人工进化丙型肝炎病毒C和E1区噬菌体展示文库的构建及初步筛选

目的构建人工进化丙型肝炎病毒(HCV)C和E1区基因噬菌体展示文库,并进行初步筛选。方法应用DNA改组技术进行不同基因型的HCV C和E1区基因的人工进化,克隆于噬菌体载体,以辅助噬菌体M13K07援救后,构建噬菌体展示文库,应用HCV C和E1区单克隆抗体进行初步筛选。随机选取筛选后的20个噬菌体克隆,用高滴度HCV阳性血清通过双抗体夹心酶联免疫吸附法进行抗原抗体结合反应,以正常人血清作为对照。结果人工进化HCV C和E1区噬菌体展示文库库容达1．64×106,重组率为0．86。以C和E1区单克隆抗体淘筛4轮,噬菌体展示文库得到特异性富集。筛选获得12个阳性克隆。结论所构建的噬菌体展示文库的库容和多样性符合筛选的要求。筛选获得的阳性克隆具有较好的抗原抗体结合反应活性,表现出较好的亲和力。

The construction and primary screening of a phage display library of HCV C and E1 genes evolved with an artificial pattern

OBJECTIVES: To construct and screen a primarily phage display library of HCV C and E1 genes evolved with an artificial pattern. METHODS: Two genes of about 1 kb with different genotypes were evolved by DNA shuffling. The re-assembled HCV C and E1 genes were cloned into a phage vector. After being rescued with helper phage M13KO7, a phage display library was constructed. Then the library was screened with anti-C and E1 McAb. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out on twenty individual phage clones selected randomly to detect their binding and reactive activity with high-titer HCV-positive sera. Normal sera were used as controls. RESULTS: The phage display library of HCV C and E1 genes which evolved with an artificial pattern was constructed. Their capacity amounted to 1.64 x 10(6), and 86 percent of the clones contained C and E1 genes. After four rounds of panning, the phage library was specifically enriched. Twelve positive clones were successfully screened. CONCLUSION: The capacity and diversity of the constructed library are enough for screening. The results demonstrate the superiority of the specific binding and reactive activity and affinity of the 12 phage clones from the HCV positive sera.