pSNAV_2-CTGF重组质粒构建及其在人胚肾293细胞中的表达

CONSTRUCTION OF ADENO ASSOCIATED VIRUS RECOMBINANT PLASMID OF CONNECTIVE TISSUE GROWTH FACTOR AND ITS EXPRESSION IN HEK293 CELLS

目的构建含人结缔组织生长因子(CTGF)的腺相关病毒(AAV)表达载体,并观察其在人胚肾293(HEK293)细胞中的表达。方法以含有人CTGF基因序列的质粒pCMV-SPORT6为模板,应用PCR克隆出CTGF基因,采用分子克隆的方法把CTGF克隆到AAV表达载体pSNAV2.0上构建重组质粒pSNAV2-CTGF。把重组质粒转染HEK293细胞,用免疫荧光法对重组质粒目的蛋白的表达进行研究,MTT法检测细胞培养上清液中CTGF蛋白的生物学活性。结果成功构建完全正确的CTGF基因序列的AAV表达载体pSNAV2-CTGF,将其转染HEK293细胞后,免疫荧光检测到目的蛋白的表达,转染后的细胞上清具有促使成纤维细胞增殖的生物学活性。结论成功构建了真核表达载体pSNAV2-CTGF,转染HEK293细胞后能够表达具有生物学活性的CTGF蛋白。

Objective To construct a recombinant plasmid of adeno associated virus （AAV） expression vector containing human connective tissue growth factor （CTGF） and to study its expression in human embryonic kidney 293 cells （HEK293）. Methods CTGF gene was cloned from pCMV-SPORT6 by PCR, then ligated with pSNAV2. 0 to form recombinant plasmid pSNAV2-CTGF and transducted into HEK293 cells. Immunofluorescence was used to detect the synthesis of CTGF protein in the HEK293 cells, MTT was used to detect the biological activity of CTGF in the supernatant of culture medium after transfection. Results A perfect CTGF gene was ligated to pSNAV2.0, after transfected into HEK293 cells, the expression of CTGF protein was observed and detected by immunofluorescence. The conditioned medium after transfection showed the biological activity of stimulating the proliferation of fibroblast. Conclusion The eukaryotic expression vector, pSNAV2-CTGF, is constructed, which can express CTGF protein with biological activity after transfection with HEK293 cells.