重组人抗乙型肝炎病毒表面抗体的筛选及序列分析

目的克隆、筛选人抗乙型肝炎病毒表面抗体(抗-HBs),并对其基因序列进行分析。方法从抗-HBs抗体阳性血液中分离淋巴细胞,提取总RNA,合成cDNA,以此为模板,PCR扩增轻链和重链抗体可变区基因;应用已构建的抗体支架载体,构建重组表达载体,转化毕赤酵母X33,建立相应的酵母表达文库;甲醇诱导表达,Westernblot及ELISA筛选抗-HBs抗体阳性菌株;PCR扩增DNA片段,进行测序及Blastn比对,并与已发表的抗-HBs抗体序列进行同源性分析。结果重链及轻链抗体重组表达载体经双酶切鉴定,证明构建正确;经Westernblot及ELISA筛选,获得了具有HBsAg结合活性的完整轻重链抗体;轻链可变区与已发表序列核苷酸同源性为93%,而氨基酸同源性仅为88%,尤其CDR3变异较大;重链可变区与已发表序列同源性较低,CDR3区基因长度及序列均有较大差别。结论应用毕赤酵母表达体系筛选,获得了新的抗-HBs抗体。

Screening and Gene Sequencing of Recombinant Human Anti-HBs Antibody

Objective To clone and screen human anti-HBs antibody and analyze its gene sequence. Methods Lymphocytes were isolated from anti-HBs positive blood samples, from which total RNA was extracted for amplification of VH and Vκ genes by RT-PCR. Insert the amplified VH and Vκ genes into expression vectors pPICZαCH and pPICZαCκ respectively, and transform the constructed recombinant plasmids to Pichia pastoris for expression under induction of methanol. Screen positive recombinants by Western blot and ELISA for amplification of DNA fragments by PCR. Sequence the amplified DNA fragment and analyze its homology to that of published anti-HBs by using Blastn software. Results Restriction analysis proved that the recombinant plasmids for expression of H and κ chain antibodies were constructed correctly. Intact H and κ chain antibodies with antigen-binding activity were screened by Western blot and ELISA. The homology of nucleotide sequence of variable region of κ chain to that of published anti-HBs antibody was 93%, while that of amino acid sequence was only 88%, indicating significant variation especially in CDR3 region. However, the variable region of H chain showed low homology to that of published anti-HBs antibody, and both length and sequence of genes in CDR3 region of the former were significantly different from those of the latter. Conclusion A novel anti-HBs antibody was screened by using Pichia pastoris expression system.