【作者】 常彩芳；

【导师】 冯国和；

【作者基本信息】 中国医科大学 ， 内科学传染病学， 2010， 硕士

【摘要】 目的构建乙脑病毒(Japanese encephalitis virus, JEV)非结构(non-structure, NS)4A蛋白编码基因重组子,将其转染中华仓鼠卵巢(Chinese hamster ovary, CHO)细胞并用间接免疫荧光法探讨该重组子的表达特征,为进一步研究JEV NS4A蛋白的生物学功能奠定基础。材料与方法一、实验材料(一)细胞、质粒及菌株CHO细胞购自中国科学研究院上海细胞库,生长于37℃,5%CO2及含10%胎牛血清(Fatal calf serum, FCS)的Dulbecco’modified eagle medium (DMEM)高糖培养液中；含FLAG片段的JEV NS4A蛋白编码基因重组质粒被命名为pJNS4A由本实验室构建；真核表达载体pcDNA3.1(+)购自Invitrogen公司,用于JEV NS4A蛋白编码基因重组子的构建；大肠杆菌JM109与DH5a购自Takara公司,用于重组质粒构建、转化。(二)试剂及抗体RNA抽提试剂(Code No.D312), RT-PCR试剂盒(Code No.DR027A),DNA片断纯化试剂盒(Takara, product No.DV807), DNA A尾试剂盒(Takara, product No.D404),琼脂糖凝胶DNA纯化试剂盒(Takara,product No.DV805),DNA连接试剂盒(Takara,product No.D6023),质粒小剂量提取试剂盒(Takara,productNo.DV801A),限制性内切酶EcoRI、BamHI均购自Takara公司,用于重组质粒的构建和酶切鉴定。脂质体(Lipofectamine2000)购自Invitrogen公司,用于转染CHO细胞。氨苄青霉素购自Sigma公司,琼脂糖购自Takara公司,用于质粒的转化和电泳分析；DMEM高糖培养液,10%FCS,青霉素购自海科隆公司,用于CHO细胞的培养；兔抗鼠IgG Fc(一抗),购自Cell Signaling公司,山羊抗兔(荧光二抗)购自北京中杉金桥公司,用于质粒转染后的免疫荧光检测。二、实验方法(一) JEV NS4A蛋白编码基因重组质粒构建与鉴定以JEV SA14-14-2株Total RNA为模板,运用RT-PCR方法扩增JEV NS4A基因片段,并在该基因片段的5’端加入FLAG序列,再在其两侧加入起始密码子与终止密码子,最后在其两侧加入BamHI/EcoRI酶切位点。将反转录引物：5’GAATTCTTAGGCTCCTGCACAAGCT ATGAC3’及限制性内切酶EcoRI(-GAATTC-)合成至JEV NS4A编码基因5’末端。PCR法扩增前向引物5’GGATCCATGACTAAAAAACCAGGAGGGC3’,将BamHI(-GGA TCC-)合成至5’末端,反向引物为上述反转录引物。1%琼脂糖凝胶电泳并回收最终PCR产物,后者与pMD19-T simple连接构建重组子pMD-JNS4A。使用PrimeSTAR(?) HS DNA Polymerase (CodeNo.DR0--10S),对重组质粒CTB756-T-7为模板,以CTB906 F(5’CGGGATCCATGG ACTAC AAGG ACGACGATGAC AAGATGTC AGCCGTTAG CTTCATAG3’)为引物进行PCR扩增。将PCR扩增产物使用TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No. DV805A)进行切胶回收约900bp的片段后,使用BamHI/EcoRI进行酶切,经乙醇沉淀后,命名为Insert DNA；将质粒pcDNA3.1(+)使用BamHI/EcoRI进行酶切,然后使用TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code--No.DV805A)切胶回收约5.4kbp的DNA片段后,命名为Vector DNA;使用TaKaRa DNA Ligation Kit (Code No. D6022)中的Solution I将Insert DNA和Vector DNA连接后,热转化至E.coli Competent CellJM109(Code No.D9052)中,涂布平板,37℃过夜培养。挑选阳性菌落植菌,提取质粒命名为CTB906-2,使用引物T7、BGHrev对上述质粒进行测序,结果正确。转化pJNS4A于大肠杆菌DH5α,小剂量提取质粒pJNS4A,并用BamHI/EcoRI酶切进行核酸电泳鉴定。(二)pJNS4A稳定转染CHO细胞采用脂质体稳定转染法即Lipofectamine 2000将pJNS4A转染CHO细胞,同时设转染空载体pcDNA3.1(+)的CHO细胞与未转染的正常CHO细胞为阴性对照。具体方法为：1、转染实验前在6孔板中培养CHO细胞,待细胞生长浓度约90%-95%,即每500ul含0.5-2.0×105／个。配制脂质体／质粒混合物：溶液A、B：分别将重组质粒pJNS4A和空载体pcDNA3.1(+)各8μg(每孔)加入DMEM培养液(不含胎牛血清)500ul中溶液总体积各为250μl(每孔),轻轻混匀,标记；溶液C：将20μl脂质体加入500μlDMEM培养液中,共四管,轻轻混匀,室温孵育5分钟；分别将A与C,B与C液体混合,室温放置20分钟(这时溶液会变浑浊)以形成脂质体／质粒混合物；弃去6孔板中的原液,向6孔板中逐滴加入上述混合液体,边加边摇动培养板,每孔2ml,做标记。在37℃,5%的CO2中培养4小时后,改用无血清无抗生素的DMEM培养液继续培养。2、筛选6孔板培养24小时后,改为24孔板继续培养。用含G418浓度依次为100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900ug／mL的DMEM培养。每种浓度设3个孔,转染空质粒的CHO细胞及正常CHO细胞为对阴性照组。培养15 d后,以能杀死所有正常CHO细胞的最低G418的浓度作为本实验的最佳筛选浓度即为100 ug／m L,维持培养。(三)免疫荧光检测将转染pJNS4A的CHO细胞、转染空载体的CHO细胞及正常的CHO细胞接种于6孔板中的盖玻片,以盖满玻片不溢出为度,标记。于37℃,5%CO2的条件下常规培养待细胞密度达到约60%-70%时,4%多聚甲醛固定细胞1 h,0.1%Triton X.100打孔10 min,10%BSA封闭1 h,兔抗鼠IgG Fc分别按1：800稀释,4℃孵育过夜。