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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。
OBJECTIVE: To construct human anti-HEV phage antibody library and screen human-neutralizing anti-HEV monoclonal antibody (mAb). Methods: Venous blood was collected from 6 HE patients with positive anti-HEV antibodies, lymphocytes were isolated and total RNA was extracted and reverse transcribed. A group of human IgGFab gene-specific primers were used to amplify the genes encoding IgGκ0 light chain and heavy chain Fd fragment, respectively. The kappa light chain and the Fd gene were cloned into the corresponding sites of the phage vector pComb3 and transformed into E. coli XL1 Blue by electroporation. Then the supernatant was infected with helper phage VCSM13 to construct a human anti-HEV phage antibody library. Using a unique 5-round screening method (gradually reducing the amount of antigen coating, stringent elution conditions), the phage antibody library was screened with 4 recombinant phage antibody pools of HEV representative strains containing neutralizing epitopes immobilized on solid phase Phage antibodies were identified by ELISA. Results: The kappa light chain gene library with 1.9 × 10 7 recombination rate and 80% capacity was constructed by several electroporation. The Fab gene library with the capacity of 1.8 × 10 7 recombination rate was 20%. The HEV representative strain neutralizing antigen epitopes ORF2 recombinant mixed antigen specific panning sieve 5 times, the specific enrichment. ELISA screening of the fifth round of screening products, obtained 4 HEVORF2 recombinant mixed antigen with high affinity Fab phage antibody, may be neutralizing antibodies. Conclusion: Human anti-HEV phage antibody library was successfully constructed and human anti-HEV specific phage antibody was obtained.