兔抗M13多抗说明书
载体选择
1、pCMVp-NEO-BAN载体特点: 该真核细胞表达载体分子量为6600碱基对，主要由CMVp启动子、兔β-球蛋白基因内含子、聚腺嘌呤、氨青霉素抗性基因和抗neo基因以及pBR322骨架构成，在大多数真核细胞内都能高水平稳定地表达外源目的基因。
更重要的是，由于该真核细胞表达载体中抗neo基因存在，转染细胞后，用G418筛选，可建立稳定的、高表达目的基因的细胞株。
插入外源基因的克隆位点包括Sal1、BamH1和EcoR1位点。
注意在此载体中有二个EcoR1位点存在。
2、pEGFP, 增强型绦色荧光蛋白表达载体(Enhanced Fluorecent Protein Vector)特点: pEGFP表达载体中含有绿色荧光蛋白，在PCMV启动子驱动下，在真核细胞中高水平表达。
载体骨架中的SV40 origin使该载体在任何表达SV40 T 抗原的真核细胞内进行复制。
Neo抗性盒由SV40早期启动子、Tn5的neomycin/kanamycin抗性基因以及HSV-TK基因的聚腺嘌呤信号组成，能应用G418筛选稳定转染的真核细胞株。
此外，载体中的pUC origin 能保证该载体在大肠杆菌中的复制，而位于此表达盒上游的细菌启动子能驱动kanamycin抗性基因在大肠杆菌中的表达。
用途: 该表达载体EGFP上游有Nde1、Eco47111和Age1克隆位点，将外源基因扦入这些位点，将合成外源基因和EGFP的融合基因。
借此可确定外源基因在细胞内的表达和/或组织中的定位。
亦可用于检测克隆的启动子活性(取代CMV启动子,Acet1-Nhe1)。
3、pEGFT-Actin, 增强型绿色荧光蛋白/人肌动蛋白表达载体特点: pEGFP-Actin表达载体中含有绿色荧光蛋白和人胞浆β-肌动蛋白基因，在PCMV启动子驱动下，在真核细胞中高水平表达。
载体骨架中的SV40 origin使该载体在任何表达SV40 T 抗原的真核细胞内进行复制。
狂犬疫苗
狂犬疫苗研究进展石家庄学院化工学院12级生物工程杨申雨20120702039摘要：狂犬病是感染中枢神经系统的一种人兽共患的急性接触性传染病，由狂犬病痛毒(Rabies Vi—rus，RV)引起，病毒粒子聚集形成胞浆内包含体，只在神经细胞胞浆和蒲肯野氏细胞里存在。
RV可分成4个血清型和2个尚待定型的病毒株。
随着RV血清型变异，目前人用和动物用狂犬病疫苗毒株已不能提供针对所有种类RV的有效保护，为了对狂犬病及其研究进展有一个全面的认识，该文主要对RV病原以及狂犬病疫苗等方面的研究进展进行了综述。
关键字：发现过程、化学结构、理化性质、接种对象、生产工艺、趋势展望发明狂犬疫苗的是著名的法国生化学家路易斯•巴斯德。
一开始，巴斯德将狂犬病毒注射到家兔的体内，让病毒经过传代，再注射到健康狗的体内，他发现：经过多次传代后，病毒的毒性大大降低。
将这种病毒注射进健康狗的体内时，狗不仅不会发病，且能对狂犬病毒产生免疫力。
这一动物实验取得成功后，巴斯德又将多次传代的狂犬病毒随兔脊髓一起取出，并进行自然干燥减毒。
然后把这种脊髓研成乳化剂，用生理盐水稀释，制成了最早的兔脑基髓制备的减毒狂犬疫苗，并在治疗人的狂犬病时取得了成功。
虽然现在科学技术不断发展，人类目前用得最多的是人用狂犬病纯化疫苗，巴斯德的疫苗早已不再使用，但是，巴斯德是第一个成功发明狂犬疫苗的人，并用他的疫苗拯救了很多狂犬病人。
一，目前，用于人和动物狂犬病预防主要使用的是常规疫苗，常规疫苗尽管安全有效，但存在一些缺点。
随着分子病毒学和疫苗学的发展，研制安全、有效、经济、使用方便的狂犬病新型疫苗已成为当前开发的活跃领域。
目前已经和正在研制开发的狂犬病新型疫苗主要有亚单位疫苗、合成肽疫苗、DNA疫苗、活载体疫苗、基因缺失疫苗、抗独特型疫苗、转基因植物可食疫苗等。
下面就近年来国内外有关方面的研究进展进行综述。
1亚单位疫苗亚单位疫苗可以分为化学亚单位疫苗和基因工程亚单位疫苗。
ALFs 抗脂多糖因子
Q2monodon [10],ALFHa-1and ALFHa-2in lobster Homarus americanus [11],EsALF-1and EsALF-2in crab Eriocheir sinensis [12,13],and ALF Sp 1and ALF Sp 2in crab Scylla paramamosain [14,15].2.Materials and methods2.1.cDNA library construction and EST analysisA full-length cDNA library was constructed from the eyestalk of a P.trituberculatus using Creator SMART cDNA Library Construction*Corresponding author.Tel./fax:þ8653282898509.E-mail address:zhxcui@ (Z.Cui).Contents lists available at SciVerse ScienceDirectFish &Shell fish Immunologyjournal homepage:w ww.el /locate/fsi1050-4648/$e see front matter Ó2012Elsevier Ltd.All rights reserved.doi:10.1016/j.fsi.2012.01.021Fish &Shell fish Immunology xxx (2012)1e 8123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110Kit (Clontech).Random sequencing of the library using universal primer M13F (Table 1)yielded 4606ESTs [22].BLASTx analysis revealed that two ESTs were homologous to ALF in shrimp Litope-naeus stylirostris (GenBank accession no.AAY33769).One EST ptcesa3_5_D01(GenBank accession no.GT563293)was selected for further cloning of ALF.2.2.Full-length cDNA sequences determination2.4.Genomic DNA ampli ficationGenomic DNA was extracted from the muscle tissue by standard phenol e chloroform method [24].To detect the genomic structure of PtALF4,two pairs of gene-speci fic primers (4_GF1and 4_GR1,4_GF2and 4_GR2;see Table 1)were designed according to the obtained cDNA sequences.The PCR was performed in a 25m l reaction volume containing 17.3m l sterile distilled H 2O,2.5m l of mM),0.5m l of dNTP (10mM),1m l U)of Taq polymerase (TaKaRa),and 30ng).The PCR temperature by 34cycles of 94 C for 30s,s,and a final extension at 72 C for cloned and sequenced according to 2.2.sample preparation(150Æ10g)were in Qingdao,China,and acclimated processing.During the experiment,once daily at