携带K5基因突变体的腺病毒构建及K5蛋白表达  

Construction of recombinant adenovirus carrying mutant human plasminogen Kringle 5 gene and expression of K5 protein

作　　者：刘岩[1] 刘芳[2] 叶迅[2] 董继斌[3] 陆琴[2] 李秋华[1] 陈红专[2] 

机构地区：[1]复旦大学附属华山医院眼科,上海200040 [2]上海交通大学医学院药理学教研室,上海200025 [3]复旦大学药学院生物化学教研室,上海200032

出　　处：《复旦学报（医学版）》2008年第6期852-856,共5页Fudan University Journal of Medical Sciences

基　　金：上海市自然科学基金项目(03Z214116)

摘　　要：目的对血管生成抑制因子纤溶酶原Kringle 5(K5)基因进行突变,构建于重组腺病毒中,并观察突变后K5蛋白的表达情况。方法用重叠延伸技术对K5基因Furin蛋白酶水解识别位点进行定点突变,分别构建携带K5基因和突变后的K5m基因表达框的腺病毒载体Ad-EF1-αK5/K5m;用RT-PCR法比较腺病毒感染TC-1细胞后K5和K5m mRNA的表达;用免疫沉淀和Western blot法比较腺病毒感染TC-1细胞后K5蛋白和突变后K5蛋白的表达。结果成功对K5基因进行点突变,携带K5突变基因腺病毒K5蛋白表达量明显多于携带未突变K5基因腺病毒。结论对K5基因Furin蛋白酶水解识别位点进行定点突变能提高腺病毒介导的K5蛋白分泌表达量,为进一步以腺病毒为载体携带K5基因在眼部新生血管异常增生性疾病的实验研究提供基础。Objective To construct the recombinant adenovirus containing wild or mutant type of human plasminogen Kringle 5 （K5） gene, then observe the secreted expression of K5 protein. Methods The mutant site was induced by site directed mutagenesis at the Furin protease recognizing site; the wild and mutant type of K5 gene were cloned into the genome of replication-defective adenovirus by virus recombination technology; the K5 RNA expression was evaluated by RT-PCR~ the K5 protein secreted expression was determined by immunoprecipitation and Western blot. Results The mutant K5 gene was correctly constructed. The recombinant adenoviruses Ad-EFla-K5/K5m were constructed successfully and the quantity of K5 protein expressed by Ad-EFla-K5m was much larger than that of Ad-EFla-K5. Conclusions The mutagenesis of K5 gene at the Furin protease recognizing site can improve the quantity of K5 protein expression. This study may provide basis for the further use of recombinant adenovirus vectors containing K5 gene therapy in neovaseular ocular disease.

关 键 词：腺病毒 KRINGLE5 Furin蛋白酶 

分 类 号：Q754[生物学—分子生物学]