FITC标记山羊抗兔IgG按1：00稀释浓度,于37℃避光孵育1 h,50%甘油封片后于荧光显微镜下观察荧光标记。结果1、JEV NS4A蛋白编码基因构建(1)以JEV SA14-14-2株Total RNA为模板,经重组构建含有FLAG片段的JEV NS4A蛋白编码基因重组子pJNS4A。经酶切鉴定表明：重组质粒经BamHI/EcoRI酶切释出的插入子与pJNS4A经相同酶切释出插入子的分子量大小相一致,均约为900bps,使用引物T7、BGHrev对上述质粒进行测序,含有JEVNS4A蛋白编码基因片段,证明质粒构建成功。(2)将pJNS4A在感受态大肠杆菌中扩增,提取质粒RNA,行核酸电泳对其鉴定。可见在约900bps的位置上有一明显条带,证明质粒构建成功。2、JEV NS4A蛋白编码基因重组质粒转染CHO细胞免疫荧光鉴定转染pJNS4A的CHO细胞可见存在较显著绿色荧光标记,主要分布在胞膜。在正常CHO细胞及转染空质粒的CHO细胞中未见特异性绿色荧光标记,仅见散在的非特异性绿色荧光杂质。结论1、构建的重组子pJNS4A含有JEV NS4A编码基因。2、转染了重组子pJNS4A的CHO细胞能够稳定表达JEV NS4A,其主要分布在胞膜。更多还原

【Abstract】 The recombinant plasmid pJNS4A was constructed by RT-PCR method. The plasmid was transfected into China hamster ovary(CHO) by Lipofectamine 2000. The coding protein expression and distribution was detected by immunoflurescence. This study will lay the foundation for the interaction between no-structural proteins of Japanese encephalitis virus(JEV), and virus virulence.Materials and Methods1. Materials(1) Cells, plasmids and strainsChinese hamster ovary cells were purchased from shanghai cell bank of chinese academy of science and grown at 37℃in Dulbecco’modified eagle medium(D-MEM) supplemented with 10% fatal calf serum(FCS) and 5%CO2.BALB/c mice were purchased from shanghai animal center of chinese academy of science.The recombinant with gene encoding pJNS4A was constructed by us before and named pJNS4A, eukaryotic expression vector pcDNA3.1(+) was purchased from Invitrogen company, which was used for construction of fusion gene encoding JEV NS4A protein.Escherichia coli JM109 and DH5a were purchased from Takara company and were used for construction and transformation of recombinant plasmid.(2) Main reagents and antibodyRNAiso Reagent(Takara, Code No.D312), High Fidelity PrimeScriptTM RT-PCR Kit(Takara, Code No.DR027A), DNA Fragment Purification Kit Ver.2.0(Takara, Code No.DV807), DNA A-Tailing Kit(Takara, Code No.D404), Agarose Gel DNA Purification Kit Ver.2.0(Takara, Code No.DV805), DNA Ligation Kit＜Mighty Mix>(Takara, Code No.D6023), Plasmid Minidose Abstraction Kit(Takara, product No. DV801 A), restriction endonuclease EcoRI、BamHI and NotI were all purchased from Takara company, which were used construction and identification of recombinant plasmid. Lipofectamine 2000 was purchased from Invitrogen company and was used for transfection into CHO cells. Ampicillin was puchased from Sigma company, Agarose was purchased from Takara company, they were all used for transformation and electrophoretic analysis of plasmid. Dulbecco’modified eagle medium,10% fatal calf serum, penicillin and streptomycin were all purchased from Hyclone company and used for cultivation of CHO cells. DYKDDDDK Tag Antibody(Binds to same epitope as Sigma’s Anti-FLAG M2 Antibody) was purchased from Santa company, Fluorescein-Conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) was purchased from beijing Zhongshan