night.60crabs were experiment.The crabs were and each contained 20individuals.100m l live V.alginolyticus resus-7.0,106CFU/ml)at the arthrodial leg were used as challenge group.an injection of 100m l PBS were groups,respectively.The injected tanks and three individuals were point of 3,6,12,24and 32h post-from the last walking leg using an equal volume of anticoagulant sodium citrate,336mM NaCl,pH 7.0)[25].Samples were imme-for 5min to collect the hemocytes.eyestalk and muscle from three determine the tissue distribution transcript expressiontissues,including hemocytes,muscle of untreated crabs,and transcript in hemocytes chal-determined by quantitative real-tissues and hemocytes was according to the manufacture ’s cDNA was synthesized using and oligo dT with 2m g of by DEPC-treated water for the assay was carried out in an ABI System (Applied Biosystems).The 4_cR;see Table 1)were used to amplify the corresponding product.The b -actin from P.trituberculatus [26],ampli fied with primers Actin-F and Actin-R (Table 1),was chosen as a reference gene for internal standardi-zation.DEPC e water for the replacement of cDNA template was used as negative control.The PCR was carried out in a total volume of 20m l,containing 10m l of 2ÂSYBR Premix Ex Taq Ô(TaKaRa),0.4m l 50ÂROX Reference Dye,4m l of the diluted cDNA mix,0.4m l of each primer (10m M),and 4.8m l of sterile distilled H 2O.The PCR program was 95 C for 5min,followed by 40cycles of 95 C for 5s4_GR1CGCCGAAACGCTTAGAAATAC Genomic cloning 4_GF2GACGCTCTGAAGGACTTTATG Genomic cloning 4_GR2ATAGTATCACATTCACAGTCAGGC Genomic cloning Actin-F TCACACACTGTCCCCATCTACG Real-time RT-PCR Actin-R ACCACGCTCGGTCAGGATTTTCReal-time RT-PCR1-3_RE-F GGATCCCAGTATGAGGCTCTGGTAARecombinant expression 1_RE-R CTCGAGTCAGAAGACGACACAATACTTAC Recombinant expression 3_RE-R CTCGAGTTAGTTATTCAGCCACAGAGAA Recombinant expression 4_RE-F GGATCCGGGTGGCTAGACATTGTAAAAG Recombinant expression 4_RE-RCTCGAGTTAGCGGTGGTTGAGCCAGGGTRecombinant expressionThe Bam HI and Xho I sites are underlined.Y.Liu et al./Fish &Shell fish Immunology xxx (2012)1e 82111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240and60 C for31s.Each sample was run in triplicate along with the internal control gene.To confirm that only one PCR product was amplified and detected,dissociation curve analysis of amplification products was performed at the end of each PCR reaction.After the PCR program,data were analyzed with ABI7300SDS software (Applied Biosystems).Fold change for the gene expression relative to controls was determined by the2ÀDD Ct method[27].All data were given in terms of relative mRNA expression as meanÆS.E.The results were subjected to one-way analysis of variance(one-way ANOVA)using SPSS13.0,and the P values less than0.05and0.01 were considered statistically significant.2.7.Expression and purification of recombinant PtALF1,PtALF3and PtALF4Three pairs of gene-specific primers,1-3_RE-F and1_RE-R,1-3_RE-F and3_RE-R,and4_RE-F and4_RE-R,were designed to amplify the sequences encoding the mature peptides of PtALF1, PtALF3and PtALF4,respectively(Bam HI and Xho I sites are under-lined,see Table1).The purified PCR products were inserted into pMD18-T simple vector,and digested completely by restriction enzymes Bam HI and Xho I(NEB),then subcloned into the Bam HI/ Xho I sites of expression vector pET-32a(þ)(Novagen).The recombinant plasmids pET-32a-PtALF1,pET-32a-PtALF3and pET-32a-PtALF4were transformed into E.coli BL21(DE3)-pLysS(Nova-gen)and subjected to DNA sequencing,respectively.The pET-32a vector