Construction of eukaryotic expression vector carrying human TSLC1 gene and its expression in Hep
DC-SIGN真核表达载体的构建及其稳定转染HeLa细胞系的建立
DC-SIGN真核表达载体的构建及其稳定转染HeLa细胞系的建立王欣;詹媛;王琰;周恩民;丁铲;谭磊;陆凤;徐乐乐;仇旭升;宋翠萍;孙英杰;廖瑛;茅翔【摘要】为研究新城疫病毒(Newcastle disease virus, NDV)与树突状细胞特异性粘附分子-3-结合非整合素分子(dendritic cell specifi c intercellular adhesion molecule-3-grabbing non-integrin,DC-SIGN)的相互作用对NDV感染树突状细胞(dendritic cell,DC)的影响,本研究拟构建能够稳定表达DC-SIGN蛋白的细胞系,为研究DC-SIGN蛋白与病毒蛋白互作提供基础.以小鼠DCs提取的cDNA为模板,通过PCR方法获得DC-SIGN基因,连入真核表达载体,并命名为pcDNA-DC-SIGN,利用LipofectamineTM2000转染HeLa细胞,G418进行药物压力筛选,经RT-PCR和Western blot鉴定,获得了一株可以高效表达DC-SIGN蛋白的HeLa 细胞,且经过多次传代后仍然可以稳定表达DC-SIGN,说明该细胞系构建成功.%To investigate the infl uence of the interaction between Newcastle diseases virus (NDV) protein with dendritic cells specifi c adhesion molecules - 3 - grabbing non-integrin (DC-SIGN) on NDV infection of dendritic cell (DC), we generated a Hela cell line stably expressing DC - SIGN protein for studying DC - SIGN protein and virus protein interactions. The cDNA was extracted from mouse DCs and used as template. The DC - SIGN gene was amplifi ed in polymerase chain reaction (PCR) for construction of eukaryotic expression vector pcDNA-DC-SIGN, which was then transfected into HeLa cells using LipofectamineTM 2000. A HeLa cell line effi ciently expressing DC - SIGN protein was identifi ed through RT-PCR and Western blot, and the specifi c Hela cell line was stable after many passages.【期刊名称】《中国动物传染病学报》【年(卷),期】2015(023)003【总页数】5页(P12-16)【关键词】树突状细胞特异性粘附分子-3-结合非整合素分子;新城疫病毒;树突状细胞;HeLa细胞【作者】王欣;詹媛;王琰;周恩民;丁铲;谭磊;陆凤;徐乐乐;仇旭升;宋翠萍;孙英杰;廖瑛;茅翔【作者单位】西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海200241;中国农业科学院上海兽医研究所,上海 200241;西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海200241【正文语种】中文【中图分类】S852.659.5树突状细胞特异性粘附分子-3-结合非整合素分子（dendritic cell specific intercellular adhesion molecule-3 grabbing non integrin，DC-SIGN），又称CD209，由美国科学家在研究人类免疫缺陷病毒（Humanimmunodeficiency virus，HIV）感染机制过程中发现。
南极鱼Calmodulin基因在ZF4细胞中抗寒功能的分析
基因组学与应用生物学，2020年，第39卷，第11期，第4947-4952页研究报告Research Report南极鱼基因在Z F4细胞中抗寒功能的分析胡瑞芹w李根芳w陈良标@1上海海洋大学，海洋生物科学国际联合研究中心，上海,201306; 2上海海洋大学，水产科学国家级实验教学示范中心，上海,201306; 3上海海 洋大学，水产种质资源发掘与利用教育部重点实验室，上海,201306* 通信作者，***************.cn摘要为探究南极鱼Co/motWi/i基因在低温适应下的作用与应用，本研究从南极鱼m aw;-的CDNA文库中克隆得到CaM基因，通过构建真核表达载体并转染到ZF4细胞中，在低温胁迫下检 测细胞的存活率。
结果表明，南极鱼CaM基因能够在ZF4细胞中表达，并分布在细胞质中；在低温胁迫下,与对照组相比，过表达南极鱼基因的细胞的LDH毒性明显低于对照组，并且细胞活性明显高于对照 组。
因此，低温胁迫下南极鱼CaM基因的过表达能够显著提高ZF4细胞的抗寒能力。
本研宄结果证明了 一种候选的抗寒基因，为鱼类的抗寒育种提供了理论依据。
关键词南极鱼基因，低温胁迫，ZF4细胞，LDH细胞毒性，细胞活性Analysis on Cold Resistance of Antarctic Fish Calmodulin Gene in ZF4 CellsHu Ruiqin1Z3Li Genfang1,2,3Chen Liangbiao旧*1International Research Center for Marine Biosciences at Shanghai Ocean University, Shanghai, 201306; 2 National Demonstration Center for Experimental Fisheries Science Education, Ministry of Science and Technology, Shanghai Ocean University, Shanghai, 201306; 3 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306*Corresponsingauthor,***************.cnDOI: 10.13417/j.gab.039_004947Abstract To explore the role and application of Calmodulin gene in Antarctic fish under low temperature adaptation,the CaM gene was cloned from the cDNA library of Antarctic fish Dissostichus mawsoni,and the eukaryotic expression vector was constructed and transfected into ZF4 cells to examine the survival rate of the cells under low temperature stress.The results showed that the Antarctic fish CaM gene could be expressed in ZF4 cells and distributed in the cytoplasm.Under low temperature stress,compared with the control group,the LDH toxicity of the cells overexpressing the Antarctic fish CaM gene was significantly lower than that of the control group,and the cell activity was significantly higher.Therefore,the overexpression of CaM gene in Antarctic fish under low temperature stress can significantly improve the cold resistance of ZF4 cells.The results of this study demonstrate a candidate cold-resistant gene that provides a theoretical basis for cold-resistant breeding of fish.Keywords Antarctic fish CaM gene,Low temperature stress,ZF4 cells,LDH cytotoxicity,Cell viability温度是限制物种分布、影响个体生长和决定生 殖周期的一个基本环境因子。
