的最小内弯曲半径在内的有关钢板冷弯的资料。
1.6 这些材料仅适于熔焊，熔焊时应采用适合于该钢种级别及使用条件的焊接工艺。
1.7 如本标准与专用材料标准的要求不一致，则以专用材料标准的要求为准。
1.8 买方可以规定不否定本标准或专用材料标准任何条款的附加要求。
这些附加要求必须纳入采购订货单说明中，并取得供方同意。
1.9 为了确定供应材料是否与标准及材料标准的规定极限值相一致，各数值右面的有效位数应按E29推荐的圆整方法进行圆整。
1.10 以英寸-磅单位或SI单位表示的数值均可作为标准值。
本文中SI单位表示于括号内。
由于各单位制所表示的数值不能与另一种单位制进行精确换算，因此，每一种单位制必须独立使用。
1.11 本标准及所引用的材料标准均采用英寸-磅和SI两种单位，但除非订货单中规定采用“M”标志（SI单位），材料将以英寸-磅单位供货。
2 引用标准3 术语3.1 本标准专用术语说明：3.1.1 封顶钢---因受早期压盖过程而使沸腾作用受到限制的沸腾钢。
压盖可用机械方法，在瓶口式锭模顶部采用一个重的金属槽，也可用化学方法对敝口式锭模中钢水的顶部加铝或硅铁。
3.1.2 卷板—以卷材形式加工为成品钢板的热轧钢。
关于第二十条的英语作文
关于第二十条的英语作文英文回答：The 20th Amendment to the United States Constitution, adopted on February 6, 1933, addressed two main issues: the "lame duck" period and the start of presidential and congressional terms. Prior to the 20th Amendment, the president-elect and members of Congress elected in November did not take office until the following March 4th. This created a period of time where the outgoing administration, known as the "lame duck" period, had limited authority and effectiveness.To remedy this, the 20th Amendment established January 20th as the date for the inauguration of the president and vice president. This change aimed to reduce the "lame duck" period and allow the new administration to start promptly. The amendment also moved the start of congressional terms from March 4th to January 3rd, aligning the beginning of both the executive and legislative branches.The 20th Amendment had several significant implications. First, it strengthened the authority of the outgoing president during the "lame duck" period by ensuring that he or she would retain full powers until January 20th. Second, it eliminated the potential for mischief or inaction during the "lame duck" period as the outgoing administration would have less time to make significant policy decisions. Third, it allowed for a smoother transition of power between administrations and ensured that the new government could begin its work promptly.中文回答：第二十条修正案。
DisiTest 20 磨碎试验仪说明书
DisiTest 20The DisiTest 20 is a very easy-to-use disintegration tester with two independent stations.It has been specifically designed for use in the quality and production control of normal, plain coated, delayed release coated tablets and gelatin capsules in full compliance with current Pharmacopeia regulations. Simple operation, reliable mechanics and high-end key components make the DisiTest 20 the most user-friendly and reliable disintegration tester in its class.• 2 independent stations• Each station can be controlled, started and stopped individually• Baskets lower / raise automatically at start / completion of a test• Pause & Resume test function to visually check disintegration status• Large universal icon-based display for temperature control• Integrated display with simple control buttons for each station• Reliable basket design with quick-center coupling• Rigid one-piece water bath with rounded corners for easy cleaning• Precise temperature control with state-of-the-art immersion heater / circulator• Powerful circulation ensures +/- 0.07°C temperature stability• Built-in bath temperature sensor• Integrated electronic calibration functions• DIN 12876-1 Class 1 safety rating• Fully USP (chapters 701 and 2040) and EP (chapter 2.9.1) compliant design> DisiTest 20Disintegration TesterSimplicity maximizedUser-friendly operationUsing intuitive language-independent buttons, the num-ber of operator actions necessary to perform a test are kept to a minimum. Testing time can be quickly set in hours:minutes (hh:mm) or minutes:seconds (mm:ss) mode. Thereafter, it is only necessary to press the start key to automatically