without insert fragment was selected as a negative control, which could express a thioredoxin(Trx)with6ÂHis-tag in the prokaryotic expression system.After sequencing to ensure in-frame insertion,positive transformants of PtALF1,PtALF3,PtALF4and negative control were incubated in LB medium(containing100m g/ ml ampicillin)at37 C with shaking at220rpm.When the culture reached OD600of0.5e0.7,isopropyl-b-D-thiogalactosidase(IPTG) was added to thefinal concentration of1mM,and incubated for additional4h under the same conditions.Cells were harvested by centrifugation at8000g for5min at4 C,resuspended in buffer I (50mM sodium phosphate,300mM NaCl,pH7.0),and sonicated at 4 C for30min in a combination of2s sonication and2s interval under180W power.The cell lysates were centrifuged at8000g for 10min at4 C to collect the inclusion bodies.The inclusion bodies were washed twice with buffer I,then washed twice with buffer II (50mM sodium phosphate,300mM NaCl,2M urea,pH7.0),and dissolved in buffer III(50mM sodium phosphate,300mM NaCl, 8M urea,pH7.0).The recombinant PtALF1(designated as rPtALF1), PtALF3(rPtALF3),PtALF4(rPtALF4)and negative control sample (rTrx)were purified by TALON Metal affinity resins(Clontech) under denaturing conditions.The purified proteins were refolded in gradient urea e TSB glycerol buffer(50mM Tris e HCl,50mM NaCl,10%glycerol,1%glycine,1mM EDTA,0.2mM oxidized glutathione,2mM reduced glutathione, a gradient urea concentration of6,5,4,3,2,1,0M urea in each gradient,pH8.0;each gradient at4 C for12h).Then the resultant proteins were separated by15%sodium dodecyl sulfate-poly-acrylamide gel electrophoresis(SDS-PAGE),and visualized with Coomassie brilliant blue R250.The purified protein solutions were concentrated with Microsep Advance Centrifugal Devices(3kD,Pall corporation)based on the manufacturer’s instructions.The concen-tration of rPtALF1,rPtALF3,rPtALF4and rTrx was measured by BCA (bicinchoninic acid)Protein Assay Kit(Beyotime),respectively.2.8.Antimicrobial activityAntimicrobial activity was measured against two Gram-negative bacteria V.alginolyticus L59and Pseudomonas aeruginosa P25,two Gram-positive bacteria Micrococcus luteus M2and Staphylococcus aureus S7,and one fungus Pichia pastoris GS115, using a liquid phase assay modified from that of Rathinakumar et al.[28].The minimal inhibitory concentration(MIC)was determined as methods of Hancock(http://cmdr.ubc.ca/bobh/methods/). V.alginolyticus L59,P.aeruginosa P25,M.luteus M2,S.aureus S7and P.pastoris GS115were grown in TSB medium at28 C,TSB medium at37 C,LB medium at37 C,LB medium at37 C and YPD medium at28 C to mid-logarithmic phase and diluted with Tris e HCl (50mM,pH8.0)to103CFU/ml,respectively.In sterile96-well plates,50m l of rPtALF1,rPtALF3or rPtALF4in1/2-fold serial dilu-tion with Tris e HCl(50mM,pH8.0)were added into the wells.The wells with50m l of Tris e HCl(50mM,pH8.0)and50m l of rTrx diluted with Tris e HCl(50mM,pH8.0)were used as blank group and negative control,respectively.And then50m l of cell suspension (1Â103CFU/ml)were added into the wells and mixed.The96-well plates were incubated at corresponding temperatures for up to3h, and150m l of corresponding growth medium were added,then the mixtures were allowed to recover overnight.Absorbance at600nm for Gram-positive bacteria or560nm for Gram-negative bacteria and fungus of each well was determined using a precision micro-plate reader.The assay was performed with triplicates in three independent experiments.The minimum inhibitory concentration (MIC)value