表达抗G250人源性抗体增殖缺陷型腺病毒的构建  

Construction of a replication - deficient adenovirus expressing anti -G250 fully human antibody

作　　者：方琳[1] 程乾[1] 李望[1] 刘俊杰[1] 郑骏年[1] 

机构地区：[1]徐州医学院江苏省肿瘤生物治疗重点实验室,江苏徐州221002

出　　处：《徐州医学院学报》2013年第1期1-6,共6页Acta Academiae Medicinae Xuzhou

基　　金：国家自然科学基金（81101702）;江苏省自然科学基金（BIL2011207,BK2009089）;江苏省“六大人才高峰”项目（2011-ws-069）;江苏省高校自然科学研究重大项目（12K,JA320001）

摘　　要：目的构建表达抗G250人源性抗体的增殖缺陷型腺病毒。方法通过重叠延伸PCR、常规PCR方法，分别获得G250重链的可变区和恒定区（GH）eDNA序列及G250轻链（GL）eDNA序列，并克隆至pCLON12-IRES载体上，构建质粒pCLON12-GL-IRES—GH。酶切质粒pCLON12-GL—IRES—GH回收GL—IRES—GH片段，克隆至腺病毒载体pDC315，构建pDC315-GL—IRES—GH（pDC315-G250）。将pDC315-G250与腺病毒包装骨架质粒pBHGE3共转染HEK293细胞，重组包装表达G250抗体的增殖缺陷型腺病毒AdDC315一G250。纯化病毒表达的G250抗体，免疫印迹实验检测该抗体能否与细胞表面抗原特异性结合。结果PCR及测序结果表明成功构建质粒pCLON12-GL—IRES—GH及腺病毒穿梭质粒pDC315-G250。PCR鉴定表明重组腺病毒AdDC315-G250含有目的基因，病毒包装成功。免疫印迹实验结果显示，纯化的抗体在G250抗原阳性的细胞中能检测到目的条带。结论成功构建表达抗G250人源性抗体的增殖缺陷型腺病毒。Objective To construct a replication - deficient adenovirus expressing anti - G250 fully human antibod- y. Methods GH cDNA coding for the heavy chain of G250 antibody and GL cDNA coding for the light chain of G250 antibody were amplified by overlap PCIl and conventional PCR using specific primers, and then GH and GL gene were cloned into pCLON12 （IRES） resulting in the plasmid pCLON12 -GL -IRES -GH. The correct construction of this plasmid was confirmed by DNA sequencing. The fragment （2 724 bp） of GL -IRES -GH released from pCLON12 -GL - IRES - GH with Eeot/I and Xho I was sub - cloned into the same sites of pDC315, resulting in the plasmid pDC315 - GL - IRES - GH （ also named pDC315 - G250）. The correct construction of this plasmid was confirmed by DNA se- quencing. In order to construct recombinant adenovirus, the plasmid pDC315 -（;250 and adenovirus packaging plasmid pBHGE3 were co -transfected into HEK293 cells. The AdDC315 -（;250 viral plaques appeared 9 -12 days after infec- tion. Western blotting assay was done to identify the specific binding between the G250 antibody mediated by adenovirus and cell surface （;250 antigen. Results pCLON12 - GL - IRES - GH and pDC315 - G250 were constructed as de- scribed in methods. Both PCR and sequence assay confirmed the correct construction of these vectors. We constructed a replication- deficient adenovirus AdDC315 -（;250 which earried the anti -G250 fully human antibody expressing cas- sette. To verify the sequences of this adenovirus, DNA was extracted from purified viruses and analyzed by PCR. The aimed band in G250 - positive cell lines was detected by Western blotting. Conclusion We had suceessfully constructed the replication - deficient adenovirus AdDC315 - G250 which expressed the anti - G250 fully human antibody.

关 键 词：肾肿瘤 G250 抗体 增殖缺陷型腺病毒 重组腺病毒 

分 类 号：R392.12[医药卫生—免疫学]