大鼠神经母细胞特异性转移因子基因重组腺病毒载体的构建及鉴定  

Construction and identification of a recombinant adenovirus vector expressing rat achaete-scute homology 1

作　　者：袁静[1] 葛坚[2] 俞建雄[3] 

机构地区：[1]武汉大学人民医院眼科中心,湖北省武汉市430060 [2]中山大学中山眼科中心眼科学国家重点实验室,广东省广州市510060 [3]武汉大学人民医院普外科,湖北省武汉市430060

出　　处：《中国组织工程研究》2015年第41期6699-6705,共7页Chinese Journal of Tissue Engineering Research

基　　金：中山大学中山眼科中心眼科学国家重点室开放课题(303060202400306)“ASCL1信号在诱导神经干细胞向视神经细胞定向分化中的作用及机制”;国家自然科学基金青年基金(81100664)“17β-E2调控BMSCs向神经细胞分化及在青光眼视神经再生中的作用和机制”;武汉市青年科技晨光计划项目(2014070404010222“miR-124调控诱导神经干细胞向视神经细胞定向分化和在青光眼视神经再生中的作用及机制”;武汉大学自主科研项目学科交叉类(2042014kf0259)“复合TFPI-2多肽凝胶药物控释系统在青光眼小梁切除手术中的应用及作用机制研究”~~

摘　　要：背景:研究表明,神经母细胞特异性转移因子(ASCL1)是神经发育过程中的关键调控因子,因此通过对成体细胞进行ASCL1基因修饰,有可能将成体细胞转分化为神经细胞,为视神经再生治疗提供新策略。目的:构建携带鼠ASCL1基因的重组腺病毒表达载体,获得重组腺病毒pA d-rat-ASCL1-EGFP,以期为进一步研究ASCL1基因的功能奠定基础。方法:用XhoⅠ和EcoRⅠ将目的基因ASCL1及带有增强型绿色荧光蛋白表达盒的腺病毒穿梭载体p Yr-adshuttle-4进行双酶切;回收酶切质粒的目的片段进行连接,产物转化至DH5α感受态;提取质粒酶切鉴定正确后测序。然后通过LR体外同源重组将rat-ASCL1表达框构建至pA d/PL-DEST腺病毒表达载体,PacⅠ酶切线性化后用脂质体法转染HEK293细胞进行包装、扩增,采用PCR法对重组腺病毒进行鉴定,TCID50法测定病毒滴度,荧光显微镜观察病毒感染效率。结果与结论:通过酶切鉴定和DNA测序鉴定,证实重组腺病毒载体pA d-rat-ASCL1-EGFP构建正确,PCR鉴定扩增出862 bp的目的条带,TCID50法测定病毒滴度为2×1010pfu/mL。荧光显微镜观察HEK293细胞的病毒感染效率为80%以上。提示含ASCL1基因的重组腺病毒载体构建成功,获得的病毒具有高滴度,高效感染率,为下一步进行ASCL1基因功能研究和视神经再生治疗研究奠定了基础。BACKGROUND： Previous studies have suggested that achaete-scute homology 1（ASCL1） plays a key role in the neuronal commitment. Therefore, somatic cells may directly differentiate into neurons by gene transfection of ASCL1, which will provide new therapeutic strategies for optic nerve regeneration. OBJECTIVE： To construct a recombinant adenovirus vector expressing rat ASCL1 gene for further research of ASCL1 gene function. METHODS： The rat ASCL1 gene and advenovirus shuttle plasmid（p Yr-adshuttle-4） which contained enhanced green fluorescent protein（EGFP） reporter gene were cleaved by restriction endonuclease Xho I and Eco R I. The target gene fragments were connected together to generate a recombinant plasmid p Yr-ads-4-rat-ASCL1 and then transfected into E.coli DH5α. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequence reaction. The plasmid p Yr-ads-4-rat-ASCL1 and p Ad/PL-DEST were reconstructed by homologous recombination processes to obtain rat ASCL1 recombinant adenovirus vector. The plasmid p Yr Ad-r ASCL1 was linearized by Pac I and subsequently transfected into HEK293 cells for packaging and amplification. Rat ASCL1 gene in the recombinant adenoviruses were identified by PCR. Virus titer was determined by tissue culture infectious dose 50. Infection efficiency was monitored by EGFP expression. RESULTS AND CONCLUSION： Restriction endonuclease digestion and DNA sequencing showed that the recombinant adenovirus vector p Ad-rat-ASCL1 was constructed correctly. The positive amplification bands of 862 bp could be seen in PCR analysis. The virus titer reached 2×1010 pfu/m L. Infection efficiency of recombinant adenovirus in HEK293 cells was more than 80%. The results indicate that the recombinant the adenovirus vector containing ASCL1 with high titer and infection efficiency has been successfully constructed, which can be helpful for further research of the function and clinical application of ASCL1 gene for optic nerve regeneration.

关 键 词：干细胞 腺病毒科 绿色荧光蛋白质类 视神经 组织工程 培养 神经母细胞特异性转移因子 重组腺病毒载体 增强型绿色荧光蛋白 病毒滴度 视神经再生 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]