pSNAV2CTGF重组质粒构建及其在人胚肾293细胞中表达.docVIP

pSNAV2CTGF重组质粒构建及其在人胚肾293细胞中的表达 【摘要】 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。 【关键词】 结缔组织生长因子 腺相关病毒 质粒 基因转染 椎间盘 ［ABSTRACT］ObjectiveTo 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). MethodsCTGF 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. ResultsA 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. ConclusionThe eukaryotic expression vector, pSNAV2CTGF, is constructed, which can express CTGF protein with biological activity after transfection with HEK293 cells. ［KEY WORDS］ connective tissue growth factor; adeno associated virus; plasmid; transfection; intervertebral disc 结缔组织生长因子（CTGF）是一种富含半胱氨酸的分泌性多肽,是CCN (CTGF、Cef10/ cyr61 和Nov 的缩写) 家族中的成员，参与很多正常的生理过程，如受精卵的植入、胎盘形成、胚胎发育，也参与一些病理过程如伤口愈合、纤维变性等［1］。CTGF还可以刺激许多细胞合成细胞外基质（ECM）蛋白。在纤维化疾病如肝纤维化、肾纤维化、肺纤维化等已经对其进行了大量研究，但其在椎间盘领域中的研究还处在起步阶段。为了研究其在逆转腰椎间盘退变过程中的作用，本研究构建了重组质粒pSNAV2CTGF，并对其在人胚肾293（HEK293）细胞中的表达进行观察。 1 材料和方法 1.1 材料 携带人CTGF cDNA的pCMVSPORT6质粒购于Openbiosystems公司，腺相关病毒（AAV）表达载体pSNAV2.0购于本元正阳公司，HEK293