【摘要】 目的　在原核表达系统中对HCVE1基因进行克隆和高效表达 ,并初步探讨表达产物在抗 HCV筛选中的作用。方法　通过RT PCR法从HCVRNA阳性的血清标本中扩增出HCVEI片段 ,并在扩增用的引物中引入克隆所需的酶切位点 ,克隆产物经XbaⅠ和EcoRⅠ酶切后 ,在T4DNA连接酶的作用下克降入经同样双酶切的原核表达载体PMS 31b中 ,并用此连接产物转化大肠埃希菌POP2 136 ,挑取具有氨苄抗性的菌落扩大培养后提取质粒进行酶切鉴定 ,鉴定出的阳性菌经42℃诱导后用SDS PAGE检查其表达状况 ,并用Westernblot鉴定表达产物的特异性 ,同时用初步纯化的表达产物用ELISA法检测病人血清。结果　经SmaⅠ酶切后PCR产物被切成 144bp和 35 6bp的两个片段 ,在具有氨苄抗性的菌中提取的质粒经SmaⅠ和XbaⅠ酶切后产生了一 35 6bp的片段 ;42℃诱导后在SDS PAGE中出现了一条相对分子质量为 310 0 0的条带 ,其表达量约为 17% ,经West ernblot鉴定后在该处出现了阳性信号 ;ELISA实验显示在抗HCV阳性的血清中的阳性率约为2 9 8% (2 6 /90 ) ,而在抗HCV阴性的血清中其阳性率约为 3 9% (3/76 )。结论　通过RT PCR方法将HCVE1基因从HCVRNA阳性的血清标本中调出 ,构建了HCVE1的原核表达载体—PMS E1,其表达量为 17% ,表达产物具有良好的特异性 ,有?更多还原

【Abstract】 Objective To express the HCV E1 gene in \%E.coli\% cells and to demonstrate its clinical significance in detection of anti\|HCV E1 antibodies.Methods The expression vector was constructed by ligation of HCV E1 sequence,which was amplified by RT\|PCR methods from 50 μl of HCV RNA positive serum using primers specific to the HCV E1 sequence,to the prokaryotic expression vector PMS\|31b transfected POP2136 at 16 ℃ for 16 hours. The recombinant plasmid was screened out and characterized by restriction enzyme analysis.The bacteria containing the recombinant plasmid was induced at 42 ℃ for 4 hours,and the recombinant protein was visualized by SDS\|PAGE.The specificity of the recombinant protein was determined by Western blot assay.After purification of the expressed protein,this protein was coated on the plate with the concentration of 2 μg/ml in pH 9\^6 buffer at 4 ℃ for overnight,and the serum specimen was tested at the dilution of 1∶20 by ELISA.Results There were 2 fragments could be seen on the SDS\|PAGE after digestion of the RT\|PCR product with Sma Ⅰ.And there emerged one fragment of 356 bp after digesting the recombinant plasmid with Sma Ⅰ and Xba Ⅰ.A band of 30 000 could be seen on the SDS\|PAGE after the induction of bacteria containing the recombinant plasmid pMS\|E1 at 42 ℃ for 4 hours.The Western blot assay showed that the expressed band could react with the anti\|HCV positive serum.The ELISA result indicated that there were 28\^9% (26/90) anti\|HCV positive serum were anti\|HCV E1 positive,but 3\^9%(3/76) were positive in the anti\|HCV negative serum.Conclusion The HCV E1 sequence from HCV RNA positive serum has been expresed in \%E.coli\%.The expression rate is about 17% of the total protein of the bacteria.This protein possessed good specificity and may be used in the diagnosis of HCV infection.更多还原