小鼠次级淋巴组织趋化因子成熟肽基因/腺相关病毒重组载体的构建  

TO CONSTRUCT A RECOMBINANT ADENO-ASSOCIATED VIRUSVECTOR CARRYING THE GENE ENCODING THE MATURE PEPTIDE OFSECONDARY LYMPHOID-TISSUE CHEMOKINE

作　　者：梁春敏[1,2] 钟翠平[1] 郑宁[1] 吴欣[3] 孙瑞霞[3] 刘银坤[3] 叶胜龙[3] 

机构地区：[1]复旦大学上海医学院人体解剖学与组织学胚胎学系 [2]皖南医学院组织学胚胎学教研室,芜湖241001 [3]复旦大学肝癌研究所

出　　处：《解剖学报》2004年第2期147-151,共5页Acta Anatomica Sinica

基　　金：国家 8 63基金资助项目 ( 2 0 0 1AA2 1713 1) ;国家自然科学基金资助项目 ( 3 0 3 70 748) ;复旦大学"985工程"重中之重学科项目;"2 11工程"肿瘤学科建设项目基金资助

摘　　要：目的　构建含小鼠次级淋巴组织趋化因子 (SLC)cDNA的重组腺相关病毒载体。　方法　以C5 7BL 6J小鼠的淋巴组织为材料抽提总RNA ,用RT PCR方法克隆小鼠SLC的成熟肽基因。PCR产物回收后经EcoRⅠ和XhoⅠ双酶切 ,插入pAAV IRES hrGFP质粒 ,用磷酸钙法 ,与pAAV RC和pHelper三者共同转染HEK2 93细胞 ,包装成重组的病毒颗粒 ,并通过绿色荧光蛋白 (GFP)报告基因荧光检测及抽提病毒DNA ,进行PCR扩增等进一步证实重组病毒的形成。　结果　约 5 0 0bp的小鼠SLC成熟肽基因被成功克隆 ,序列分析表明 ,所克隆的SLC基因与基因库注册的相同 ,重组载体的酶切及测序鉴定表明SLC基因被定向插入。重组载体转染HEK2 93细胞 ,经荧光显微镜和病毒DNA的PCR检测 ,证实已完成对重组病毒的包装 ,并表达GFP。　结论　成功构建了SLC成熟肽基因重组腺相关病毒载体。Objective To construct a recombinant AAV vector modifed by mouse secondary lymphoid tissue chemokine(SLC) gene. Methods The total RNA of C57BL/6J-mice lymph nodes was extracted and the gene,encoding the mature peptide of SLC,was cloned by RT-PCR.The EcoRⅠ-XhoⅠ fragment of the PCR product was inserted into pAAV-IRES-hrGFP vector,an ITR-containing plasmid,which co-transfect HEK 293 cells with pAAV-RC and pHelper to produce viral particles,following the calcium phosphate-based protocol.Viral titer was determined directly by calculating the fraction of fluorescent cells in microscopic fields.SLC was cloned from virus DNA to test the rAAV. Results The PCR product was observed about 500*!bp long by DNA electrophoresis.Nucleotide sequence analysis indicated that the sequence of the cDNA was exactly the same as that registered in the gene bank and the SLC gene was inserted into the vectors.The GFP was expressed and could be detected using fluorescence microscopy.PCR from the virus DNA also confirmed the rAAV.Conclusion The recombinant AAV vector was constructed successfully.This research will facilitate the further study of the immunobiological characteristics of SLC in the immuno-therapy agaist tumors.

关 键 词：小鼠 次级淋巴组织趋化因子 成熟肽基因 腺相关病毒 重组载体 构建 

分 类 号：R392.11[医药卫生—免疫学]