Huang Chen-Ji, Peng Hwei-Ling, Cheng Chih-Yu
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan.
J Biomed Biotechnol. 2011;2011:359042. doi: 10.1155/2011/359042. Epub 2011 Aug 29.
In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W(84), P(95), P(110), or V(129). The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W(84)S, P(110)S and V(129)L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM(-1). Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.
为提高诊断的敏感性，对含有丙型肝炎病毒核心区I（氨基酸残基1至123）的重组克隆进行随机诱变。通过反向ELISA对616个突变体进行菌落筛选，获得了5个敏感性更高的突变体。对这些突变体的序列分析显示，突变集中在W(84)、P(95)、P(110)或V(129)。随后，使用6 M尿素溶解在大肠杆菌BL21(DE3)中过表达的这些重组蛋白的包涵体，然后通过逐步透析进行复性。与未折叠的野生型抗原相比，复性后的M3b抗原（W(84)S、P(110)S和V(129)L）的抗原性增加了66%，结合能力为0.96，亲和力为113 μM(-1)。此外，生产需求降低33%表明M3b是抗丙型肝炎病毒抗体检测的潜在替代品。