company, they were all used for immunofluorescence.2. Methods(1) The recombinant construct of JEV NS4A protein encoding geneTotal RNA of SA14-14-2 strain in Japanese encephalitis virus is as templates, using RT-PCR to amplify JEV non-structura protein 4A genes and addin initiation codon and termination codon to the Bilateral genes; Bilateral genes are admoved BamHI/EcoRI Enzyme Cutting site Reverse transcription primer:5’GAATTCTTA GGCTCCTGCACAAGCTATGAC 3’, restriction incision enzyme EcoRI(-GAA TTC-) is synthetic in 5’terminatio PCR amplification prorsad primer:5’GGATCCATGA CTAAAAAACCAGGAGGGC 3’,BamHI(-GGA TCC-) is synthetic in 5’terminatio, reverse primer is primer above-mentioned.Electrophoresis using 1% Agarose Gel and retrieve PCR product, latter and pMD19-T simple bond to build recon pMD-JNS4A.Use PrimeSTAR(?) HS DNA Polymerase(Code No.DR010S)to undertake PCR amplification of pMD-JNS4A in according to CTB 906F(5’CGGGATCCAT GGACTACAAGGACGACGATGACAAGATGTCAGCCGTTAGCTTCATAG3’)as templates. PCR amplifi-cation product is named Insert DNA after carried out to cutting glue and retrieved about 900bp fragment using TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No. DV805A) with used BamH I/EcoRI to realized enzyme incise and undertaked ethanol precipitation. Plasmid pcDNA3.1(+) is named Vector DNA. after Using BamH I/EcoR I to proceed enzyme incise and using TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut glue and retrieve about 5.4kbp DNA fragments; Insert DNA and Vector DNA then are spread plate and stay overnight cultivation in 37℃after using Solution I in TaKaRa DNA Ligation Kit (Code No. D6022) to couple with them followed thermo-conversion to E.coli Competent Cell JM109 (Code No. D9052), then choosing male bacterial colony to plant and extraction for plasmid named pJNS4A. Using primer BGHrev to proceed sequencing of above-mentioned plasmid, result is well. Transform pJNS4A to escherichia coli DH5a and extract low dose plasmid followed identification by BamH I/EcoR I enzyme incise and Electrophoresis.(2) PJNS4A transiently transfected into CHO cellsTransfection:Before the experiment in 6-well plates in cultured CHO cells, the concentration of cell growth to be about 90%-95% per 500ul containing 0.5-2.0×105/ month.The preparation of liposome/plasmid mixture:solution A:The recombinant plasmid pJNS4A and empty vector pcDNA3.1 (+) of 8μg (per hole) by adding DMEM culture medium 500ul, the solution for the total volume of 250μl (per hole) and gently mixeduniform; Solution B:liposome by adding 500μl DMEM will 20μl culture medium, a total of four, gently mixing at room temperature, incubated 5min; will be diluted recombinant plasmid pJNS4A,250μl diluted liposome mixing gently, incubated for 20min at room temperature(where the solution becomes turbid) to form a liposome/ plasmid mixture; with serum-free