特异性抗SSA/Ro噬菌体抗体的制备及其基因序列分析

Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis

目的 由已构建的人源单链可变区噬菌体抗体库中制备出抗SSA/Ro单克隆抗体 ,并对这些抗体的基因序列进行分析。方法 将冻存的抗体库菌种复苏制备噬菌体抗体库。用PCR、基因序列分析及酶切的方法对其进行鉴定。以组织培养皿法对该噬菌体抗体库进行富集筛选 ,用ELISA方法对富集后次级抗体库进行鉴定。制备单克隆抗体并鉴定其特异性 ,对单克隆抗体的可变区基因序列进行测序分析。结果 所制备的噬菌体抗体库的滴度为 1 6× 10 12 cfu/ml,外源基因的插入重组率为 80 % ,插入片断为人抗体可变区基因 ,且该抗体库具有良好的多样性。富集筛选回收的噬菌体数逐渐增加 ,富集后的抗SSA/Ro噬菌体抗体次级库吸光度 (A)值为富集前的 2 8倍 ,且该次级库有良好的抗SSA/Ro特异性。经富集制备出 5个特异性抗SSA/Ro单克隆抗体 ,这些单克隆抗体重链及轻链可变区基因分别与VH1、VH3、VH4、Vκ1、Vκ2、Vκ3基因家族有高度同源性。结论 本实验室构建的scFv噬菌体抗体库可以用于特异性抗体的筛选及单克隆抗体的制备。制备出的抗SSA/Ro单克隆抗体可变区基因序列与人胚系基因比较有突变 。

Objective To prepare monoclonal antibodies(mAbs) specific for SSA/Ro antigen from the phage-displayed human single-chain Fv (scFv) antibody library constructed previously, and analyze its gene sequence. Methods Frozen strain of Escherichia coli TG1 containing pHEN2-scFv was defrosted and infected by helper phage to produce scFv phage antibody library. 10 random clones from the primary library were checked for the presence of inserts by PCR screening. Four clones containing exogenous gene with correct length were selected randomly to undergo gene sequencing. The diversity of the library was monitored by digesting the PCR products of 8 different phagemids encoding scFv genes. The phage antibody library was biopanned for 3 cycles, using purified SSA/Ro antigen. After biopanning, the binding characterization and the specificity of the enriched library to SSA/Ro antigen was assayed by ELISA. mAbs were obtained from this enriched library and ELISA was used to detect the binding characterization and the specificity of these mAbs, using different purified antigens. The variable region of the monoclonal antibody undergoes sequencing. Results A phage scFv library with the titer of 1.6×10 12 cfu/ml was established. Sample screening showed that eight of the ten clones contained scFv fragments. The insertion and recombination rate of exogenous gene was 80%. These 8 clones were also analyzed by restriction enzyme analysis to assess the diversity of the library. Each of the 8 clones showed a distinct restriction pattern. Sequencing results demonstrated that the genes of scFv were the human antibody genes, and the gene ligating and cloning were correct and successful. The library was subjected to 3 rounds of rescue and panning. A secondary phage antibody library against SSA/Ro was generated and the interest phages were obviously enriched. The 3rd panned library showed good reaction to the SSA/Ro antigen with higher OD value than unenriched library demonstrated by ELISA. The result of ELISA showed that the enriched library reacted specifically to SSA/Ro and had no cross-reactivity to the other autoantigen. 5 mAbs specific for SSA/Ro antigen could be selected from 52 clones, and the gene sequences of their VH and VL were coded by VH1, VH3, VH4, Vκ1, Vκ2, and Vκ3 family genes with somatic mutations. Conclusion The primary scFv phage antibody library fits the need for later operations, and this library was enriched successfully. Enriched scFv phage antibody library is specific for SSA/Ro antigen, from which 5 monoclonal anti-SSA/Ro phage antibodies can be obtained successfully. The sequences of these mAbs show somatic mutations comparing with germline. It may help understand the pathogenesis of some antibody-mediated diseases.