CAJ | 학술논문

目的在原核表达系统中对HCV E1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HCV RNA阳性的血清标本中扩增出HCV EI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Xba I和EeoR I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POP2136，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Western blot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Sma I酶切后PCR产物被切成144 bp和356 bp的两个片段，在具有氨苄抗性的菌中提取的质粒经Sma I和Xba I酶切后产生了一356 bp的片段；42℃诱导后在SDS-PAGE中出现了一条相对分子质量为31 000的条带，其表达量约为17％，经West-ern blot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29.8％(26/90)，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HCV E1基因从HCV RNA阳性的血清标本中调出，构建了HCV E1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，有望应用于抗HCV的检测中。
목적재원핵표체계통중대HCV E1기인진행극륭화고효표체，병초보탐토표체산물재항-HCV사선중적작용。방법통과RT-PCR법종HCV RNA양성적혈청표본중확증출HCV EI편단，병재확증용적인물중인입극륭소수적매절위점，극륭산물경Xba I화EeoR I매절후，재T4DNA련접매적작용하극강입경동양쌍매절적원핵표체재체PMS-31b중，병용차련접산물전화대장애희균POP2136，도취구유안변항성적균락확대배양후제취질립진행매절감정，감정출적양성균경42℃유도후용SDS-PAGE검사기표체상황，병용Western blot감정표체산물적특이성，동시용초보순화적표체산물용ELISA법검측병인혈청。결과경Sma I매절후PCR산물피절성144 bp화356 bp적량개편단，재구유안변항성적균중제취적질립경Sma I화Xba I매절후산생료일356 bp적편단；42℃유도후재SDS-PAGE중출현료일조상대분자질량위31 000적조대，기표체량약위17％，경West-ern blot감정후재해처출현료양성신호；ELISA실험현시재항HCV양성적혈청중적양성솔약위29.8％(26/90)，이재항HCV음성적혈청중기양성솔약위3.9％(3/76)。결론통과RT-PCR방법장HCV E1기인종HCV RNA양성적혈청표본중조출，구건료HCV E1적원핵표체재체-PMS-E1，기표체량위17％，표체산물구유량호적특이성，유망응용우항HCV적검측중。
Objective To express the HCV E1 gene in E. coli cells and to demonstrate its cli.nical 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 2μg/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 SDSPAGE after digestion of the RT-PCR product with Sma I And there emerged one fragment of 356 bp af ter 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 antiHCV E1 positive, but 3.9 % (3/76) were positive in the anti-HCV negative serum. Conclusion The HCV El sequence from HCV RNA positive serum has been expresed in E. coli. The expression rate is about 179% of the total protein of the bacteria. This protein possessed good specificity and may be used in the diagnosis of HCV infection.