was recorded as the range between the highest concentration of the protein where microbial growth was observed and the lowest concentration that caused100%inhibition of microbial growth[29].3.Results3.1.cDNA cloning and sequence analysis of PtALF4The complete cDNA sequence of PtALF4was obtained by over-lapping the corresponding ESTs with the amplified fragments. Sequence analysis showed that PtALF4was consistent with the characteristics of all the other ALF family members.The nucleotide and deduced amino acid sequences were shown in Fig.1,and the sequence data were deposited in GenBank under the accession number JF756050.The full-length cDNA sequence of PtALF4was1353bp.It con-tained a50-untranslated region(UTR)of80bp,30-UTR of895bp and an open-reading frame(ORF)of378bp encoding125deduced amino acids(Fig.1).The poly(A)tail was found in PtALF4,while no canonical polyadenylation signal-sequence(AATAAA)was detec-ted.The putative signal peptides were located at the N-terminus, which was cleaved at amino acid positions25e26.The estimated molecular weight of mature PtALF4(100amino acids)was 11.20kDa and its theoretical isoelectric point was9.07.BLAST analysis revealed that the deduced amino acid sequence matched a variety of ALFs previously submitted to GenBank. However,the sequence similarities were relatively low.PtALF4dis-played38%amino acid identity with Macrobrachium rosenbergii ALF isoform3(MrALF3,ADI80708),37%with L.stylirostris ALF(LstALF, AAY33769),27%with PtALF3and20%with PtALF1.Multiple align-ment of15decapod ALF proteins revealed that all these ALFs con-tained a signal peptide and an LPS-binding domain(Fig.2). Moreover,they all possessed two conserved cysteine residues which could form a disulfide bridge.The positively charged amino acid residues were major clustered within the disulfide loop.A consensus pattern of W(T)CPG(S)WT(A)was also detected in all ALFs.3.2.Genomic organization of PtALF4The amplified genomic DNA fragment of PtALF4was2379bp, and deposited in GenBank under accession number JF756054.By aligning with the corresponding cDNA sequences,the exon e intronY.Liu et al./Fish&Shellfish Immunology xxx(2012)1e83241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370boundaries of PtALF4were determined (Fig.3).The genomic sequence contained three exons (49,137and 774bp)interrupted by two introns (704and 522bp).The sequences coding for the LPS-binding domain were present in exon2.All splice sites followed the canonical GT/AG splicing recognition rule.Several tandem repeats were found in both introns.One pure dinucleotide repeat (AT)20and one 16bp tandem repeat (TAAACTTAACCTAACC)3were observed in intron1,two stretches of pure dinucleotide repeats (GT)44and (TC)43were identi fied in intron2(Fig.3).3.3.Tissue distribution of PtALF4transcriptQuantitative real-time RT-PCR was employed to quantify mRNA expression of PtALF4in the tissues of healthy crabs,including hemocytes,gill,hepatopancreas,eyestalk and muscle (Fig.4).ThemRNA transcript could be detected in all the examined tissues with signi ficant variation.The highest expression level was showed in eyestalk which was 13.31-fold (P <0.01)higher than that in hemocytes,while moderate expression in gill and hemocytes,lower expression in hepatopancreas and muscle.3.4.Temporal change of PtALF4transcript after V.alginolyticus challengeThe temporal mRNA expression of PtALF4transcript in hemo-cytes post V.alginolyticus challenge was shown in Fig.5.During the first 3h after V.alginolyticus challenge,the expression was induced