TP-PCR法构建抗人CD28嵌合抗体双启动子昆虫杆状病毒重组转移载体  

Construction of a Recombinant Baculovirus Transfer Vector with Two Promoters Expressing the Anti-human CD28 Chimeric Antibody by Using TP-PCR Method

作　　者：朱艳[1] 陈永井[2] 邱玉华[2] 郑峰丰[1] 朱江[1] 

机构地区：[1]苏州大学生命科学学院,苏州215006 [2]苏州大学医学生物技术研究所,苏州215007

出　　处：《生物工程学报》2005年第5期832-836,共5页Chinese Journal of Biotechnology

基　　金：江苏省自然科学基金资助项目(No.BK2004203)。~~

摘　　要：为构建能同时表达抗人CD28嵌合重链和嵌合轻链基因的双启动子杆状病毒转移载体,利用PCR法扩增出抗人CD28鼠源性单抗的可变区基因和人IgG1的恒定区基因,再采用一种不需要限制性核酸内切酶和连接酶的新方法———三引物PCR(TP_PCR)法将两者拼接后分别插入杆状病毒转移载体pAcUW3的ph和p10启动子后,并通过酶切、电泳、PCR扩增和测序对重组体进行了鉴定。研究结果表明,TP_PCR法快速、方便、准确,无需设计外源的DNA序列,就能构建完好的融合表达基因。该转移载体的构建成功为嵌合抗体基因在昆虫细胞中的表达奠定了基础。CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p 10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.

关 键 词：杆状病毒 转移载体 CD28单克隆抗体 嵌合抗体 TP-PCR 

分 类 号：Q782[生物学—分子生物学]