抗戊型肝炎病毒噬菌体抗体库的构建与筛选

目的 :构建人抗戊型肝炎病毒 (HEV)噬菌体抗体库 ,筛选人源中和性抗HEV的单克隆抗体 (mAb)。方法 :取抗HEV抗体阳性的 6例HE患者静脉血 ,分离淋巴细胞 ,提取细胞总RNA后逆转录。用一组人IgGFab基因特异性引物 ,分别扩增IgGκ0轻链与重链Fd段基因。将κ轻链与Fd段基因先后克隆入噬菌体载体pComb3的相应位点 ,经电穿孔法转化大肠杆菌XL1 Blue ,再以辅助噬菌体VCSM13超感染 ,构建人抗HEV噬菌体抗体库。采用独特的 5轮筛选法 (逐渐降低抗原包被量 ,严格洗脱条件 ) ,以固相化的 4种含中和抗原表位的HEV代表株ORF2重组混合抗原 ,筛选人噬菌体抗体库 ,并以ELISA鉴定噬菌体抗体。结果 :经数次电转化构建了容量为1.9× 10 7重组率为 80 %的κ轻链基因库 ;容量为 1.8× 10 7重组率为 2 0 %的Fab基因库。以含中和抗原表位的HEV代表株ORF2重组混合抗原特异淘筛 5次 ,出现特异富集。ELISA鉴定第 5轮筛选产物 ,得到 4株与HEVORF2重组混合抗原具有较高亲和力的Fab噬菌体抗体 ,可能为中和抗体。结论 :成功地构建了人抗HEV噬菌体抗体库 ,并获得人源抗HEV特异性噬菌体抗体。

Construction and screening of hepatitis E virus-specific phage antibody combinatorial library

AIM: To construct HEV-specific phage combinatorial anti-body library and screen anti-HEV antibodies with neutralizing activity from the library. METHODS: The total RNA was extracted from B-lymphocytes of 6 HE patients. Kappa chain and Fd segment of IgG gene were amplified respectively by RT-PCR using a set of Fab-specific primers. The amplified gene were inserted successively into vector pComb3 and electrotransformed E. coli XLI-Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV-specific phage anti-body library. RESULTS: Fab displayed on the surface a as fusion protein with the N terminal of coat protein III, and 1. 8 x 10(7) clone library was established. Specific antibodies to HEV ORF2 recombinant antigen were acquired after five rounds of panning with HEV ORF2 recombinant antigen including neutralizing epitope. CONCLUSION: Four clones exhibited specific binding to HEV ORF2 recombinant antigen including neutralizing epitope is identified by ELISA. The results show that we have got the recombinant phage antibodies.