【摘要】 通过融合乙型肝炎病毒(HBV)PreS2基因及大肠杆菌β-半乳糖苷酶基因,使HBVPreS2蛋白在大肠杆菌中得到高效表达,推算所表达的融合蛋白占菌体可溶性蛋白的15％左右。经检测表明,表达产物可与抗PreS2单克隆和多克隆抗体反应,并能与多聚人血清白蛋白相结合,同时还具有较强的β-半乳糖苷酶活性,可用于抗PreS2抗体的血清学诊断。利用β-半乳糖苷酶底物类似物亲和层析的方法对融合蛋白进行一步纯化,通过Western印迹及N端氨基酸测序对纯化产物进行鉴定,表明虽然在裂解细菌的同时已加入蛋白酶抑制剂,但仍有相当一部分(>50％)的融合蛋白已在PreS2与β-半乳糖苷酶之间发生断裂,对此现象进行了讨论。更多还原

【Abstract】 The Pres2 region of the hepatitis B virus genome was fused to the lacZ gene of E.coli, and the in-frame fusion gene was efficiently expres-sed in E.coli under the control of a synthetic tac promoter, producing a PreS2-β-galactosidase fusion protein which comprised up to 15% of the total soluble protein in the cell lysate. As shown in ELISA tests,the expressiqn product with high β-galactosidase activity reacted specifically with monoclonal and polyclonal anti-PreS2 antibodies and with polymeri-zed human serum albumin. The fusion protein was then purified through a substrate analogue affinity chromatography and its N-terminal sequ-ence was determined. It was found that a substantial part of the fusion protein was cleaved at a site just between the PreS2 and the β-galactosi-dase segments. The possible cause and effect of the cleavage is discussed.更多还原