丙型肝炎病毒基因组部份NS5区蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组ＮＳ５区Ａ段和部份Ｂ段蛋白。ＮＳ５Ａ蛋白溶于水，可经过ＧＳＴ亲和层析柱纯化；ＮＳ５Ｂ蛋白不溶于水，经ＰＢＳ－Ｔｒｉｔｏｎ Ｘ－１００洗涤，尿素溶解后，通过离子交换柱纯化。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ分析，ＮＳ５Ａ蛋白除在５７ｋＤ左右有条带外，还有不同程度的降解产物；ＮＳ５Ｂ蛋白主要在５８ｋＤ左右有条带出现。为查明表达蛋白抗体在病人血清中的分布，取９

EXPRESSION OF HEPATITIS C VIRUS NS5 GENE IN E.coli

epatitis C virus (HCV) genome NS5A and part of NS5B proteins were expressed in Ecoli using prokaryotic expression Vectors PGEX3X or PGEX2T. The soluble NS5Aglutathione S-transferase(GST) gene fusion protein were purified from bacterial lysates byaffinity chromatography using prepacked glutathione sepharose CL 4B column. The insolubleNS5B fusion protein was treated by PBS/TritonX- 100 and urea,then purified through ionexchange chromatography. The expression products were analysed by SDS-PAGE and Westemblot. The MS5A protein was unstable,besides the 57k D fusion protein,there were some degradation products.NS5B protein was mainly seen at 58K D.The expression products wereused to check the positive rate among sera which had been tested by C33c and C22 antigens.64.8% was anti NS5A positive and 57.7% was anti NS5B positive among anti-C33c andanti -C22 positive sera. The conformity rate between anti-NS5A antibody and anti-NS5Bantibody was 75.5%.