王志毅 刘杞 万泽生 张定凤 王志毅 刘杞 张定凤 重庆医科大学病毒性肝炎研究所; 万泽生 第三军医大学微生物教研室 中华肝脏病杂志 1999 0 7 3


    关键词：抗体，单克隆 噬菌体 乙型肝炎表面抗体 期刊 zhgzbzz 0 实验研究 fur -->


    

摘要 目的 构建含抗-HBs的人源性噬菌体抗体库。方法 用RT-PCR技术，从自然感染的抗-HBs阳性的两名献血员的外周血淋巴细胞中，扩增出人抗体轻链k基因和重链Fd基因，将二基因先后克隆入PComb3Hss载体中，转化大肠杆菌，再用辅助噬菌体感染。结果 构建了库容量为1.5×10⁵ 的轻链基因抗体库，轻链基因插入率为57%和库容量为5×10⁵ 的人Fab抗体库，轻、重链基因插入率为50%。经辅助噬菌体感染，得到噬菌体滴度为5×10¹⁴ CFU/ml的人源性噬菌体抗体库。 结论 成功构建了含抗-HBs的人源性噬菌体抗体库，为进一步筛选HBsAg的人Fab噬菌体抗体奠定了基础。

    

Construction of humancombinatorial immunoglobulin library containing HBsAb on the surface of phage

WANG Zhiyi, LIU Qi, WAN Zesheng, et al.

    Institute for Viral Hepatitis ,Chongqing University of Medical Sciences ,74 Lin JiangRoad,Chongqing 400010

Abatract Objective To construct a humanrecombinant immunoglobulin library containing HBsAb. Methods The mRNA isolated fromperipheral blood lymphocytes of two blood donors with HBsAb were reversely transcribed tothe first strand cDNA with oligo-(dT) primer. Human immunoglobulin k light chains andheavy chains Fd genes were amplified respectively by PCR with designed oligo nucleotideprimers and the first strand cDNA templates. The k light chain genes were first clonedinto PComb3Hss vector to construct a human recombinant light chain library. The heavychain genes were subsequently inserted into the corresponding sites of k-PComb3Hss plasmidto generate a combinatorial k-Fd-PComb3Hss plasmid. The plasmid transformed into E.Coli.XLI-Blue and the E.Coli.XLI-Blue infected by help phage. Results The constructedlight chains library size was 1.5×10⁵ , and the recombinant freguency of kchain genes was 57% and the human combinatorial Fab immunoglobulin library was 5×10⁵ members and the recombinant freguency of Fab genes was 50%. Finally, the titer of phagesin the phage antibody library containning HBsAb was 5×10¹⁴ CFU/ml. ConclusionOur work in constructing human immunoglobulin combinatorial library containning HBsAb willbe beneficial to further study on selecting Fab phage antibodies of HBsAg from thelibrary.

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