小鼠B7-H4重组腺病毒的构建及鉴定  

Construction and identification of recombinant adenoviral vector encoding B7-H4 gene

作　　者：闫佩毅[1] 姚航平[1] 钱韵[1] 古丽娟[1] 陶岚[1] 张立煌[1] 

机构地区：[1]浙江大学医学院免疫学研究所,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2008年第5期499-505,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家重点基础研究发展计划资助项目(2003CB515503)

摘　　要：目的:利用AdEasyTMXL system构建携带小鼠B7-H4基因的重组腺病毒,并鉴定其生物学活性。方法:以RT-PCR方法从C57小鼠肺组织中扩增B7-H4全长基因并克隆到T载体,然后测序鉴定。经Xhol和EcoR双酶切后接入pshuttle-CMV穿梭载体(简称PSC),构建重组腺病毒的穿梭质粒PSC-mB7-H4,并电转化至BJ5183-AD-1感受态细菌,经筛选获得携带mB7-H4的重组腺病毒的质粒pmB7-H4/Ad。将此质粒转化人胚肾细胞(AD-293)产生复制缺陷的重组腺病毒mB7-H4/Ad。以RT-PCR及Western blot方法检测被mB7-H4/Ad感染的AD-293细胞中B7-H4 mRNA和蛋白的表达,并观察重组腺病毒感染的小鼠成纤维细胞(L929)对T细胞增殖和细胞因子表达的影响。结果:获得序列准确的mB7-H4基因并成功构建重组腺病毒表达质粒,转化AD-293细胞获得重组腺病毒;细胞生物学实验证实,该重组腺病毒具有抑制CD3单抗诱导的T淋巴细胞的增殖和细胞因子的分泌。结论:成功构建具有免疫抑制功能的mB7-H4重组腺病毒。Objective： To construct the recombinant adenovirus containing B7-H4 gene with AdEasyTM XL system and to identify its biological activities. Methods： The full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol Ⅰ and EcoR Ⅴ the B7-H4 gene was inserted into pshuttle-CMW（PSC）. Pme Ⅰ linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac Ⅰ linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested. Results： The full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination,and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production. Conclusion： The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.

关 键 词：T淋巴细胞/免疫学 重组 遗传 腺病毒科/遗传学 转染 细胞增殖 细胞 培养的 质粒 

分 类 号：R346[医药卫生—基础医学]