流感病毒受体结合表位真核载体的构建及表达 (1)
论著流感病毒受体结合表位真核载体的构建及表达罗俊1,王欢1,戴军1,左斌1,裴德翠1,丰峰1,李婉宜1,邝玉1,王保宁1,蒋忠华1,李虹2,李明远1*(1.四川大学华西基础医学与法医学院微生物学教研室,四川成都610041;2.四川大学华西第二医院港大联合研究中心) 摘要 目的 构建甲型流感病毒血凝素(H A )信号肽(SP)与HA 唾液酸受体结合部位(RB)所含T 、B 表位的基因片段(RBT 、R BB)真核表达质粒,研究其在真核细胞中的表达与免疫特性。
方法 通过重叠延伸PCR 法(o ver lap PCR)将HA 的SP 分别与R B 、RBT 、RBB 通过一段多肽接头Gly4Ser 融合为SP RB 、SP RBT 和SP R BT ,将其分别插入pcD N A 3.1(+)载体,构建质粒,鉴定正确后转染M DCK 细胞,RT PCR 、免疫荧光、M T T 检测该质粒的表达和分泌。
结果 融合基因真核表达质粒构建成功,在M DCK 细胞中成功表达,转染细胞培养上清能刺激淋巴细胞增殖。
结论 SP RB 、SP RBT 、SP RBB 真核表达载体成功构建,表达产物能刺激淋巴细胞增值,为流感病毒唾液酸受体结合部位的T 、B 细胞表位免苗研究提供初步依据,也为流感核酸疫苗的研制奠定了基础。
关键词 流感病毒;T 细胞表位;B 细胞表位;免疫荧光技术;淋巴细胞增殖试验中图分类号 R373.13 文献标识码 A 文章编号 1673 5234(2011)01 0008 4[J our nal of Pathogen B iology .2011Jan;6(1):8-11.]C onstruction of eukaryotic expression plasmid for receptor binding of influenza virus and their expression LU O Jun 1,WANG H uan 1,DAI Jun 1,ZU O Bin 1,PEI Dei cui 1,FENG Feng 1,LI Wan yi,KU ANG Yu 1,WANG Bao ning 1,JIANG Zhong hua 1,LI H ong 2,LI M ing yuan 1 (1.D ep ar tment of M icrobiology ,West China School of Pr eclinical and For ens ic M edicine,Sichuan Univ er sity ,Chengdu 610041,China;2.T he J oint R esearch Center of W es t China Seco nd Univ er s ity H os p ital of S ichuan Univ er sity and the Faculty of M edicine,Univer sity of H ong K ong )Abstract Objective T o const ruct eukary otic ex pression vector s fo r the signal pept ide (SP )of hemagg lutinin (HA )o f influenza A vir us and its acylneuraminate recepto r binding (RB)sites incor po rating gene segments o f T and B epitopes (RBT and RBB,respectively)and to observe the expressio n o f t arg et pr otein by and immunolog ical characterist ics o f eu kar yot ic cells. M ethods A polypept ide linker Gly4Ser w as used to splice SP w ith RB,RBT ,and RBB by ov erlap P CR to r espectively construct SP RB,SP RBT ,and SP RBT fusio n g enes.T hese g enes w ere inserted into eukar yo tic expres sio n plasmids pcD NA 3.1(+).A fter identification,pcDN A3.1(+)/SP R B,pcDN A3.1(+)/SP RBT ,and pcDN A 3.1(+)/SP R BB wer e tr ansfected into M D CK cells.T he ex pr essio n o f these g enes was analy zed w ith RT P CR,immunoflu o rescence assay ,and lym phocyt e pr oliferation test with M T T . Results pcDN A 3.1(+)ex pr essio n vector s incor po ra ting SP RB,SP RBT ,and SP RBT fusion genes wer e successfully constr ucted.T he fusion pro tein w as detect ed in M DCK transfected with pcDN A 3.1(+)/SP RB,pcDN A 3.1(+)/SP RBT ,or pcD NA 3.1(+)/SP RBB and st imulated lym phocyte pro liferatio n. C onclusion Eukar yo tic ex pr essio n plasmids fo r SP RB,SP RBT and SP RBT fusion genes wer e successfully constructed and t he ex pression pro ducts stimulated lympho cyte pro liferatio n,establishing a solid foundation for fur ther study of receptor binding,immuno lo gical study o f T and B cell epitopes o f influenza v ir us,and study of DN A vaccines.Key words Influenza virus;T cell epito pe;B cell epitope;immunofluo rescence assay ;lymphocyte pro liferatio n test流感病毒的高度变异性常导致流感的流行,因此如何获得高效及安全的流感疫苗是科学工作者关注的课题。
Bax、Bad基因真核表达质粒构建及转染人胚肾细胞后的翻译水平观察
Bax A Bad基因真核表达质粒构建及转染人胚肾细胞后的翻译水平观察王孜恒一2，周敬伟一2，侯筱宇一2(1徐州医科大学，江苏徐州221000；2江苏省脑病生物信息重B实验室)摘要：目的构建Bcl-2相关X蛋白(Bax)、Bcl-2相关细胞死亡因子(Bad)基因真核表达质粒pcDNA3.1-Bax、pcDNA3.1-Bad,并观察两质粒转染至人胚肾293(HEK293)细胞后Bax,Bad的表达。
方法采用TRIzol试剂提取大鼠脑组织总RNA,利用反转录一聚合酶链反应(RT-PCR)扩增Bax和Bad的互补DNA(cDNA)序列，将cDNA分别克隆至pcDNA3.1载体上。
采用脂质体转染法将序列正确的pcDNA3.1-Bax和pcDNA3.1-Bad真核表达质粒分别转染至HEK293细胞，免疫印迹法鉴定Bax,Bad在细胞中的表达。
结果双酶切和DNA测序分析显示Bax和Bad基因片段成功插入pcDNA3.1载体中，并且与其cDNA(Genbank：NM_017059.2和AF031227.1)序列完全一致。
免疫印迹显示pcDNA3.1-Bax,pcDNA3.1-Bad真核表达质粒能够在HEK293细胞中高效表达。
结论pcD-NA3.1-Bax、pcDNA3.1-Bad真核表达质粒构建成功，Bax、Bad在HEK293细胞中成功过表达。