and increased to 1.77-fold compared with that in the control group.Then,the transcript expression decreased slightly at 6h post-injection.As time progressed,the expression level wasup-Fig.1.Nucleotide and deduced amino acid sequences of PtALF4from Portunus trituberculatus .Numbers on the right of the sequence give the positions of the last nucleotide and amino acid on each line,respectively.The putative signal peptides are underlined.The LPS-binding domains are shadowed.Two conserved cysteine residues are boxes.The stop codons are indicated by asterisks(*).Fig.2.Multiple alignments of PtALF4with other known ALFs.Amino acid residues that are conserved in at least 60%of sequences are shaded in dark,and similar amino acids are shaded in gray.Numbers on the right indicate the amino acid position of the different sequences.The signal peptides are boxed.The LPS-binding domains are enclosed with bracket.The conserved cysteine residues are marked with arrowheads below the alignment.The species and the GenBank accession numbers are as follow:Portunus trituberculatus PtALF1(ADU25042),P.trituberculatus PtALF3(ACS45385),Scylla paramamosain SpALF1(ABP96981),Scylla serrata SsALF (ACH87655),Fenneropenaeus indicus FiALF (ADE27980),Macrobrachium rosenbergii MrALF (AEP84102),Penaeus monodon PmALF3(ABP73289),Litopenaeus schmitti LscALF (ABJ90465),Procambarus clarkii PcALF (ADX60063),Penaeus monodon PmALF6(ADM21460),Eriocheir sinensis EsALF-1(ABG82027),Eriocheir sinensis EsALF-2(ACY251862Q 1),Scylla paramamosain SpALF2(AEI88034)and Litopenaeus stylirostris LstALF (AAY33769).Y.Liu et al./Fish &Shell fish Immunology xxx (2012)1e 84371372373374375376377378379380381382383384385386387388389390391392393394395396397398399400401402403404405406407408409410411412413414415416417418419420421422423424425426427428429430431432433434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476477478479480481482483484485486487488489490491492493494495496497498499500that that sion fold Fig.3.repeats are 024681012141618Hemocytes Gill Hepatopancreas Eyestalk MuscleTissue distribution of PtALF4T h e r e l a t i v e e x p r e s s i o no f P t A L F 4Fig.4.Tissue distribution of PtALF4transcript was measured by SYBR Green RT-PCR.Vertical bars represent the mean ÆS.E.(n ¼3).The transcript levels in gill,hepato-pancreas,eyestalk and muscle are normalized to that in hemocytes.Signi ficant differences across hemocytes are indicated with two asterisks at P <0.01.Time post V ibrio alginolyticus challenge (h)Fig.5.Temporal expressions of PtALF4transcript in hemocytes after V.alginolyticus challenge were measured by SYBR Green RT-PCR.Vertical bars represent the mean ÆS.E.(n ¼3).Samples challenged with PBS were adopted as control (white bars).Signi ficant differences across control in the same time of sampling are indicated with an asterisk at P <0.05,and two asterisks at P <0.01.Y.Liu et al./Fish &Shell fish Immunology xxx (2012)1e 85501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630BL21(DE3)-pLysS.After IPTG induction,the whole cell lysate and insoluble fraction analyzed by SDS-PAGE revealed rPtALF1,rPtALF3 and rPtALF4were mainly expressed as insoluble proteins and accumulated in inclusion bodies.They had distinct bands with molecular weight of about26,30and29kDa,respectively (Fig.6A e C),which were in accordance with the predicted molecular mass of fusion proteins.The purified and refolded rPtALFs were of the same molecular weight.Meanwhile,the transformant with pET-32a vector was induced and a unique21kDa expressed product