3D_5卵巢癌抗独特型单链抗体基因克隆及表达

CLONING AND EXPRESSING OF 3D_5 ANTI-IDIOTYPIC ANTIBODY SeFv GENES IN E.COLI

目的：降低３Ｄ＿５抗独特型抗体鼠源性，促进临床应用。方法：用ＰＣＲ及基因重组技术，扩增并构建单链可变区抗体基因，与表达载体连接后转化大肠杆菌。结果：ＥＬＩＳＡ检测３７个克隆，其中１７个表达产物与相应抗体呈特异免疫反应；重组体限制性内切酶酶切分析及ＰＣＲ扩增均见特异ＤＮＡ条带。结论：克隆表达了３Ｄ＿５抗独特型单链抗体基因，为进一步人源化改造奠定了基础。

Ohjective:In order to reduce the immunogenicity. we cloned and expressed 3D5ovarian car-cinoma Ab2singlechain Fv(SeFv)genes in E.coli. Methods: Using RT-PCR. the variable regiongeneS of heavyand light chains(VH and VL)of Ab2s hnve heen obtained from3D5 hybridomas. The VHand VL hxve been asxembled with a flexible linker.sequence to encode ScFv sequencesThe latter hasbeen cloned into bacterial expression vectors to produce protein. kesults . Expressed scFv proteins from44%of the checked clones were shown to react with the primaryantibody(Ab1).TheDNAfragmentswerefound to be thesamesize as that of the posltive control when theseelonedDNAwere digested withrestriction enzymes or amplified by PCR. Conclusion. The success of tHis study may not only pltqy a rolefor genetically manipulating the Ah2 but alsogive a clue for an active immunotherapy of ovariancancer.