人重组pcDNA6.2/生长分化因子5质粒的构建及鉴定

Construction and identification of human recombinant pcDNA6.2/growth differentiation factor-5 plasmid

背景:生长分化因子5是一种刺激肌腱、韧带塑型,增强愈合反应有效的生长因子,在活性组织工程肌腱构建中有重要作用。目的:构建人重组pcDNA6.2/生长分化因子5质粒,为转染基质干细胞向肌腱诱导分化实验奠定基础。设计、时间及地点:细胞基因工程体外实验,于2007-08/12在哈尔滨医科大学遗传学教研室和分子生物学教研室完成。材料:根据Genbank(NM000557)中人生长分化因子5的序列化学合成一对引物,从人胎儿软骨组织提取总RNA。方法:通过反转录-聚合酶链反应得到人生长分化因子5完整成熟肽基因。将所得基因片段插入克隆载体pcDNA6.2并转化入JM109菌株,提取重组质粒,PCR鉴定、酶切鉴定并测序。主要观察指标:反转录-聚合酶链反应检测结果,PCR及SalⅠ和BamHⅠ双酶切鉴定结果,重组质粒测序与Genbank中序列比较结果。结果:电泳显示,反转录-聚合酶链反应产物为一长约380bp的带,阳性克隆质粒经PCR电泳及双酶切均出现约380bp的片段。测序表明与Genbank中的序列完全相符。结论:成功构建出人重组pcDNA6.2/生长分化因子5质粒,为组织工程化肌腱过程中种子细胞的制备提供转染质粒。

BACKGROUND： Growth differentiation factor-5 is an kind of growth factors that may stimulate the molding of tendon and ligament, in addition, reinforce the healing reaction. It plays an important role on the construction of active tissue engineering tendon. OBJECTIVE： To construct human recombinant pcDNA6.2/growth differentiation factor-5 plasmid and provide a basis for inducing bone marro-derived stromal stem cells to differentiate into tendon. DESIGN, TIME AND SETTING： The experiment, cell gene engineering trial in vitro, was completed in Department of Genetics and Department of Molecular Biology, Harbin Medical University from August to December in 2007. MATERIALS： A couple of primers were chemosynthesized according to the human growth differentiation factor-5 sequence reported in Genbank （NM000557）. Total RNA was extracted from human fetus cartilage tissue. METHODS： The mature peptide gene of human growth differentiation factor-5 was gained by reverse transcription-polymerase chain reaction （RT-PCR）, and was cloned into plasmid pcDNA6.2, then transformed into E.coli JM109. The plasmid of the positive clone was analyzed by restriction endonuclease mapping, PCR electrophoresis and DNA sequence analysis. MAIN OUTCOME MEASURES： RT-PCR test results, identification results of PCR, Sal I and BamH I, the result of sequence assay compared with the sequence in Genbank. RESULTS： Agarose gel electrophoresis showed that the product of RT-PCR was about 380 bp, PCR and double enzyme digestion of the recombinant plasmid were consistent with it. The result of sequence assay was in agreement with the reported human growth differentiation factor-5 sequence in Genbank. CONCLUSION： The human recombinant eukaryotic expression plasmid pcDNA6.2/growth differentiation factor-5 can be successfully constructed. It is a promising plasmid to transfect seed cells for tissue engineering tendon.