representing Trx was detected and purified from the IPTG induced whole cell lysate(Fig.6D).The concentration of the rPtALF1,rPtALF3 and rPtALF4protein was1.65,2.15and3.08mg/ml,respectively.3.6.Antimicrobial activities of rPtALF1,rPtALF3and rPtALF4Antimicrobial activities and MIC of rPtALFs were determined and summarized in Table2.No significant antimicrobial activity of rTrx was observed.The purified rPtALF3could inhibit all the tested Gram-negative,Gram-positive bacteria and fungus with highest activity against Gram-negative bacteria V.alginolyticus L59and P.aeruginosa P25(MIC value of0.29e0.58m M).The rPtALF1and rPtALF4proteins displayed inhibitory activities against Gram-negative bacteria and fungus P.pastoris GS115,but no obvious antibacterial activity was observed against Gram-positive bacteria M.luteus M2and S.aureus S7.4.DiscussionPtALF4is distinct from the previously cloned PtALF1and PtALF3 from the same crab species[21],yet it is clearly members of the ALF family.The deduced protein sequence of PtALF4shares the conserved structures with other ALFs[11,15,17],including the signal peptide,the LPS-binding domain and two conserved cysteine residues at the both ends of the domain.All these structures especially the positively charged amino acids in the disulfide loop are important for ALFs biological activities[8,9,30].It is apparent that PtALF4and PtALF1-3are transcribed from separate genomic loci and are not the result of differential splicing. Consistent with that of PtALF1-3,the genomic organization of PtALF4is formed by three exons and two introns.SuchgenomicFig.6.SDS-PAGE analysis of rPtALF1(A),rPtALF3(B),rPtALF4(C),and rTrx(D).Lane M:protein marker(kDa);lane A1:negative control for rPtALF1without IPTG induction;laneA2:IPTG induced rPtALF1;lane A3:purified rPtALF1;lane B1:negative control for rPtALF3without induction;lane B2:induced rPtALF3;lane B3:purified rPtALF3;lane C1:negativecontrol for rPtALF4without induction;lane C2:induced rPtALF4;lane C3:purified rPtALF4;lane D1:negative control for rTrx without induction;lane D2:induced rTrx;lane D3:purified rTrx.Y.Liu et al./Fish&Shellfish Immunology xxx(2012)1e86631632633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738739740741742743744745746747748749750751752753754755756757758759760[12,14,15,17,35],exhibits a broad spectrum antimicrobial activity toward Gram-positive,Gram-negative bacteria and also fungus.However,no obvious anti-Gram-positive bacterial activity is observed in rPtALF1and rPtALF4treatment groups.Such functional diversity is also found in recombinant EsALF-1and EsALF-2proteins [12,13].Consistent with that reported in Brogden [36]and Imjongjirak et al.[15],we also presume the differences of the charged amino acids (e.g.arginine and lysine)could account for the observed different antimicrobial activities among the recombinantPtALF proteins.As the first report of antimicrobial properties of ALFs from P.trituberculatus ,our results indicate PtALF1,PtALF3and PtALF4would provide candidate promising therapeutics or agents in crab disease.Acknowledgmentsby the National Natural Science and the Chinese National ‘863’Dr.Zhaoxia Cui.D.Antimicrobial peptides in insects;Immunol 1999;23:329e 44.of cationic antimicrobial peptides in innatee 10.S.Anti-LPS factor:an anticoagulantactivation of coagulation system.e 23.T,Tanaka S,Iwanaga S,Ohashi K,et al.of Limulus anticoagulant (anti-LPS factor)(LPS).J Biochem 1985;97:1611e 20.N,Amato SF,Black KM,Kirsch SJ,et al.by Limulus antilipopolysaccharide e 13.R.Crystal structure