Cloning of rat C-sis gene and construction of its eukaryotic expression vector

doi:10.3969/j.issn.2095-4344.2016.49.018

Abstract

Abstract:

BACKGROUND: C-sis proto-oncogene can promote tissue repair by inducing cell proliferation and inhibiting cell apoptosis. Therefore, C-sis may play a positive role in the repair of damaged liver tissue and the treatment of fulminant hepatic failure.
OBJECTIVE: To construct pcDNA3.1/C-sis eukaryotic expression vector and detect its expression in BRL cells (the normal liver cells of rats) and rat liver cells in vivo.
METHODS: The full-length coding sequence of C-sis gene was cloned through real time-PCR. pcDNA3.1/C-sis eukaryotic expression vector was constructed and sequenced, followed by transfected into BRL cells using liposome and injected into the rat liver via tail vein. Finally, its expression in BRL cells and rat liver cells in vivo was identified by fluorescence quantitative PCR and western blotting.
RESULTS AND CONCLUSION: (1) The full length of encoding region of C-sis gene was successfully cloned. Sequencing proved that pcDNA3. 1/C-sis recombinant eukaryotic expression vector was constructed successfully. (2) The expression of C-sis was increased after transfected into BRL cells and rat liver. (3) These results provide basis for the subsequent study of the effect of C-sis gene on fulminant hepatic failure in rats.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Models, Animal, Genes, Liposomes, Transfection, Tissue Engineering

CLC Number:

R318

Sun Guo-fang, Ding Hao. Cloning of rat C-sis gene and construction of its eukaryotic expression vector[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(49): 7418-7424.

Figures/Tables 1

2.1 造模方法的改进首先克隆了大鼠C-sis基因，并构建pcDNA3.1/C-sis真核表达载体，通过酶切测序以证实C-sis基因克隆成功与否。然后将pcDNA3.1/C-sis转染进入大鼠肝脏细胞，并通过RT-PCR和Western Blot检测C-sis基因在大鼠正常肝细胞株BRL细胞和在体大鼠肝脏细胞中的表达。 2.2 模型稳定性结果显示，pcDNA3.1/C-sis真核表达载体构建成功，且将其转染入BRL细胞及导入在体大鼠肝脏，RT-PCR和Western Blot均显示C-sis的表达增加。C-sis能在肝脏稳定高表达，这为后续研究C-sis对暴发性肝衰竭的治疗作用提供了稳定模型。 2.3 大鼠C-sis基因的RT-PCR扩增大鼠C-sis基因通过特异性引物扩展的PCR产物只有一条亮带，位于726 bp的位置。根据数据库GenBank所收录的C-sis的基因序列(NM_24628)，所得产物大小与预期值相符合，见图1。 2.4 真核表达载体pcDNA3.1/C-sis的鉴定限制性内切酶EcoRⅠ、XbaⅠ酶切重组质粒pcDNA3.1/C-sis，用1%琼脂糖凝胶电泳检测，初步鉴定重组质粒pcDNA3.1/ C-sis构建成功，见图2。经南京金斯瑞公司测序，与Pubmed数据库中GenBank所给序列(No：NM24628)对比同源性达到100%，进一步证实重组质粒pcDNA3.1/C-sis构建成功。 2.5 真核表达载体pcDNA3.1/C-sis的结构图如图3所示，dh-1015即为C-sis基因，其余所示为酶切的位点。 2.6 pcDNA3.1/C-sis转染BRL细胞后C-sis mRNA的表达与空白对照组和空载质粒组比较，C-sis质粒组的C-sis mRNA表达量有明显增加，见图4。 2.7 pcDNA3.1/C-sis转染BRL细胞后C-sis蛋白的表达与空白对照组、空载质粒组比较，C-sis质粒组的C-sis蛋白表达量有明显增加，见图5。 2.8 pcDNA3.1/C-sis导入大鼠肝脏后C-sis mRNA的表达实验结果由荧光定量PCR分析软件BIO-RAD CFX Manager自动进行统计和计算。与空白对照组和空载质粒组比较，C-sis质粒组的C-sis mRNA表达量有明显增加，见图6。 2.9 pcDNA3.1/C-sis导入大鼠肝脏后C-sis蛋白的表达与空白对照组、空载质粒组比较，C-sis质粒组的C-sis蛋白表达量有明显增加，见图7。"

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