抗戊型肝炎病毒噬菌体抗体库的构建与筛选  

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

作　　者：魏华[1] 张建琼[1] 吕海芹[2] 孟继鸿[2] 鲁晓瑄[3] 谢维[1] 

机构地区：[1]东南大学医学院遗传学研究中心 [2]东南大学医学院病原生物学与免疫学系,江苏南京210009 [3]东南大学医学院江苏省基因诊断与治疗医学重点实验室,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2003年第5期473-475,485,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省科委自然科学基金资助项目 (No .BK2 0 0 0 2 2 )

摘　　要：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。AIM: To construct HEV specific phage combinatorial antibody 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. κ 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 XL1 Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV specific phage antibody library. RESULTS: Fab displayed on the surface as fusion protein with the N terminal of coat protein Ⅲ, and 1.8×10 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.

关 键 词：噬菌体展示 抗体库 戊型肝炎病毒 

分 类 号：Q781[生物学—分子生物学]