lower the basket into the test medi-um and begin or repeat a test. During testing, elapsed time or remaining time is displayed. If required, the test can be paused to visually check the disintegration status. Paused tests can be resumed in case not all tablets have disintegrated. When the set testing time has been reached, the basket automatically raises out of the test medium. To alert the operator on completion of a test, an audible alarm signal is given.Easy handlingFrom test setup to cleaning – the DisiTest 20 has been optimized for fast and ergonomic handling. The robust baskets with quick-center coupling can be conveniently exchanged within seconds and are easy to clean. Temperature controlThe DisiTest 20 incorporates a powerful immersion heater / circulator module with adjustable flow that guarantees highest temperature stability (+/- 0.07°C) of the water bath. Currently measured temperature and set target temperature are continuously displayed on the integrated 3.25" LCD screen. Three control buttons and on-screen prompts make setup and operation easy and straightforward. To simplify cleaning, the entire module can be quickly removed.A safe investmentUser-settable high-temperature safety prevents over-heating and provides low-liquid level protection. The unique design of the combined heater / circulator module with DIN 12876-1 Class 1 safety rating features an inte-grated pump and heater coil protection. Electronic cali-bration functions for temperature and stroke height are readily available and ensure full regulatory compliance.> Basket with quick-center coupling> Immersion heater / circulator modulewith integrated temperature sensor20012-0103/06-2015Your local sales & service partner3025 4110 26。
Coxsackievirus A6 and Hand, Foot, and Mouth Disease, Finland
Coxsackievirus A6 and Hand, Foot, and Mouth Disease, Finland Riikka Österback, Tytti Vuorinen, Mervi Linna, Petri Susi, Timo Hyypiä, and Matti WarisDuring fall 2008, an outbreak of hand, foot, and mouth disease (HFMD) with onychomadesis (nail shedding) as a common feature occurred in Finland. We identified an un -usual enterovirus type, coxsackievirus A6 (CVA6), as the causative agent. CVA6 infections may be emerging as a new and major cause of epidemic HFMD.Hand, foot, and mouth disease (HFMD) is a common childhood illness characterized by fever and vesicular eruptions on hands and feet and in the mouth (Figure 1). It is caused by members of the family Picornaviridae in the genus Enterovirus . Complications are rare, but pneumonia, meningitis, or encephalitis may occur. Outbreaks of HFMD have been mainly caused by 2 types of enterovirus A spe-cies, coxsackievirus (CV) A16 (CVA16) or enterovirus 71 (1). Some outbreaks have been associated with CVA10, but only sporadic cases involving other members of the entero-virus A species have been reported (2,3).During fall 2008, a nationwide outbreak of HFMD oc-curred in daycare centers and schools in Finland, starting in August and continuing at least until the end of the year and possibly into the following year. From vesicle fluid speci -mens of hospitalized children, we identified the etiologic agent as coxsackievirus A6.The StudyIn August 2008, vesicle fluid specimens were collected from 2 children and 1 parent with HFMD at the Central Hospital of Seinäjoki, Southern Ostrobothnia. Specimens were sent to the Department of Virology, University of Turku, for identification of the causative agent. After detec -tion of CVA6 in these index cases, the virus was also found in specimens obtained from the Pirkanmaa Hospital Dis-trict (Tampere), Turku University Hospital (Turku), Pori Central Hospital (Pori), and Central -Ostrobothnia Central Hospital (Kokkola) (Table).Nucleic acids were extracted from specimens by usingthe NucliSens EasyMag automated extractor (bioMèrieux,Boxtel, the Netherlands). When the extracts were analyzedfor enteroviruses by using real-time reverse transcriptase–PCR (RT-PCR) specific for the 5′ noncoding region (NCR)of picornaviruses (4), amplicons with melting points indis-tinguishable from each other and typical to enteroviruseswere obtained.To identify the enterovirus type in the specimens, RT-PCR, specific for a partial sequence of the viral protein 1 (VP1) region, was performed by using the COnsensus-DEgenerate Hybrid Oligonucleotide (CODEHOP) Prim-ers (bioinformatics.weizmann.ac.il/blocks/codehop.html) (5). The amplicons were separated by agarose gel electro-phoresis, purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), and sequenced in the DNA Sequencing Service Laboratory of the Turku Centre for Biotechnology. The virus type in the 3 index specimens, 3 samples of vesicular fluids, and 1 throat swab was success -fully identified with sequencing and BLAST (/BLAST) analysis as CVA6. Phylogenetic rela-tionships of the sequences were examined by using CVA6 (Gdula strain), CVA16 (G10), and enterovirus 71 (BrCr) prototype strains as well as selected clinical CVA6 isolates obtained from GenBank. Sequence alignments were gen-erated with the ClustalW program (/clust-alw), and the phylogenetic tree was computed by using the Jukes-Cantor algorithm and the neighbor-joining method. Phylogenetic analyses were conducted by using MEGA4 software () and the bootstrap con-sensus tree inferred from 1,000 replicates (6) (Figure 2).Author affiliations: University of Turku, Turku, Finland (R. Öster -back, T. Vuorinen, P . Susi, T. Hyypiä, M. Waris); and Central Hospi -tal of Seinäjoki, Seinäjoki, Finland (M. Linna)DOI: 10.3201/eid1509.090438Figure 1. Vesicular eruptions in A) hand, B) foot, and C) mouth of a 6.5-year-old boy from Turku, Finland, with coxsackievirus (CV) A6 infection. Several of his fingernails shed 2 months after the pictures were taken. D) Onychomadesis in a 10-year-old boy from Seinäjoki, Finland, 2 months after hand, foot and mouth disease with CVA6 infection. Photographs courtesy of H. Kujari (A–C) and M. Linna (D).Phylogenetic analysis placed all CVA6 strains from the HFMD outbreak in 1 cluster (97%–100% identity), whereas the nucleotide identities between those isolates and CVA6 prototype strains Gdula, CAV16, G-10, and en-terovirus 71 BrCr were 82.5%–83.2%, 55.6%–56.6%, and 55.6%–57.3%, respectively. The closest preceding CVA6 strain was isolated from cerebrospinal fluid in the United Kingdom in 2007 and had 92%–94% nucleotide identity with the strains described here (7).To improve the detection of the novel CVA6 strains in clinical specimens, we designed specific VP1 primers from the aligned sequences. CVA6vp1 reverse primer (5′-ACTCGCTGTGTGATGAATCG-3′) and CVA6vp1 for-ward primer (5′-CGTCAAAGCGCATGTATGTT-3′) gen-erated a 199-bp amplicon. First, cDNA was synthesized in a 20-μL reaction mixture containing 1 μmol/L CVA6vp1 reverse primer, 2.5 mmol/L of each dNTP, 20 U Rever-tAid H Minus M-MuLV reverse transcriptase (Fermentas, St. Leon-Rot, Germany), reaction buffer (Fermentas), 4 U RiboLock RNase inhibitor (Fermentas), and 5 μL RNA in-cubated at 42ºC for 1 h. Then, 5 μL of cDNA was added to 20 μL of master mixture containing 0.4 μmol/L each of the CVA6vp1 primers and Maxima SYBR Green qPCR Master Mix (Fermentas). PCR with melting curve analy-sis was performed in a Rotor-Gene 6000 real-time