CAJ | 학술논문

目的：研究在大肠埃希菌（ Escherichia coli， E?coli）表达系统中表达和纯化7型庚型肝炎病毒（GB virus C genotype 7， GBV?C G 7）非结构蛋白NS3的149个氨基酸（NS3?149），并对其进行反应原性鉴定。方法将NS3?149基因克隆入原核表达载体pET?28a，并转化入BL21，利用异丙基?B?D?硫代吡喃半乳糖苷（ IPTG）诱导其表达。用Ni2＋柱对融合蛋白NS3?149进行纯化，最后经Western 印迹检测其抗原特异性和反应原性。结果①成功构建了pET?28a?NS3?149重组质粒，该重组质粒转化入BL21后，融合蛋白NS3?149表达的最佳条件为在32℃条件下， A600 nm等于0?8时，用1 mmol／L 的 IPTG 诱导6 h 后，融合蛋白表达量达到最高。经过SDS?PAGE分析，融合蛋白以包涵体的形式表达，其大小约27 kD，与预期大小基本一致；②用感染GBV?C 7型的人阳性血清经Western印迹鉴定，融合蛋白NS3?149具有较好的反应原性，另外，其与GBV?C容易发生共同感染的HIV或HCV阳性血清无非特异性交叉反应，说明了融合蛋白NS3?149具有较好的特异性。结论本实验在大肠埃希菌中表达并纯化并获得具有良好反应原性的GBV?C 7型融合蛋白NS3?149，为进一步对GBV?C进行抗体检测的流行病学调查研究奠定了重要的前期工作基础。
목적：연구재대장애희균（ Escherichia coli， E?coli）표체계통중표체화순화7형경형간염병독（GB virus C genotype 7， GBV?C G 7）비결구단백NS3적149개안기산（NS3?149），병대기진행반응원성감정。방법장NS3?149기인극륭입원핵표체재체pET?28a，병전화입BL21，이용이병기?B?D?류대필남반유당감（ IPTG）유도기표체。용Ni2＋주대융합단백NS3?149진행순화，최후경Western 인적검측기항원특이성화반응원성。결과①성공구건료pET?28a?NS3?149중조질립，해중조질립전화입BL21후，융합단백NS3?149표체적최가조건위재32℃조건하， A600 nm등우0?8시，용1 mmol／L 적 IPTG 유도6 h 후，융합단백표체량체도최고。경과SDS?PAGE분석，융합단백이포함체적형식표체，기대소약27 kD，여예기대소기본일치；②용감염GBV?C 7형적인양성혈청경Western인적감정，융합단백NS3?149구유교호적반응원성，령외，기여GBV?C용역발생공동감염적HIV혹HCV양성혈청무비특이성교차반응，설명료융합단백NS3?149구유교호적특이성。결론본실험재대장애희균중표체병순화병획득구유량호반응원성적GBV?C 7형융합단백NS3?149，위진일보대GBV?C진행항체검측적류행병학조사연구전정료중요적전기공작기출。
Objective To express and purify the GB virus C genotype 7 ( GBV?C G 7 ) non?structural protein NS3?149 in Escherichia coli ( E?coli) , and analyze its reactogenicity?Methods The NS3?149 gene (447 bp) of GBV?C was amplified by RT?PCR, and it was cloned into the pro?karyotic expression vector pET?28 a?The recombinant vector pET?28 a?NS3?149 was transformed into E?coli cells and induced by Isopropyl?D?1?Thiogalactopyrano?side ( IPTG);then inclusion body was lysed, and purified via Ni?affinity chromatography;finally, the reactogenicity of recombinant NS3?149 was detected by western blotting?Results DNA sequencing confirmed that GBV?C?NS3?149 gene had been accurately cloned into pET?28 a?The best expression condition for recombinant NS3?149 gene involved induction with 1mM IPTG for 6 hours at 32℃ and the A600 nm value of 0?8?SDS?PAGE indicated that the expressed recombinant NS3?149 protein was inclusion bodies, and the size of recombinant NS3?149 protein was approximately 27 kD;②Western blot analyses showed that the recombinant NS3?149 protein possessed reactogenicity by human positive sera infected with GBV?C 7?In addition, recombinant NS3?149 protein has specificity; it can’ t cross reaction that human positive sera infected with HIV or HCV?Conclusion GBV?C recombinant NS3?149 protein with re? actogenicity was expressed and purified in E?coli, the foundation of assay could be potentially used for epidemiology investigation of the GBV?C infection.