of an endotoxin-crab,Limulus anti-LPS factor,at 1.5A 6.T,Iwanaga S.Primary structure ofAmerican horseshoe crab,Limulus poly-e 30.V,Tassanakajon A.Locali-(ALFPm3)in tissues of the black tiger of its binding properties.Dev A,Moulin G,Tassanakajon A,et al.NMRfactor from shrimp:model Biopolymers 2009;91:207e 20.R,Hirono I,Aoki T,Tassanakajon A.in the black tiger shrimp Penaeus monodon Org 2004;61:123e 35.N,Smith CM,Shields JD,Small HJ,et al.the American lobster Homarus americanus :response to Vibrio fluvialis D Genomics Proteomics 2008;3:263e 9.H,Gai Y,et al.Molecular cloning,genomicof an antilipopolysaccharide factor from .Dev Comp Immunol 2008;32:784e 94.J,Gai Y,Qiu L,et al.The second anti-with antimicrobial activity from Erio-2010;34:945e 52.Tassanakajon A,Sittipraneed S.Anti-mud crab Scylla paramamosain :molecular the antimicrobial activity of its synthetic 2007;44:3195e 203.A.Molecular cloning,genomicactivity of a second isoform of anti-the mud crab,Scylla paramamosain .Fish cloning and expression pro file ofin Chinese shrimp (Fenneropenaeus e 8.LP,Zhang T,Zhao XF,et al.An anti-swamp cray fish,Procambarus clarkii ,in vitro and in vivo .Fish Shell fish Immunol [18]Muroga K,Suzuki K,Ishimaru K,Mogami K.Vibriosis of swimming crab Por-tunus trituberculatus in larviculture.J World Aquacult Soc 1994;25:50e 4.[19]Wang GL,Shan J,Chen Y,Li Z.Study on pathogens and pathogenesis ofemulsi fication disease of Portunus trituberculatus .Adv Mar Sci 2006;24:527e 31.[20]Yue F,Pan L,Miao J,Zhang L,Li J.Molecular cloning,characterization andmRNA expression of two antibacterial peptides:crustin and anti-lipopolysaccharide factor in swimming crab Portunus trituberculatus .Comp Biochem Physiol B Biochem Mol Biol 2010;156:77e 85.[21]Liu Y,Cui Z,Luan W,Song C,Nie Q,Wang S,et al.Three isoforms of anti-lipopolysaccharide factor identi fied from eyestalk cDNA library of swim-ming crab Portunus trituberculatus .Fish Shell fish Immunol 2011;30:583e 91.Table 2Antimicrobial activities and minimal growth inhibition concentrations (MIC)of rPtALF1,rPtALF3and rPtALF4.MicroorganismsMIC (m M)rPtALF1rPtALF3rPtALF4Gram-negative bacteria Vibrio alginolyticus L590.50e 1.010.29e 0.58 1.66e 3.32Pseudomonas aeruginosa P25 1.01e 2.010.29e 0.5826.57e 53.14Y.Liu et al./Fish &Shell fish Immunology xxx (2012)1e 87761762763764765766767768769770771772773774775776777778779780781782783784785786787788789790791792793794795796797798799800801802803804805806807808809810811812813814815816817818819820821822823824825826827828829830831832833834835836837838839840841842843844845846847848849850851852853854855856857858859860861862863864865866867868869870871872873874875876877878879880881882883884885886887888889890[22]Liu Y,Cui Z,Song C,Wang S,Li Q.Multiple isoforms of immune-related genesfrom hemocytes and eyestalk cDNA libraries of swimming crab Portunus tri-tuberculatus.Fish Shellfish Immunol2011;31:29e42.[23]Thompson JD,Gibson TJ,Plewniak F,Jeanmougin F,Higgins DG.The ClustalXwindows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res1997;25:4876e82.[24]Sambrook J,Russell DW.Molecular cloning:a laboratory manual.New York:Cold Spring Harbor Laboratory Press;2001.[25]Rodriguez J,Boulo V,Mialhe E,Bachere E.Characterisation of shrimp hae-mocytes and plasma components by monoclonal antibodies.J Cell Sci1995;108:1043e50.[26]Cui Z,Liu Y,Luan W,Li Q,Wu D,Wang S.Molecular cloning and character-ization of a heat shock protein70gene in swimming crab(Portunus tritu-berculatus).Fish Shellfish Immunol2010;28:56e64.