抗bFGF McAb制备、鉴定及轻链可变区基因克隆和序列分析

目的以重组人碱性成纤维细胞生长因子为抗原免疫小鼠,建立多株稳定分泌bFGF单克隆抗体(McAb)的杂交瘤细胞.并对bFGF单克隆抗体(McAb)可变区基因进行扩增及序列分析.方法采用Western blot法和免疫沉淀法对抗体特异性和Ig类型进行鉴定,同时,应用分子生物学技术,对Vk基因的DNA片段进行克隆,并对4a2Vk基因进行序列测定.结果bFGF单克隆抗体可特异性结合人重组bFGF抗原,且均为IgM.由杂交瘤4a2、3d3细胞株扩增Vk基因是由330个碱基组成,编码110个氨基酸残基.通过国际联机检索进行EMBL和Kabat基因库扫描发现,4a2VkDNA仅与Ig同源,符合小鼠Ig的Vk基因特征;同源性为90%～98%,应归属于Ig的Vk基因.结论杂交瘤4a2、3d3细胞株可稳定分泌bFGF单克隆抗体,并获得其轻链可变区基因,为进一步构建和表达单链Fv(ScFv)抗体打下良好的基础.

Preparation and sequence analysis of Vk gene of anti-human bFGF McAb

objective: Five hybridoma cell lines secreting anti-bFGF mAb have been developed from immunized Balb/c mice with recombinant human bFGF, by cell fusion. The variable region of K chain (Vk) genes was isolated by RT-PCR and sequencd. Methods:Two of these hybridoma cell lines were identified by using westem blot analysis. The recombinant vectors containing 4a2 Vk gene insert were identified by sequencing with Sanger's dideoxy-mediated chain termination method and computer-assisted comparative sequence analysis. Results: It showed that Vk gene consisted of 333bp encoding 111 amine acid resides. Comparing with EMBL and Kabat database, the Vk was in agree ment with the characterization of DNA sequence present in the mouse Ig Vk region. Conclusion:The success of cloning of the Vk gene of 4a2 McAb lay a good foundation for the consruction and expression of single chain variable fragment of 4a2 McAb.