关键词:Bcl-2相关X蛋白；Bcl-2相关细胞死亡因子;真核表达质粒;人胚肾细胞doi：10.3969/j.issn.1002-266X.2019.30.006中图分类号:R743.31文献标志码:A文章编号:1002-266X(2019)30-0021-04Construction of eukaryotic expression plasmid of Bcl-2-assaciated X protein and Bcl-2-associated agonist of cell death and its expression in human embryonic kidney cellsWANG Ziheng1,ZHOU Jingwei,HOU Xiaoyu(1Xuzhou Medical University,Xuzhou221000,China)Abstract:Objective To construct the eukaryotic expression plasmids pcDNA3 .1-Bax and pcDNA3 .1-Bad for Bcl-2-assaciated X protein(Bax)and Bcl-2-associated agonist of cell death(Bad),and then to observe their expression in human embryonic kidney293(HEK293)cells.Methods Total RNA of rat brain tissues was extracted by using trizol reagent,cDNA sequences of Bax and Bad were amplified by reverse transcription-polymerase chain reaction(RT-PCR),and then were cloned into pcDNA3.1vectors.The constructed eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad were transfected into HEK293cells by liposome transfection,respectively.The expression of pcDNA3.1-Bax and pcD-NA3 .1-Bad plasmids in cells was identified by Western blotting.Results Double enzyme digestion and DNA sequencing results showed that Bax and Bad gene fragments were successfully inserted into pcDNA3.1vector,and were completely consistent with its cDNA sequences(Genbank:NM_017059.2and AF031227.1).Western blotting showed that the eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad were highly expressed in HEK293cells.Conclusion The eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad are successfully constructed,and Bax and Bad are highly expressed in HEK293cells.Key words:Bcl-2-associated X protein;Bcl-2-associated agonist of cell death;eukaryotic expression plasmids;human embryonic kidney cells凋亡最早是由英国病理学家Kerr JF教授于1972年根据细胞形态学改变提出的概念，是指细胞在接受外界信号刺激后启动的主动的程序性细胞死亡过程［］。
里氏木霉外源表达载体的构建_英文_
Constructi on of Exogenous Expression Vector of Tri -choder ma reeseiZHANG X i ao-xuan 1,WANG Ao -xue2*1.Chengdong Co llege ,Nort heast Agricultural Univ ers ity ,Har b in 150030;2.Co llege of Lif e Sc iences ,Nort heas tAgricult ural Universit y ,Har -bin 150030Abstract [Objecti ve]The st udy w as t o construc ta ex ogenous expression v ector f or Tri chode r m a ree se i .[M et ho d]U sing CBH I pro moter and t er m inat or of T.ree se i strain 40359,we cons tructed an expr ession vec t or o f T.ree se i s train 40359f or expressing Hp t gene and got s ix strains capab l e o f grow ing on basic m ediu m c ontaining 175mg /L of hygro myc i n B ,f urt her c onduc t ed hygr omycin resis t ance tes.t [R es ults ]In co mpar-i son w ith t he ori g inal strain(w il d t ype),hygro myc in r esis t ance t he six engineer ed strains was increased by 75%;t he hygro myc i n resis t ance c oul d inherit st ab l y .[Co ncl usio n]Our results laid bas is f or bi o l o g i c al study on T.ree se i atm ol e cul a r and genetic a lly engineeri n g levels .Key wo r ds T ri cho de r m a ree se i ;Vec t or ;Genetic transf or mati o nReceived :February 18,2011 Accept ed :March 21,2011*C orresponding author .E -mai:l wangaoxue @yahoo .co mC BH I pro mot er fro m Tri choder m a reese i is a very strong pro m oter that is usually used for vector constructi o n f or gene-t i c m i provement of T.reese i ,i .e .,inserting t he gene of inter -est(GOI)int o t he space bet w een CBHI promot er and ter m-i nat or sequence .The reco mbined frag ment is transf or med into fil a ment ous fungi prot oplas,t and located and int egrat ed into chromoso m e by us i n g t he m ethod of t argeted gene replace -ment and int egrati o n .This makes the GO I dri v en by t he strong pro mot er CBH I and the required homologous or heter -ologous prot ein expressed secret ory under the induction of t he l e ader peptide of C BH I .This method has been successf ull y used f or construct engineered filament ous fungistai n s overex -pressing various t arget prot eins .For inst ance ,MU Jing -yu e t a l .