[27]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method.Methods2001;25: 402e8.[28]Rathinakumar R,Walkenhorst WF,Wimley WC.Broad-spectrum antimicrobialpeptides by rational combinatorial design and high-throughput screening:the importance of interfacial activity.J Am Chem Soc2009;131:7609e17. [29]Casteels P,Ampe C,Jacobs F,Tempst P.Functional and chemical character-ization of hymenoptaecin,an antibacterial polypeptide that is infection-inducible in the honeybee(Apis mellifera).J Biol Chem1993;268:7044e54.[30]Ried C,Wahl C,Miethke T,Wellnhofer G,Landgraf C,Schneider-Mergener J,et al.High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor.J Biol Chem1996;271:28120e7.[31]Tharntada S,Somboonwiwat K,Rimphanitchayakit V,Tassanakajon A.Anti-lipopolysaccharide factors from the black tiger shrimp,Penaeus monodon are encoded by two genomic loci.Fish Shellfish Immunol2008;24:46e54. [32]Yedery RD,Reddy KVR.Identification,cloning,characterization andrecombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab,Scylla serrata.Fish Shellfish Immunol2009;27: 275e84.[33]Keller R.Crustacean neuropeptides:structures,functions and comparativeaspects.Experientia1992;48:439e48.[34]Nagoshi H,Inagawa H,Morii K,Harada H,Kohchi C,Nishizawa T,et al.Cloning and characterization of a LPS-regulatory gene having an LPS binding domain in kuruma prawn Marsupenaeus japonicus.Mol Immunol2006;43: 2061e9.[35]Somboonwiwat K,Marcos M,Tassanakajon A,Klinbunga S,Aumelas A,Romestand B,et al.Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor(ALF)from the black tiger shrimp Penaeus monodon.Dev Comp Immunol2005;29:841e51.[36]Brogden KA.Antimicrobial peptides:pore formers or metabolic inhibitors inbacteria?Nat Rev Microbiol2005;3:238e50.Y.Liu et al./Fish&Shellfish Immunology xxx(2012)1e8 8891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928。
原位杂交原理及具体操作
原位杂交原理及具体操作原位杂交实验原理与方法一、目的本实验的目的是学会原位杂交的使用方法。
了解各种原位杂交的基本原理和优缺点。
二、原理原位杂交组化（简称原位杂交，in situ hybridization histochemistry ；ISHH）属于分子杂交的一种，是一种应用标记探针与组织细胞中的待测核酸杂交，再应用标记物相关的检测系统，在核酸原有的位置将其显示出来的一种检测技术。
原位杂交的本质就是在一定的温度和离子浓度下，使具有特异序列的单链探针通过碱基互补规则与组织细胞内待测的核酸复性结合而使得组织细胞中的特异性核酸得到定位，并通过探针上所标记的检测系统将其在核酸的原有位置上显示出来。
当然杂交分子的形成并不要求两条单链的碱基顺序完全互补，所以不同来源的核酸单链只要彼此之间有一定程度的互补顺序（即某种程度的同源性）就可以形成杂交双链。
探针的种类按所带标记物可分为同位素标记探针和非同位素标记探针两大类。
目前，大多数放射性标记法是通过酶促反应将标记的基因掺入DNA 中，常用的同位素标记物有3H、35S、125I 和32P。
同位素标记物虽然有灵敏性高，背底较为清晰等优点，但是由于放射性同位素对人和环境均会造成伤害，近来有被非同位素取代的趋势。
非同位素标记物中目前最常用的有生物素、地高辛和荧光素三种。
探针的种类按核酸性质不同又可分为DNA探针、cDNA探针、cRNA探针和合成寡核苷酸探针。
cDNA 探针又可分为双链cDNA探针和单链cDNA 探针。
原位杂交又可分为菌落原位杂交和组织原位杂交。
菌落原位杂交( Colony in situ hybridization ) 菌落原位杂交是将细菌从培养平板转移到硝酸纤维素滤膜上，然后将滤膜上的菌落裂菌以释出DNA。
将NDA烘干固定于膜上与32P 标记的探针杂交，放射自显影检测菌落杂交信号，并与平板上的菌落对位。
组织原位杂交( Tissue in situ hybridization ) 组织原位杂交简称原位杂交，指组织或细胞的原位杂交，它与菌落的原位杂交不同。
MUM1抗体试剂免疫组织化学说明书
MUM1抗体试剂（免疫组织化学）说明书【产品名称】通用名称MUM1抗体试剂（免疫组织化学）英文名称：FLEX Monoclonal Mouse Anti-Human MUM1 Protein, Clone MUM1p, Ready-to-Use (Dako Omnis)【包装规格】12mL【预期用途】体外诊断用途。
在常规染色（如：HE染色）基础上进行免疫组织化学染色，为医师提供诊断的辅助信息。
MUM1抗体试剂（免疫组织化学），预期与Dako Omnis全自动免疫组化染色系统配合用于免疫组化（IHC）分析。
该抗体可标记MUM1蛋白，MUM1蛋白表达于生发中心明区的B细胞亚群（代表B细胞分化晚期）、浆细胞和活化T细胞。
其结果可辅助血液淋巴肿瘤的分类以及淋巴系统恶性肿瘤的亚型分类（1, 2）。
临床上解释任何染色或其缺失都应辅以使用适当对照的形态学研究，并应由有资质的病理学家结合患者临床病史和其他诊断检测进行评价。
该抗体预期在使用非免疫组化法的常规病理学染色对肿瘤进行初步诊断的基础上使用。
【检验原理】抗原别名：IRF4（干扰素调节因子4）、ICSAT（活化T细胞干扰素保守序列结合蛋白）和PIP（PU.1相关元件）（3）MUM1蛋白（多发性骨髓瘤癌基因-1）是一种由MUM1基因编码的50 kDa蛋白质，在多发性骨髓瘤（MM）中观察到由于一种罕见的t（6;14）（p25;q32）染色体易位，导致MUM1基因与Ig重链基因座并置（4, 5）。
Mum1-蛋白缺陷小鼠无法形成生发中心、脾和固有层缺乏浆细胞，因而血清免疫球蛋白水平显著降低，难以产生可检测的抗体应答、T淋巴细胞毒性反应或抗肿瘤反应（1）。
MUM1蛋白的表达并不依赖于t（6;14）（p25;q32）易位，可在多发性骨髓瘤、淋巴浆细胞淋巴瘤（LPL）、弥漫大B细胞淋巴瘤（DLBCL）和活化T细胞中检测到。
MUM1蛋白表达对浆细胞分化无特异性（1-3, 6）。