兔—人嵌合型抗-HEV IgM质控物及耐RNase病毒样颗粒的双质粒双表达研究

【作者】 张括；

【导师】 李金明； 王露楠；

【作者基本信息】 中国协和医科大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 第一部分兔-人嵌合型戊型肝炎病毒IgM抗体质控物的构建目的本研究拟用化学交联方法构建一种兔-人嵌合型戊型肝炎病毒（Hepatitis Evirus,HEV）IgM抗体作为抗-HEV IgM检测质控物。方法基因重组蛋白NE2作为抗原免疫家兔,获得兔抗-HEV IgG,酶免方法检测其滴度和活性后,蛋白A亲合层析纯化IgG,十二烷基磺酸钠聚丙烯酰胺凝胶电泳（Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis,SDS-PAGE）分析IgG纯度,紫外分光光度计测定260/280nm吸光度并计算抗体浓度。EDC（1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,1-乙基-3[3-二甲基氨基丙基]碳二亚胺）交联兔抗-HEV IgG与人IgM。抗-HEV IgM酶联免疫测定（enzyme-linkedimmunosorbent assay,ELISA）试剂盒同时检测嵌合抗体和阳性血清,并比较两者滴度。嵌合抗体稀释后,放置于不同温度条件下,保存不同时间后,ELISA检测稳定性。结果未纯化的兔抗-HEV IgG滴度为1∶100,000,纯化后,SDS-PAGE电泳结果显示轻链和重链两条带（25kD和50kD）,没有杂带,达到电泳纯,抗体浓度达10mg/ml。嵌合抗体与人抗.HEV IgM阳性血清滴度均为1∶160,说明其同抗原的亲和性与真实的人类样本相似。稳定性实验表明,此嵌合抗体在室温、4℃可至少保存两个月,而在较低温度如-20℃、-70℃和反复冻融条件下不稳定。结论本研究用化学交联方法成功构建了抗-HEV IgM嵌合抗体质控物,在国内外尚属首次,其克服了传统血清质控物的缺点,根据已建立起的方法,可以构建一系列应用于免疫测定检测不同病毒特异IgM抗体的阳性质控物。第二部分双质粒双表达构建内含长片段RNA的病毒样颗粒目的本研究拟通过改变大肠杆菌噬菌体MS2-RNA 19mer茎环结构（包装位点,pac site）的数量、位置和亲和力,构建能够表达内含长片段嵌合体RNA耐核糖核酸酶病毒样颗粒（virus-like particles,VLPs）的双质粒原核表达系统,探讨茎环结构在表达内含更长片段RNA的VLPs时所起的作用。方法利用pET-28b和pACYCDuet-1构建两种双质粒表达系统,质粒之一为本室已经构建好的内含MS2成熟酶蛋白和衣壳蛋白的质粒pET-MC。另一质粒的构建方法分别如下:（1）设计均含有PacI酶切位点的上下游引物,且下游引物含有茎环结构的突变体（C-variant）,扩增HIV gag序列,单酶切连接目的序列与表达载体pACYC-3V（内含3段SARS-CoV基因、C-variant、1段HCV基因和2段H5N1基因）,构建重组载体pACYC-3V-gag。（2）设计均含有PacI酶切位点的上下游引物,且下游引物含有茎环结构的突变体,扩增HIV gag序列,单酶切连接目的序列和表达载体pACYC-pol（内含HIV pol序列及一个C-variant）,构建重组载体pACYC-pol-gag。双质粒共转化入表达菌株BL21（DE3）。IPTG诱导表达,超声碎菌,DNaseⅠ和RNase A双酶消化,氯化铯密度梯度离心,超声处理液透析过夜后丙烯葡聚糖凝胶层析纯化得到VLPs。电镜观察后RT-PCR验证包装外源RNA的长度。结果成功构建了重组载体pACYC-3V-gag和pACYC-pol-gag并得到两VLPs。丙烯葡聚糖凝胶层析纯化后,VLPs OD₂₆₀（0.3）高于OD₂₈₀（0.15）。DNaseⅠ和RNase A双酶消化结果表明,所包装的RNA具有耐RNase和DNase消化的特性。RT-PCR验证第一种VLPs中包装了2,698bp的目的序列,第二种VLPs逆转录后扩增出了pol和gag的部分片段,分别为600bp和450bp,间接说明VLPs能够包装3,250bp的外源RNA。结论本研究将MS2 RNA突变型包装位点的数量增加至两个并改变其在外源RNA中的位置,利用双质粒双表达系统可表达内含2.7～3.2kb外源RNA片段的耐RNase的VLPs,初步阐明了包装位点的数量和位置在外源RNA包装中的作用。更多还原