instru-ment (Corbett Research, Mortlake, Victoria, Australia) byDISPATCHESTable. Laboratory findings in clinical specimens and epidemiologic data for patients with CVA6 infections, Finland, 2008*City or place, identification Samplingdate Sex/age, y Specimen type Disease or signsCVA6-VP1RT-PCR VP1 sequence5ƍ NCRsequenceSeinäjokiFin/Se8717 2008 Aug M/1.3 Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Se8781 2008 Aug F/34 Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Se8841 2008 Aug M/0.9 Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Se8865 2008 Aug † Feces HFMD Pos NR CVA6 Fin/Se8913 2008 Sep M/10 Throat swab HFMD Pos NR CVA6 Fin/Se8925 2008 Sep M/0.9 Throat swab HFMD Pos NR CVA6 Fin/Se8926 2008 Sep † Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Se8927 2008 Sep F/1.3 Throat swab HFMD Pos NR CVA6 Fin/Se8928 2008 Sep † Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Se8931 2008 Sep M/0.8 Throat swab NA Pos NR CVA6 TurkuFin/Tu8859 2008 Sep M/5.8 Throat swab Fever, tonsillitis Pos CVA6 CVA6 Fin/Tu8866 2008 Sep M/3.1 Throat swab HFMD Pos NR CVA6 Fin/Tu81027 2008 Oct M/1.8 Throat swab HFMD Pos NR CVA6 Fin/Tu81042 2008 Oct M/1.8 Throat swab Fever, eczema Pos NR CVA6 Fin/Tu81038 2008 Oct F/2.2 Throat swab Fever, seizure Pos NR CVA6 Fin/Tu81274 2008 Nov M/6.5 Vesicle fluid HFMD Pos ND CVA6 Fin/Tu81309 2008 Dec F/3.7 Vesicle fluid HFMD Pos ND CVA6 Fin/Tu81321 2008 Dec F/1.2 Throat swab HFMD Pos ND NR Fin/Tu963 2009 Jan M/1.2 Throat swab HFMD Pos ND CVA6 Fin/Tu/IB 2009 Feb F/5.7 Nail Recent HFMD Pos ND CVA6 PoriFin/Po8959 2008 Oct M/4.8 Throat Fever, vomiting Neg ND CVA6 Fin/Po81375 2008 Dec M/0.5 Vesicle fluid HFMD Pos ND CVA6 Fin/Po81376 2008 Dec † Throat HFMD Pos ND CVA6 Fin/Po81324 2008 Dec M/10 Feces Fever, eczema Pos ND NR Fin/Po81325 2008 Dec M/10 Feces Fever, HFMDcontactPos ND NR TampereFin/Ta8966 2008 Sep M/3.1 Tracheal aspirate NA Pos NR CVA6 Fin/Ta81145 2008 Sep M/2.1 Feces NA Pos NR NR Fin/Ta81074 2008 Oct M/0.3 Tracheal aspirate NA Pos ND CVA6 Fin/Ta81125 2008 Oct F/0.7 Vesicle fluid HFMD Pos CVA6 CVA6 Fin/Ta81126 2008 Oct M/0.3 Vesicle fluid HFMD Pos NR CVA6 Fin/Ta81216 2008 Nov F/2.0 Throat swab NA Pos ND CVA6 Fin/Ta81252 2008 Nov M/6.1 Throat swab NA Pos ND CVA6 KokkolaFin/Ko937 2009 Jan M/10 Vesicle fluid HFMD Pos ND CVA6 *CVA6, coxsackievirus A6; VP1, virus protein 1; RT-PCR, reverse transcriptase–PCR; NCR, noncoding region; HFMD, hand, foot, and mouth disease; pos, positive; neg, negative; NA, not available; ND, not done; NR, no result (VP1 sequencing was attempted without result).†Same patient as on the line above.Coxsackievirus A6, Finlandusing the following cycling conditions: initial denaturation at 95ºC for 10 min, 45 cycles at 95ºC for 15 s, 60ºC for 30 s, and at 72ºC for 45 s, followed by generation of melting curve from 72°C to 95°C with temperature increments of 0.5°C/s. Partial 5′ NCR sequence of the strains in clinical specimens was determined as described (4) and compared with the known sequences by using BLAST (/Blast.cgi).During autumn 2008, a total of 47 acute-phase speci-mens, including 12 vesicle fluid samples, 23 throat swabs, 2 tracheal aspirates, 5 fecal samples, and 5 cerebrospinal fluid specimens from 43 patients yielded amplicons with similar melting points as the originally identified CVA6 strains in 5′ NCR RT-PCR. All specimens were subjected to the specific CVA6-VP1 real-time RT-PCR, and a positive result was obtained for 11 vesicle fluid samples, 14 throat swabs, 2 tracheal aspirates, and 4 fecal samples (Table). The virus in 1 throat swab was identified as CVA6 from the result of 5′ NCR sequencing alone. None