antibiotic-free D-MEM culture medium wash cells three times within a short time before the replacement of transfected Iml37℃temperature and a good pre-serum-freeantibiotics, D-MEM culture medium; to 6 well plate by adding 500μl drop of liposome/plasmid mixture, while increases while shaking culture plate; in 37℃,5% of the CO2 in the culture for 4 hours in serum-free antibiotic-freeDMEM culture medium.Filter:6-well plates were incubated for 24 hours to 24-well plates, containing the concentration of G418 was followed by 50,100,200,300,400,500,600,700,800,900 mg/ L of DMEM culture.The concentration of each set three holes, and the remaining three holes for normal cell control.Determined after 15 d culture can kill all cells in the lowest concentration of G418 as the best screening test concentration, that is, the maintenance of 100 m L train.(3) Immunofluorescence assay Trypsin digestion and transfected CHO cells and normal cells seeded in 6-well its the coverslip to the slide covered with no overflow of degrees at 37℃,5% CO2 under the conditions of normal culture when cell density reached About 60% of a 70%,4% paraformaldehyde fixed cells,1 h,0.1% Triton X.100 Punch 10 min,10% BSA closed 1 h, rabbit anti-mouse IgG Fc diluted 1:800 respectively,4℃overnight incubation.FITC-Goat anti-rabbit IgG diluted 1:00 according to the concentration of dark at 37℃incubation 1 h,50% glycerol Fengpian observed under fluorescent microscope after fluorescent labeling.Results1. JEV non-structural protein NS4A coding genes and identification of recombinant plasmid(1) As Japanese encephalitis virus SAM-14-2 strain Total RNA for template, we obtain the recombinant pJNS4A though RT-PCR, The outcome of sequencing analysis is in line with the gene order which have published, enzyme cutting appraisement indicatding:The molecular weight of recombinant plasmid which enzyme cutting by BamHI/EcoRI is about 900bps, which is in line with pJNS4A be enzyme cut with the same enzyme, The sequence analysis showed that purpose genes were in their corret sites.(2) pJNS4A the emotional state will be amplified in E. coli, extracted plasmid RNA, DNA electrophoresis of their identification line.Known pJNS4A size is about 843 bps, can be seen in the corresponding position on a clear strip. pJNS4A transfected CHO cells in the immunofluorescence assay2. Immunofluorescence assayJEV NS4A protein coding recombinant plasmid transfected CHO cells can be seen more significant green fluorescent marker, mainly in the membrane. Normal CHO cells, no specific green fluorescent marker, only to see the scattered non-specific green fluorescent impurities.Conclusions1. Constructed recombinant pJNS4A containing JEV NS4A protein coding genes.2. Transfection of the JEV NS4A protein coding recombinant plasmid CHO cells stably expressing JEV NS4A protein, mainly in the membrane.更多还原