摘要
背景:低氧诱导因子1能够调控多种基因共同表达,在骨缺损部位可诱导成熟的血管生成,为各种细胞的成骨分化和成骨活动提供营养支持和代谢保证,促进骨愈合,但其真核表达载体的构建及表达却少见报道。目的:实验拟构建携带低氧诱导因子1αmu(hypoxia inducible factor 1 alpha,HIF-1α)目的蛋白和人源化海肾绿色荧光蛋白(human renilla reniformis green fluorescent protein,hrGFP)的双基因真核表达载体,并将其转染HEK293A细胞,观测其在细胞中的表达。方法:利用PCR技术定点突变目的基因供体质粒pCMV6-XL5-HIF1α携带的人HIF1α基因编码区的第402位、564位和803位氨基酸以及去掉其终止密码子,之后在基因序列前后添加新的酶切位点NotⅠ和PvuⅠ,酶切、测序检测突变情况,将正确突变的HIF1αmu定向连入腺病毒穿梭载体pShuttle-CMV-IRES-hrGFP-1中。经测序鉴定、PmeⅠ酶切线性化后转化BJ5183-AD-1电感受态细胞,通过hrGFP基因荧光表达检测转染情况。结果与结论:经基因测序证实,HIF-1α基因编码区的第402位、564位和803位氨基酸均定点突变成丙氨酸,终止密码子成功去除。经酶切鉴定及测序证实,重组腺病毒表达载体构建成功。荧光显微镜下观察表明,感染重组腺病毒的HEK293A细胞内有大量绿色荧光表达证实通过Lipofectamine 2000途径可使得重组腺病毒载体成功转染HEK293A细胞。
BACKGROUND：Hypoxia inducing factor 1 is able to regulate co-expression of various gene and induce new vascular generation in bone defects areas.It can supply nutritional support and metabolism promotion for osteogenesis differentiation andosteogenesis activity in cells and accelerate bone healing.However,studies regarding the construction and expression of hypoxia inducing factor 1 are few.OBJECTIVE：To construct a new adenovirus eukaryotic expression vector which can express mutant hypoxia inducible factor 1 α（HIF1-α） interest protein and reporter molecule of human renilla reniformis green fluorescent protein（hrGFP） and to transfect it into HEK293 cells to observe its expression.METHODS：Three aminoacid including the 402 location,the 564 location and the 803 location in gene coding region in donor vector pCMV6-XL5-HIF1α carrying HIF-1α were selected to complete site-directed mutagenesis and add new enzyme sites including Not Ⅰ and Pvu Ⅰ after removing stop codon pre and post gene sequence by polymerase chain reaction.The mutation was monitored by restriction enzyme and sequencing.The correct HIF-1α gene mutation HIF-1αmu was linked into adenovirus shuttle vector pShuttle-CMV-IRES-hrGFP-1 directionally.The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cell after sequencing identification and Pme Ⅰ restriction enzyme linearization.The transfection status was determined by hrGFP fluorescent expression.RESULTS AND CONCLUSION：Aminoacid including the 402 location,the 564 location and the 803 location in gene coding region in HIF-1α turned to alanine after rite-directed mutagenesis and removing stop codon was successful.Enzyme restrictionand sequencing confirmed that the recombinant adenoviral expressing vector was successful constructed.Results of fluorescence microscope showed there was a large number of green fluorescence expression in HEK293A cells,which confirmed that the recombinant adenovirus vector was successful
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第46期8594-8599,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省自然科学基金资助项目(项目编号:20062199)
课题名称:促进转染BMP2基因后的兔骨髓基质细胞在骨组织工程~~
