6B11卵巢癌抗独特型单链抗体表达及序列分析

CLONING AND SEQUENCE ANALYZING OF 6B11 ANTI-IDIOTYPIC ANTBODY ScFV GENES

目的：克隆和表达６Ｂ１１卵巢癌抗独特型抗体单链可变区基因，并进行序列分析。方法：采用逆转录ＰＣＲ和基因重组技术，扩增并构建６Ｂ１１鼠抗独特型抗体单链可变区基因，重组至噬菌体表面表达载体，在大肠杆菌表达，并利用双脱氧末端终止法进行核苷酸序列分析。结果：阳性克隆表达产物与卵巢癌单克隆抗体ＣＯＣ１６６－９特异结合；ＤＮＡ序列分析表明重链和轻链可变区分属小鼠免疫球蛋白重链第Ｉ亚群和轻链第Ⅱ亚群。结论：自６Ｂ１１小鼠杂交瘤细胞成功地克隆表达了该抗独特型单链抗体基因，为进一步人源化改造，推进卵巢癌的免疫治疗奠定了良好基础。

Objective:To clone and express 6Bll ovarian ctqrcinoma anti-idioiypic antihody(Ab2β)singlechain Fv(ScFv)genes.To analyze the nucleotide sequence of ScFv.Methods:Using RT-pCR,the vari-able region genes of heavy llnd light chains(VH and VL)of 6B11 were amplified and assembled with aflexible linker sequence to form ScFv genes,which were then cloneed into,bacterial expresxion vectors toproduce proteinx,Nucleotide sequences of VH and VL were analyzed.Results:Expressed proteins fromsome clones could interact specifically with the primary anti-Ovarian carcinoma monoclonal antibody(COC166-9).The analysis of nucleotide sequence indicated that 6B11 VH and VL genes belonged to themouse Ig heavy chain subgroup I and light chain subgrnup Ⅱ,respectively.Conclusion:6B11 Ab2βSeFvgenes were successfully cloned and expressed,whch may play an imporlant role in genetically manipu-lating The murine Ah2 and make it close to clinical application for immunothcrapy of ovarian cancer.