[1]from Jilin Universit y succeeded i n overexpression of As-pe rg ill u s n i g er glucose ox i d ase gene under the control of CBH I pro m oter fro m T.reese i .I n vie w of t his ,we att empt ed to ex -press hygro mycin B phos photrans f erase gene i n T.reese i by us i n g CBH I promot er and ter m i n at or sequence ,and carri e d out hygromyc i n B resist ance t est on the y i e lded resistant strains and reco mbinants ,am i i n g at prov i d ing basis for bi o log -i c al study on T.reese i at molecular and genetically engineer -i n g l e vels .Mat eri a ls and MethodsExperi m entalmateri al sStrai ns and reagents Tri chode r m a reese i strain 40359was purchas ed from Chi n a Center of IndustrialCulture Collection ;plas m id pCAM1305.1harboring hygromyc i n B phos photrans -ferase gene was preserved by our research group ;vectors pMD18and pU C19were purchas ed fro m TaKaRa ;EF Taq DNA pol y merase ,plas m id DNA isolation k it and prm i ers were purchas ed from Beijing Langang B i o t ech Co .,Ltd .;T 4-DNA ligase ,DL2000Marker ,RT -PCR k i,t CTAB ,Tris #C,l B -mer -capt oet hano,l RNase and agarose were all purchas ed fro m TaKaRa ;all the restrictive endonulceas es used were pur -chased from MB;I agarose gel extrac tion k it was purchased fro m T iangen Biot ech (Beijing)Co .,Lt d .All other reagents were home made products at analyti c pure grade .M ed i a r eci pe(for 1L) Basalmediu m:20g glucose ,15gKH 2PO 4,5g(NH 4)2SO 4,0.6g CaC l 2#2H 2O,0.6g M g -SO 4#7H 2O,0.005g FeSO 4#7H 2O ,0.0016g M n SO 4#H 2O,0.0014g ZnSO 4#7H 2O ,0.002g Co C l 2#6H 2O,pH=5.5;aut o -cl a ving at 121e f or 30m in (addition of 2%of agar f or solid m ediu m and 1%agar for sem-i soli d medium ).Regenerati o n m edia :(1)l o wer layermediu m :additi o n of1mol/L of s orb-i t oland 2%of agar into basalm ediu m;(2)upper layermed-i u m :addition of 1mol/L of sorbit ol and 1%of agar into basal m ediu m.Experi m entalmethods Pri m er s and their sequences used for PCR a mplifi cati on Based on sequence inf or mation from GenBank ,soft ware Gene runnerwas e mployed to design prm i ers as f ollo w s :amplificati o n of CBH I promoter and signalpeptide(pCBHI ):P1:Sense :5c -GC GCATGC AATTCTGGAGACGGCTT -GTT -3c :S ph ÑP2:Ant -i s ens e :5c -GCGT CGACCTCCAAGT GTTGC -CAT CGTA -3c :S a lÑamplification of C BH I t er m inator(t CBHI ):T1:Sense :5c -GCGCGGATCCAGGTCACCTTCT CCAA -CATCA -3c :Bam H ÑT2:Ant -i sense :5c -GCGCGAATT CCACGAAGAGCG -GCGATTCTA -3c :Eco R Ñamplification of Hp t :H1:Sense :5c -GCGC GTCGAC ATGCCTGAACTCAC -CGCGAC -3c S a l ÑH2:Ant -i sens e :5c -GC GC GGAT CC CGGTCGGCATC -TACT CT ATT -3c Bam H Ñ.A mplifi cati on of target frag ment (1)PCR reacti o n volu me (50L l)f or amplifying t he upstrea m nucleoti d e fragment of T.reese i cbh 1gene was composed of 312.5L mol/L dNTPs ,2.5L mol/L of prm i er ,500ng of t emplate ,3.5-3.6mmol/L of MgC l 2,2.5U EF Taq DN A poly merase .These reacti o n co mponents were first react ed at 94e f or 7m in predenat ur -ati o n before t he addition of EF Taq DNA poly meras e ,then 30cyc l e s at 94e f or 1m in ,60e f or 1m in and 72e for 2.5m in ,finally at 72e f or 10m i n .(2)PCR reaction volume(50L l)f or a mplif y ing t he downstream nuc l e otide fragment of T.reese i cbh 1gene was composed of 200L mol/L of dNTPs ,1.5L mol/L of prm i er ,500ng of te mpl a t e ,1.5-2.5mmol/L ofAgri c ultural B i o technol o gyAgricult ural Sc i e nc e&Technology ,2011,12(2):308-312Copyright k 2011,I nf or mati o n Institute ofHAAS .A ll ri g ht s reserv ed .MgC l2,1.5U EF Taq DNA poly meras e.These reacti o n co mpo-nent s were first reac t ed at94e f or7m i n predenat urationbef ore the addition of EF Taq DNA poly meras e,t hen30cycl e s at94e f or1m in,55e f or1.5m in and72e f or1.5m in,fi n ally at72 e f or10m in.(3)PCR reac tion volu me(50L l)f or a mplifying the Hp t gene was co mposed of312.5L mol/L of dNTPs,1.0 L mol/L of prm i er,500ng of t emplate,2.5mmol/L ofMgC l2, 1.5U EF Taq DNA poly merase.These reaction co mponents were first react ed at94e f or7m i n predenaturati o n bef ore t he additi o n of EF Taq DNA poly merase,then30cycles at94e f or30s,60e f or1m in and72e f or2.5m in,finally at72e f or10m in.Constr uction of expr ession vector Geno m ic DNA of T.reese i[2-3]and P l a s m i d contai n ing Hygro m ycin B phospho-transferase gene was extracted as te mplat e f or PCR amplif-i cati o n using prm i ers P1and P2t o a mplif y T.reese iCBHI pro-mot er and CBHI signal pepti d e gene;the reacti