of the CVA6-positive specimens were positive by an RT-PCR assay with CVA16- and EV71-specific primers (8). Attempts to culti-vate the virus from 8 CVA6 RT-PCR-positive specimens were unsuccessful, whereas the prototype strain could be propagated in rhabdomyosarcoma cells.Onychomadesis was 1 characteristic feature in patients during this HFMD outbreak; parents and clinicians reported that their children shed fingernails and/or toenails within 1–2 months after HFMD (Figure 1). Only a few published reports of nail matrix arrest in children with a clinical his-tory of HFMD exist in the medical literature (9–11). We obtained shed nails from 2 siblings who had HFMD 8 weeks before the nail shedding. The nail fragments were stored at –70°C for a few weeks and treated with proteinase K be-fore nucleic acid extraction. The extracts were enterovirus positive in 5′ NCR RT-PCR. The virus in one of them was identified as CVA6 by the specific RT-PCR and yielded a 5′ NCR sequence that was similar to the novel CVA6 strains.ConclusionsEnterovirus CVA6 was a primary pathogen associ-ated with HFMD during a nationwide outbreak in Finland in autumn of 2008. HFMD epidemics have primarily been associated with CVA16 or enterovirus 71 infections; those caused by enterovirus 71 have occurred more frequently in Southeast Asia and Australia in recent years (12). Re-portedly, CVA10 has been found in minor outbreaks; other coxsackievirus A types have been found in only sporadic cases of HFMD (2,3). In general, CVA6 infections have been seldom detected and mostly in association with her-pangina (13,14). In Finland, CVA6 has been identified only on 4 occasions over 8 years during enterovirus surveillance from 2000 to 2007 (15).Although the CODEHOP primers were elementary for rapid genotyping of the novel CVA6 strains, we identi-fied more viruses with the designated CVA6-VP1 specific primers. Onychomadesis was a hallmark of this HFMD outbreak. To our surprise, we detected CVA6 also in a frag-ment of shed nail. The same virus could have given rise to the outbreak in Spain in 2008 (10). Supposedly, virus rep-lication damages nail matrix and results in temporary nail dystrophy. Whether nail matrix arrest is specific to CVA6 infections remains to be shown. This study demonstrates that CVA6, in addition to CVA16 and enterovirus 71, may be emerging as a primary cause of HFMD.AcknowledgmentsWe thank Heidi Berghäll for nail samples, Harry Kujari for photographs, and Tiina Ylinen for assistance in the laboratory.Ms Österback is a doctoral candidate at the University of Turku in Finland. Her research interests are laboratory diagnostics and molecular epidemiology of viruses.References1. McMinn PC. An overview of the evolution of enterovirus 71 andits clinical and public health significance. FEMS Microbiol Rev. 2002;26:91–107. DOI: 10.1111/j.1574-6976.2002.tb00601.xCVA6 AB114096 Fin/Ta81125 FJ870502 Fin/Se8717 FJ870505 Finland 2008UK 2007Japan 1999–2003Bangladesh 1999–2002Figure 2. Phylogenetic analysis of coxsackievirus (CV) A6 partial (289 bp) viral protein 1 sequences showing the relationships between the recent clinical CVA6 samples isolated in Finland (triangles), selected CVA6 isolates from GenBank, and prototypes of CVA6, CVA16, and enterovirus (EV) 71. GenBank accession numbers are included. Scale bar indicates nucleotide substitutions per position.2. Kamahora T, Itagaki A, Hattori N, Tsuchie H, Kurimura T. Oligo-nucleotide fingerprint analysis of coxsackievirus A10 isolated in Japan. J Gen Virol. 1985;66:2627–34. DOI: 10.1099/0022-1317-66-12-26273. 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