构建携带增强型绿色荧光蛋白报告基因的重组反转录病毒表达载体pLXSN-Kozak-EGFP及鉴定  

Construction and identification of recombinant retroviral expression vector containing enhanced green fluorescent protein report gene pLXSN-Kozak-EGFP

作　　者：杨天燕[1] 王乃平[2] 王劲[1] 刘冠达[2] 韦锦斌[3] 

机构地区：[1]广西医科大学第一附属医院药剂科,广西壮族自治区南宁市530027 [2]广西中医学院药理学教研室,广西壮族自治区南宁市530001 [3]广西医科大学实验中心,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究与临床康复》2010年第37期6908-6912,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广西自然科学基金(0640125;0991158)~~

摘　　要：背景:增强型绿色荧光蛋白作为报告基因可在活细胞中表达发光蛋白。研究表明通过促进表达增强的Kozak序列可促进基因编码的蛋白质在真核细胞中的表达。目的:构建含增强型绿色荧光蛋白报告基因EGFP与促进表达增强的Kozak序列的pLXSN-Kozak-EGFP重组反转录病毒表达载体,并对此重组载体进行鉴定。方法:用聚合酶链反应方法从含增强型绿色荧光蛋白基因的表达载体pEGFP-N1上高保真克隆出EGFP基因,通过分子克隆技术将目的基因重组至反转录病毒表达载体pLXSN。用菌斑快速聚合酶链法筛选阳性重组子,针对目的基因插入载体的方向设计鉴别插入方向的引物,用聚合酶链反应法、限制性酶法鉴定目的基因及连接的正向性,并对pLXSN-Kozak-EGFP重组载体进行DNA测序分析。结果与结论:从pEGFP-N1载体中克隆出上游含Kozak序列的EGFP基因,目的条带大小约750bp。构建的pLXSN-Kozak-EGFP重组子经菌落PCR鉴定,750bp处有特异性条带。插入方向经PCR鉴定,350bp处有特异性条带。限制性内切酶法鉴定,750与6000bp处有特异性条带。DNA测序比对结果表明克隆的目的基因与Kozak-EGFP一致性为100%。结果提示实验成功构建了含EGFP报告基因与促进表达增强的Kozak序列的pLXSN-Kozak-EGFP重组反转录病毒表达载体,且插入方向为正向。BACKGROUND：Enhanced green fluorescent protein（EGFP） can express photoprotein in vital cells as a report gene.Kozak consensus sequences at translation initiation site should increase the translation efficiency in eukaryotic cells.OBJECTIVE：To construct the recombinant retroviral expression vector which contains report gene EGFP and Kozak consensus sequences increasing the translation efficiency in eukaryotic cells,and to identify this recombinant vector.METHODS：EGFP gene was high-fidelity cloned from expression vector pEGFP-N1 containing EGFP report gene using polymerase chain reaction（PCR）.Target gene was inserted to retroviral expression vector pLXSN with molecular cloning techniques.Positive recombinants were screened via colonial rapid PCR.The primers were designed through insertion site in vector in order to identify the insert direction of target gene,and PCR and restrictive enzymes digestion was used to identify its correction.Furthermore,DNA sequences of pLXSN-Kozak-EGFP were examined.RESULTS AND CONCLUSION：The EGFP gene with Kozak consensus sequences at its upper flank was cloned,with length of 750 bp.Recombinant pLXSN-Kozak-EGFP was screened by colonial rapid PCR,gained the specific product about 750 bp.Recombinant plasmid pLXSN-Kozak-EGFP was identified with PCR individually,gained the specific product about 750 bp;and insert direction was identified with PCR,gained the specific product about 350 bp.Restrictive enzymes double digested the recombinant plasmid,obtained the specific products about 750 bp and 6 000 bp.Sequences alignment indicates 100% identity between target DNA and Kozak-EGFP.The results indicate that the retroviral expression vector pLXSN-Kozak-EGFP containing EGFP report gene and Kozak sequences has been constructed successfully in this experiment,and target gene insert direction is correct.

关 键 词：增强型绿色荧光蛋白 报告基因 反转录病毒 载体 基因重组 

分 类 号：R318[医药卫生—生物医学工程]