o n products were ligat ed i n t o p MD18-T vect or;t he y i e lded vect orwas then double-digest ed by us i n g S phÑand S a lÑf or use.Likew ise,T. reese iCBH I promot erwas a mp lifi e d using prm i ers T1and T2 and ligated int o p MD18-T vector;the y i e lded vector was then double-digest ed by using Bam HÑand Eco RÑf or use.Esche-ri ch i a co l i hygromyc i n B(HygB)phosphotransferase gene was amplified using prm i ers H1and H2from pl a s m id DNA har-boring this gene,and li g at ed int o pMD18-T vec t or;t he yielded vec t or was t hen double-digest ed by us i n g S a lÑand Bam HÑf or use.The frag m ents yiel d ed above were li g at ed by usi n g T4 ligase at16e overni g h;t t he y i e lded productwas transfor med i n t o co mpet ent E.co li cells v i a heat shock method and coat ed on LB sel e cti o n pl a te cont aining Amp and HygB,the pl a tes were incubat ed at37e overni g h.t The recombi n antwas inoc-ulated i n t o LB li q ui d medium cont aini n g Amp and HygB and in-cubat ed at37e f or12-16h(200r/m in).The yiel d ed turbid bact erial solution was used to extract plas m i d DNA f or PCR and enzy mati c di g estion.The constructs consist s of CBH I promoter-CBH I signal peptide gene-Hyg gene-C BH I ter m i n at or-pUC19vector,desig-nat ed as Pcbh-HygB.Deter m i nation on the resi stance of T.reesei strain to HygB B.Se m-i solid basal medi a contai n ing25,50,75, 100,125,150,175and200mg/L of HygB were respectivel y prepared and m i x ed w ith appropriat e vol u me of T.reese i strain40359.Them ixtures were poured into the l o wer l a yerof plat e and cultured at28e for the observation of mycelial grow t h.Transfor mati on o f the pr o toplast of T.reesei strai n40359 Prot opl a st preparati o n and transfor mati o n was ref erred to the method of PenttiL¾M.et a l.[3].Appropriat e volu me of transfor mati o n reacti o n soluti o n was coated on the lower layer of regeneration mediu m and i n cubated at28e f or12-16h, then a l a yer of se m-i solid fil a ment ous f ungiMM mediu m cont a-i ning125mg/L of HygB was poured on the pl a t e used above and i n cubat ed at28e for3-4d.The Hyg B-resist ant transfor ma-nt s were sel e ct ed f or furt hermolecular identificati o n.PCR i dentifi cation of transgen i c str ai ns PCR a mplificati o n was perf or med by using genom ic DNA as t empl a t e and t he upstream and downstrea m prm i ers of Hp t as prm i ers,w ith ge-no m ic DNA of non-transf or med strain40359as negati v e con-trol and plas m id DNA as positive contro.lHpt-spec ific prm i ers:s ense:5c-ATGCCT GAACTCACCGCGAC-3c;ant-i sense:5c-CGGTCGGCATCTACT CTATT-3cFuncti onal i dentificati on o f transgenic strai ns Transgenic strains were inoculat ed on PDA media w it hout hygro mycin f or 6conti n uous generations.The yiel d ed spores were scraped int o ddH2O and coat ed on solid m edia containing diff erent concentrations of HygB;these medi a were incubat ed under dark f or12-24h,then appropriate vol u me of s em-i s olid me-dium cont aining s ame concentration of hygromycin was poured into thes e plates.Likew ise,non-transf or m ed strain 40359was used as negative control f or observing myceli a l grow t h.Results and AnalysisA mplifi cati on o f pCBH I,tCBHI and Hpt genesGenom ic DNA of T.reese i strai n YB40359was extract ed by using m i proved CTAB method(F i g.1).The y i e lded ge-nom ic DNAs were allw it h molecul a rweight hi g her t han20kb and hi g hly pure,whi c h could be used f or subsequent exper-i men.t PCR results of all the Hp t gene,t CBH I and pCBHI as-su med a singl e band,w ith Hp t gene of approxm i ately1000bp, t CBH I of approxm i ately600bp,pCBH I approxm i ately1600bp (F i g.2).M:Marker15000;lanes1-8:G enom i c DNA samples.Fi g.1E l e ctrophoresis patt ern of geno m ic DNA of T.reese i strain40359M:2000bp m arker;lanes1and2:Hyg;3-4:t C BHI;4-5:pC BH I.Fi g.2Elec tr ophores is patt ern of PCR a mp lific ati o n result sC l oning of pCBHI,t CBHI and Hpt genesThe a mplified results of pCBHI,t C BH I and Hp t genes were ligat ed into pMD18T-vect or and incubat ed at16e over-309ZHANG X i a o-xuan e t a l.Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese inigh.t The li g at ed products were transfor med i n t o E.co li viaheat shock method and coated on LB pl a t e contai n ing Amp .Follow ing incubation for another 12-16h ,t wo white col o nies f or each gene were select ed f or PCR identificati o n .The re -sults showed that all six col o ni e s produced a band consistent w ith the size of t arget genes(Fig .3).The reco mbinants were identifi e d by using double diges -tion of Kpn I and S ph ,I the digestion produc t s were identifi e d w ith agarose gel electrophoresis(F i g .4).The double endonu -c l e ase di g esti o ns all produced t w o frag ments ,t he large one is the vector bond hi g her t han2.5kb ,the s mallone is the t arget genes :l a ne 1,about 600bp of t CBH I ;lane 2,about1000bp of Hp t ;lane 3,about 1600bp of pC BH I .This suggests that the t arget genes have been insert ed int o the c l o ning vec t or and successf ull y transf or med .The sequencing results (Shanghai Sangon Bioengineeri n g Co .,Lt d .)were blast ed and f ound an identity of 100%.M :2000bp marker ;1-2:Hyg ;3-4:t CB HI ;5-6:pC BH I .F i g .3 Bac t eriu m -based PCR a mplifi c ation on recombinantsM :2000bpmarker ;1:P l a s m id cont aini n g t CBH 1gene ;2:P l a s -m i d cont aini n g Hp t gene ;3:P l a sm i d cont ainin g pCBH 1gene .F i g .4 I dentifi c ation of reco mbined plas m id by double -di g esti o nI den tifi cati on expr essi on vector by usi ng PCR amp lifi ca -ti on and double endonucl ease digesti on As i n di c ated fro m Fig .5,PCR amplification results of all pCBHI ,t CBHI and Hp t genes accord w it h t hat us ed f or liga -tion ;l a ne 4is f or a mplificati o n of pC BH I p l u s Hp t gene ,w ith a size of approxm i atel y 2600bp ;lane 5is f or a mplificati o n of Hpt plus t CBH I gene ,w it h a size of approxm i at ely 1650bp .Endonucl e ase di g esti o n on recombinant s is using Eco R Ñpro -duced a s i z e of approxm i at ely 6000bp ,accordi n g w it h t he expect ed s iz e ;double endonulcease di g esti o n on recomb-inants is using S a l Ñand Bam H Ñto i d entif y Hp t gene ,whichproduced a larger band of approxm i at ely 4800bp(pU C19+pCBH I +t CBH I )and a s maller one of approxm i atel y 1000bp (Hp t gene);double endonul c ease digestion on reco mbinants is using S ph Ñand S a l Ñto identif y pCBHI ,which produced a lar -ger band of approxm i at ely 3300bp (pUC19+Hyg+t CBHI )and a s maller one of approxm i at ely 1600bp(pCBH I );double endonulcease di g esti o n on recombinant s is us i n g Bam H Ñand PstIto i d entif y t CBH I ,which produced a l a rger band of approx -m i at ely 4300bp(pUC19+Hyg+pCBHI )and a s maller one of approxm i at ely 600bp(t CBHI ).Not e :M:2000bp marker ;1:pC BHI ;2:t C BH I ;3:Hyg ;4:pC BH I and Hyg ;5:Hyg and t C BH I .Fi g.5 PCR a mplific ati o n on ex pres s ion vectorM1:15000bp mar ker ;M2:2000bp marker ;1:Sin g l e enzymedi g estion us i n g E co R Ñ;2:Dou b l e di g estion of S a l Ñand Bam H Ñt o i d entif y Hpt ;3:Doubl e di g estion of S ph Ñand S a l Ñto identif y pCB-H I ;4:Doubl e digesti o n of Bam H Ñand Pst Ñt o identif y t CBH I .Fi g.6 I dentifi c ation of ex press i o n by double -di g esti o nTransfor mati on of T.r eesei 40359and scr eeni ng of trans -for mantsRef erring t o PenttiL ¾M(1987),2-5L lof single endonu -clease di g est ed pUC19-Hyg was m ixed w it h the prot opl a st of T.reese i strain 40359(100s L l),using non -transfor med pro -t oplast as blank contro.l After coating on regenerationmedium containing HygB and incubat ed at 28e f or 3-4d ,six tran -f or m ants capabl e of grow ing on regeneration mediu m cont a-i ning 175mg/L of HygB .These six transf or m ants had st able and l a sting hygro mycin resi s tance aft er several conti n uous gen -erati o ns of culture .For the contro,l non -transf or med prot oplast of T.reesei strain 40359di d not grow onmediu m cont ai n ing 125mg/L of hygromycin ,indicati n g t hat there is no self resist