重组质粒pCR3.1-brZPC’的构建及其体内外表达

Construction of recombinant vector pCR3.1-brZPC' and its expression in vivo and in vitro

目的 构建重组质粒 pCR3 .1 brZPC’ ,检验其在细胞及动物体内的表达。 方法 应用反转录聚合酶链反应 (RT PCR)法克隆基因 ,与真核表达载体pCR3 .1相连 ,转化感受态菌后酶切鉴定重组质粒。脂质体法转染Hela细胞 ,RT PCR法检测重组质粒在细胞中mRNA水平的表达 ;免疫荧光法检测重组质粒在细胞中蛋白水平的表达。接种BALB/c小鼠 ,一周后提取其各组织的总RNA ,RT PCR检测重组质粒在被免疫动物体内mRNA水平的表达情况。 结果 重组质粒 pCR3 .1 brZPC’能在细胞和被免疫动物体内高效表达。被免疫动物可以产生相应的抗体 ,且与该重组蛋白在细胞外结合。 结论 重组质粒 pCR3 .1

Objective: To construct recombinant vector pCR3.1-brZPC' and examine its expression in cell and in animals. Methods: To clone gene by RT-PCR. Gene is jointed with vector pCR3.1. Then recombinant vector is identified by enzyme. Recombinant vector is transfected into Hela cell with lipofectamine. Expression of mRNA and protein of recombinant vector PCR 3.1-brZPC' in Hela cell is checked either by RT-PCR or by immunofluorescence.Pick up total RNA from murine tissue one week after inoculating and check expression of mRNA of recombinant vector pCR3.1-brZPC' by RT-PCR Results: Recombinant vector pCR3.1-brZPC' can highly be expressed both in cell and in animals. Antibody can be produced by inoculated animals and can combine with protein of recombinant vector PCR3.1-brZPC' expressing in cell. Conclusion: Recombinant vector pCR3.1-brZPC' can be expressed both in cell and animals. Antibody can be produced in animals.