抗p185^(erbB2)人鼠嵌合抗体慢病毒表达载体的构建  

Construction of the Lentiviral Expression Vector for Anti-p185^(erbB2) Mouse/Human Chimeric Antibody

作　　者：刘芳[1,2] 李力[1] 张玮[1] 王琪[1] 

机构地区：[1]广西医科大学附属肿瘤医院,南宁530021 [2]广西大学农学院,南宁530005

出　　处：《生物医学工程学杂志》2013年第2期347-354,共8页Journal of Biomedical Engineering

基　　金：广西医科大学博士后启动基金资助项目(308068)

摘　　要：本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况。采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游。用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L。将其转染293T细胞,48h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达。结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础。This research was to construct the lentiviral expression vector for anti- p185^erbB2 mouse/human chimeric an- tibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes （vL and vH） coding light and heavy chain of variable regions of anti-p185^erbB2 mAb and the constant regions of human IgG1 （κ and γ1） were cloned with PCR method, The target genes were assembled by three-primers PCR method to obtain the chimeric light chain （L） and the chimeric heavy chain （H）. Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and se- quence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent mi- croscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185^erbB2 specifically. This research may lay a sound foundation for further study of anti-p185^erbB2 engineered antibody.

关 键 词：ERBB2 嵌合抗体 慢病毒载体 

分 类 号：Q78[生物学—分子生物学]