ance m ut ation inw il d t ype strai n s .I dentificati on of tr ansgeni c strai ns PCR amp lifi cati on Geno m i c DNAs of hygro m ycin -resist ant strains all assumed a s i n gl e clear and orderly band(Fig .7).310Agricultural Sc i e nc e&Technology Vo.l 12,No .2,2011This indicat es that the DNA s amples have good qualit y and could be used for subsequent PCR amplifi c ati o n .Tab l e 1Gro w th sit uation of T.see se i strains on hy gro myc in Bp l a tesHy gro m y c in c oncentration M mg /LT.see se i Z40359T.see se i4035950++++75++++100+++125++-150++-175+-200--++repr es ent s we ll gro w th ;+r epres ents poor gro w th ;-repres ents no gro w th .F i g .7 E l e ctrophoresis patt ern of geno m ic DNA of genetic a llyengineered s tr a i nAs shown in Fig .8,PCR amplification on Hp t geneshowed t hat plas m id and six resist ant transf omants all produc -ed a target band of about 1000bp ,whil e non -transfor medstrain 40359di d no.t The results well demonstrat e that Hp t gene has transf or med and i n t egrated int o T.seese i strain 40359,designed as Z40359.M :2000bp marker ;1:Pos itive contro;l 2:Negati v e contro;l3-8:Transgenic s trains .Fi g.8 PCR det ection of pUC19-Hp t transgenic strainsResi stance and stab ility of tr ansgenic T.seesei strains tohygro myci n One of the T.seese i Z40359strains was cu-lt ured on solidm ediu m for s i x conti n uous generati o ns t o det ect its resistance t o hygro mycin(Fig .9).The results sho wed t hat T.seesei Z40359strains were res i s tant to175mg/L of hygro -m ycin ,and the resist ance l a st ed f or six generations .This confir m s that T.seeseiZ40359has a heredit ary stable resis-t ance t o hygromycin.1:Gene tically engineer ed T.ree se i s train Z40359on mediu m cont aining 100mg /L of hygro m yc in B ;2:P riginal strain 40359on med i u mcont aining 100mg/L hygro myc in B ;3:G eneti c all y engineered T.ree se i s train Z40359on mediu m cont aining 175mg/L of hygr omycin B ;4:O ri g i n a l s tr a i n 40359onm ediu m cont aining 175mg/L of hygro myc in B .Fi g.9 Toleranc e o fgeneticall y engineered T.reese i strain Z40359t o hygro myc i nD i s cussi o nThe re m arkable advantages of T.seese i inc lude its well capacit y of protein synt hesis and excretion and it s eukaryoti c excretory m echanis m ,as well as its protein modification per -for mance sm i ilar w ith ma mmal s yst e m ,such as hi g h -mannose type and N -gl y cosy l a tion .Thes e advant ages hugely pro mot e the geneti c modifi c ati o n of T.seese i strain ,and that construction of strong ext ernalgene expressi o n syst e m is t he precondition of itsmol e cul a r biol o gy st udy and genetic m i provement [4].The maj o r co mponent of T.reesei -excret ed extracellular prot ein i s cellul o se(CBH I ).So f ar ,t he cellulose cont ent of produced by T.reese i reached 40g/L ,several hundredstm i es over common bacteria [4].In the present st udy ,we e m -ployed PCR technique t o cl o ne the promot er and ter m i n at or of T.reese i strain YB40359,and f urt her est ablished strong ex -ternal gene expression geneti c transf or m ation syst em in T.reese i .Our results confir med the strong f unc tion of CBH I pro m oter and also lai d basis f or molecul a r st udy and geneti cm i provement of T.reese i .References[1]MU J Y(母敬郁),WANG Q(王峤),YAN G CZ (杨纯中),e ta l .Re -co mbinant Aspe r g ill u s n i ger gl u cose ox i d as e ex press ed in Tri -chode r m a reese i (瑞氏木霉表达黑曲霉葡萄糖氧化酶)[J].Chines eJ ournal of Biot echnol o gy (生物工程学报),2006,22(1):82-86.[2]JI A NG J(冮洁),DU LX (杜连祥),LU FP(路福平),e t a l .Themethod f or chro mos o me DNA pr eparation fro m reco mbinant Tri -chode r m a t ee se i (基因工程菌里氏木霉染色体DNA 的提取方法)[J].Bi o tec hn o l o gy(生物技术),2004,14(2):24-26.[3]PE NTT I L A M,NEVALAI NEN H ,R 'TT ;M ,e ta l .A v ersatile trans -f or mation syst e m f or t he cell u lol y tic fila ment ous fungus T ri chode r -rn a reese i [J].Gene ,1987,61(2):155-164.[4]WANG DH(汪大虹),WU ZH(吴志红).Construc ti o n of het er oge -n eous genes ex pression s yst e m of fila ment ous f ungus Tri chode r m ar e ese i (丝状真菌瑞氏木霉外源基因表达系统的构建)[J].Chines e J ournal of Bioc hem i s t ry and Molecul a r B i o logy (中国生物化学与分子生物学报),2003,19(6):736-742.Respons i bl e editor :DU AN Yong -boRespons i bl e proofreader :WU X i ao -yan311ZHANG X i a o -xuan e t a l .Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese i里氏木霉外源表达载体的构建(摘要)张晓烜1,王傲雪2*(1.东北农业大学成栋学院,黑龙江哈尔滨150030;2.东北农业大学生命科学学院,黑龙江哈尔滨150030)[目的]构建里氏木霉外源表达载体。