【Abstract】 Background The aim of this study was to conduct synthetic rabbit-human antibodies conjugate as controls in immunoassays that measure specific IgM to hepatitis E virus.Methods Two New Zealand white rabbits were injected with the HEV recombinant protein NE2 with ORF2 immunodominant epitope as antigen,and the titer of antibody was detected by ELISA.Rabbit IgG was isolated from immune sera by HiTrap affinity columns chromatography on protein A-Sepharose.The purified IgG was quantified by eppendorf biophotometer,and then evaluated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis（SDS-PAGE）.Cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)（EDC） was used to conjugate IgG fraction and human IgM.To determine if the conjugate was similar to human samples, conjugate and positive control serum from real patients were all detected by anti-HEV IgM ELISA and the end point titers were compared.Finally,the stability of antibody conjugate was examined.The chimeric antibodies were diluted,the stock solution were then incubated,in duplicate,at different temperature for different times.Then samples were removed at each time point and were stored at -70℃until the completion of the examination.All samples were tested by ELISA using a HEV IgM Diagnostic Kit.Results After immunization a reasonable titer 1:100,000 of IgG antibodies was being built up in these antisera.By SDS-PAGE analysis,the major stained bands were the heavy and light chains of IgG,50KD and 25KD respectively.No other minor bands were found in sample.The concentration of IgG was about 10mg/ml.The end point titers of antibody conjugate and positive serum were all 1:160,therefore,no significant difference in the end point titer between the serum-derived and the antibody conjugate, Stability of the antibody conjugate indicated that antibody conjugate were not completely stable at temperature below 4℃,room temperature was the better temperature to keep the antibody conjugate stability.Conclusion Rabbit-human antibodies conjugate against HEV described in this study offers an alternative method to prepare positive control instead of traditional serum positive control.This is the first demonstration of the chemically conjugated method to construct anti-HEV IgM positive control.According to the established method,it could also construct a series of positive controls applicable to any immunoassays,such as ELISA,immunoblot and IFA,to detect the presence of IgM specific for a given antigen. Baekgroud This study changed affinity,number and position of MS2-RNA 19mer stem-loop（pac site） in exougenous RNA and constructed two kinds of ribonuclease resistant virus-like particles by two-plasmid system to demonstrate whether larger size RNA could be encapsulated to produce virus-like particles by increasing the amount and affinity of the pac site.Methods Two kind of two-plasmid expression systems were constructed using vectors pET-28b and pACYCDuet-1.One plasmid was plasmid pET-MC constructed by our lab with 1.7kb maturase and coat protein gene of MS2.Another two plasmids were constructed as follows:（1） HIV gag sequence was amplified using sense and reverse primers containing PacI restriction sites.The C-variant of wild-type MS2 RNA stem loop was inserted into the reverse primer.The PCR-amplified DNA fragments was ligated to pACYC-3V vector（three parts of SARS-CoV genes,C-variant,one part of HCV,two parts of H5N1）to generate recombinant plasmid pACYC-3V-gag.（2） HIV gag sequence was amplified using sense and reverse primers contained PacI restriction sites.The C-variant of wild-type MS2 RNA stem loop was inserted into the reverse primer.The PCR-amplified DNA fragment was ligated to pACYC-pol vector（C-variant,HIV-pol） to generate recombinant plasmid pACYC-pol-gag.Then both plasmids were co-transformed into E.coli strain BL21（DE3）,Expression was induced by adding IPTG. The cells were sonicated and centrifuged in order to pellet the cell debris.RNase A and DNase 1 were added in order to eliminate E.coli RNA and DNA.VLP were purified by CsCl gradient.After centrifugation,the virus-like particles band is pulled and then dialyzed against sonication buffer to move CsCl.The nuclease treated crude extract of virus-like particles may be also purified by gel exclusion chromatography using a resin such as Sephacryl S-200.Electron microscopy may be used to count the virus-like particles directly.RT-PCR was carried out using the down stream primer to verify the VLPs.Results Two expression vectors pACYC-3V-gag and pACYC-pol-gag had been constructed.Two kinds of virus-like particles were expressed in E.coli BL21（DE3）.The particles were purified by Sephacryl S-200.The OD₂₆₀ was higher than OD₂₈₀.Electron microscopy was used to observe the VLP directly.The RT-PCR results of the first VLP showed that the 2,698bp target RNA was packaged into VLP.The RT-PCR results of the second VLP showed that parts of HIV pol（600bp） and gag（450bp） were amplified,and the 3,250bb target RNA could be packaged into VLP.Conclusion By using two C-virants of wild-type stem loop and changing their position in exogenous RNA,the virus-like particles with 2.7～3.2kb exogenous RNA were constructed using two-plasmid system.Therefore,increasing the number of the C-virant of wild-type stem loop and chaging its position could improve the length of exogenous RNA packaged into virus-like particles.更多还原