重组蛋白真核表达系统构建流程
重组蛋白真核表达系统构建流程1.从细菌中提取真核表达所需的重组蛋白的基因序列。
2. Extract the gene sequence of the recombinant protein required for eukaryotic expression from bacteria.3.将基因序列插入真核表达载体中。
4. Insert the gene sequence into the eukaryotic expression vector.5.确保基因插入的正确性，通过测序鉴定。
6. Ensure the accuracy of gene insertion through sequencing identification.7.将载体导入宿主真核细胞中。
8. Import the vector into the host eukaryotic cells.9.筛选出带有重组基因的真核细胞系。
10. Select eukaryotic cell lines with the recombinant gene.11.使用适当的诱导剂刺激真核细胞进行重组蛋白合成。
12. Stimulate eukaryotic cells with appropriate inducersto synthesize recombinant proteins.13.收集真核细胞培养上清液中的重组蛋白。
14. Collect recombinant proteins from the culture supernatant of eukaryotic cells.15.对纯化的蛋白进行鉴定和检测。
16. Identify and test the purified protein.17.通过离心等方法将蛋白精制并提纯。
18. Centrifuge and purify the protein by centrifugation and other methods.19.确定蛋白的浓度和纯度。
禽流感病毒HA-M2融合表位与EGFP蛋白在鸭胚成纤维细胞中的表达
禽流感病毒HA-M2融合表位与EGFP蛋白在鸭胚成纤维细胞中的表达王善辉;王文秀;沈志强;池贤凤;高三阳【摘要】本研究选择禽流感病毒H9N2株的HA的两个保守T细胞表位基因和M2基因胞外保守序列(M2e),经人工合成并通过PCR扩增获得HA-M2融合表位基因,将其与含有增强型绿色荧光蛋白的载体pEGFP-N1连接,构建了重组真核表达质粒HA-M2-pEGFP-N1.采用脂质体法转染体外培养的鸭胚成纤维细胞(DEF)后,通过RT-PCR、直接荧光观察和间接免疫荧光检测,结果表明,成功构建了重组真核表达质粒HA-M2-pEGFP-N1,且以HA抗原表位和保守的M2e序列为基础构建的重组蛋白能够在鸭胚成纤维细胞中成功表达.本试验为研制新型的禽流感通用疫苗,进一步探索HA基因与靶细胞相互作用及AIV的感染机理等奠定了基础.%In this study, two T cell epitopes genes of hamagglutinin conserved domains of avian influenza virus H9N2 strain and Ml gene of extracellular domain were synthetized and joined as HA-M2 multiple-epitopes fusion gene of avian influenza virus by RT-PCR. The recombinant eukaryotic expression plasmid of HA-M2 fusion gene containing enhanced green fluorescent protein (EGFP) report gene was constructed, and transfected by lipofectin 10 prepared duck embryo fibroblasts (DEF). The fluorescence expression was directly detected with fluorescence microscope, and the expression of HA-M2 was tested by RT-PCR and indirect immunofluorescence assay (IFA) respectively. The results indicated the successful construction of the eukaryotic expression plasmid HA-M2-pEGFP-Nl, and showed that HA-M2 gene was expressed efficiently in transfected DEF. It provided a basis forthe study of new universal influenza vaccines and further research on interaction of HA gene and its target cells and as for infection mechanismof AIV.【期刊名称】《中国畜牧兽医》【年(卷),期】2012(039)011【总页数】4页(P27-30)【关键词】禽流感;融合表位基因;增强型绿色荧光蛋白;鸭胚成纤维细胞【作者】王善辉;王文秀;沈志强;池贤凤;高三阳【作者单位】山东省滨州畜牧兽医研究院,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州 256600;山东绿都生物科技有限公司,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州256600【正文语种】中文【中图分类】S858.3禽流感是由A型流感病毒引起的禽类急性呼吸道传染病。
pcDNA3.0-CBP真核表达质粒的构建及对肺腺癌SPC-A1细胞的影响
·１４２０·ｐｃＤＮＡ３．０－ＣＢＰ真核表达质粒的构建及对肺腺癌ＳＰＣ—Ａ１细胞的影响周栋魏万胜苏云峰王志强王成苟云久【摘要】目的观察外源性ＣＢＰ基因稳定转染对人肺腺癌ＳＰＣ－Ａ１细胞体外牛长的影响。
方法构建ＣＢＰ真核表达质粒ｐｃＤＮＡ３．０．ＣＢＰ，应用ｐｃＤＮＡ３．０－ＣＢＰ和窄载体质粒ｐｃＤＮＡ３．Ｏ（一），脂质体转染法转染体外培养的人肺腺癌细胞株ＳＰＣ—Ａ１，Ｇ４１８（８００ｍｓ／Ｌ）筛选出抗性克隆。
Ｗｅｓｔｅｒｎｂｌｏｔ检测转染前后ＣＢＰ蛋白水平变化，噻唑蓝（Ｍ１Ｔｒ）比色法分析细胞生长抑制作用，Ｔｒａｎｓｗｅｌｌ体外侵袭实验和Ｗｏｕｎｄ．ｈｅａｌｉｎｇ实验对细胞进行侵袭和迁移能力研究。
结果转染ＣＢＰ基因的细胞株有目的基因整合和相应蛋白高表达。
ＭＴＹ检测ｐｃＤＮＡ３．０－ＣＢＰ转染组活细胞吸光度（Ｏ．２７８７±０．０７８６）低于未转染组（０．５０８９±０．１３０１）和ｐｅＤＮＡ３．Ｏ（一）空载体转染组（０．４８０４±０．１５４７）。
ｐｃＤＮＡ３．０－ＣＢＰ转染组与两对照组吸光度的差异有统计学意义（Ｐ＜０．０１）。
细胞侵袭实验表明ｐｃＤＮＡ３．０．ＣＢＰ转染组穿透滤膜数（３．５２±０．７７）明显少于未转染组（８．１７±０．８６）和空载体转染组（８．２２±１．３０），转染组与两对照组侵袭力的差异有统计学意义（Ｐ＜０．０１）。
细胞迁移实验转染组细胞迁移数（７１．３０±７．６８）明显少于未转染组（１１９．４０±８．３８）和空载体转染组（１１１．４１±１２．ｓ６），转染组与两对照组迁移力的差异有统计学意义（Ｐ＜０．０１）。
结论外源性ＣＢＰ基因稳定转染可抑制人肺腺癌ＳＰＣ．Ａ１细胞的恶性表型，町能通过对Ｓｒｃ家族激酶（ＳＦＫｓ）的负反馈调节抑制肿瘤细胞的增殖、侵袭。
【关键词】肺腺癌；基因转染；侵袭ＣｏｎｓｔｒｕｃｔｉｏｎｏｆｐｃＤＮＡ３．０．ＣＢＰｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄａｎｄｉｔｓｅｆｆｅｃｔｓｏｎｌｕｎｇａｄｅｎｃａｒｃｉｎｏ－ｎｌａｅｅｌｌｌｉｎｅＳＰＣ．ＡｌｚＪＦ，Ｄ￡，Ｄｏｎｇ，ＷＥ＇／Ｗａｎ．ｓｈｅｎｇ，ＳＵＹｕｎ一膨，ｌｇ，ｅｔａ１．ＤｅｐａｒｔｍｅｎｔｏｆＣａｒｄｉｏｔｈｏｒａｃｉｃＳｕｒｇｅｒｙ．ＳｅｃｏｎｄＨｏｓｐｉｔａｌｏ厂ＬａｎｚｈｏｕＵｎｉｖｅｒｓｉｔｙ。