内源性血管生成抑制因子Arresten基因的克
发布时间：2018-07-31 14:12
【摘要】： 目的:构建内源性血管生成抑制因子Arresten基因的原核表达载体,优化表达条件,初步探讨表达蛋白的抑制血管活性。 方法:从GenBank库中查找Arresten基因并获取其基因序列及蛋白序列,用DNAman软件设计引物。从健康产妇胎盘组织中提取人总RNA,经逆转录—聚合酶链式反应(RT-PCR)扩增出Arresten基因,低熔点琼脂糖凝胶电泳后切胶回收纯化,纯化产物和质粒pBV220连接,并转化E.coli JM109、DH5α、BL21、BL21(DE3)感受态细胞,在氨苄阳性平板中筛选阳性菌落。重组质粒用PCR法和三种限制性内切酶BamHⅠ、EcoRⅠ和PvuⅡ(基因序列155位的酶切位点)酶切进行鉴定,并进行序列测定,命名为pBV220-Arr。构建的重组质粒pBV220-Arr转化到宿主菌——E.coli JM109、DH5α、BL21、BL21(DE3)中进行原核表达。经SDS-PAGE凝胶电泳图条带分析,并结合湿菌重筛选出最优表达菌种,进行培养温度、培养时间、表达温度、表达时间及溶解氧量五方面表达,确定最优表达体系;超声破菌,分离得到包涵体,并对其进行洗涤纯化复性,考马斯亮蓝法测定蛋白含量;鸡胚绒毛尿囊膜实验观察新生血管生长抑制情况。 结果:成功筛选出Arresten基因,通过测序结果表明在第132、148、200、236位基因序列不同;通过成功构建的重组质粒pBV220-Arr,在E.coli JM109、DH5α、BL21、BL21(DE3)中均表达出Arresten蛋白,SDS-PAGE显示表达产物分子量约为26KD,表达产物主要以包涵体形式存在;通过优化表达,结果在E.coli BL21(DE3)中30℃培养4 h,42℃诱导表达4 h,溶解氧量比例在1㑳5时表达量达到最高,表达量约占全菌蛋白的25%以上;进一步纯化表达,用2 mol/L尿素、2%的Triton X-100溶液和1M的蔗糖溶液,及少许的EDTA洗涤效果最好,表达量约占全菌蛋白的40%以上,用梯度稀释法复性得到Arresten蛋白;经活性分析证实初步复性的Arresten蛋白与阴性对照组比较统计学上有显著性差异,证明其有抑制新生血管生成的作用。 结论:应用基因工程技术,成功筛选出Arresten基因,并构建了原核表达重组体pBV220-Arr;通过优化表达,得到最优表达菌种E.coli BL21(DE3)及最优表达条件,能高效表达重组Arresten蛋白;经过用尿素与Triton X-100及蔗糖溶液综合方法洗涤纯化,纯化效果最好并获得了较纯的Arresten蛋白;经过CAM试验证明,该蛋白具有良好的抑制新生血管生成的活性;Arresten蛋白具有良好的应用前景,通过对Arresten蛋白的研究,为进一步大规模高效表达和纯化Arresten目的蛋白提供了可靠的实验方法,也为肿瘤的基因治疗研究奠定了基础。
[Abstract]:Aim: to construct the prokaryotic expression vector of endogenous angiogenesis inhibitor (Arresten) gene, optimize the expression conditions and explore the inhibition of angiogenesis activity of the expression protein. Methods: Arresten gene was searched from GenBank library and its gene sequence and protein sequence were obtained. Primers were designed by DNAman software. The Arresten gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from placental tissue of healthy puerpera. The Arresten gene was purified by gel electrophoresis with low melting point agarose gel electrophoresis. The purified product was ligated with plasmid pBV220, and transformed into E.coli JM109 DH5 伪 -BL21 BL21 (DE3) cell. Positive colonies were screened in ampicillin positive plate. The recombinant plasmids were identified by PCR and restriction endonuclease BamH 鈪，
本文编号：2155838
[Abstract]:Aim: to construct the prokaryotic expression vector of endogenous angiogenesis inhibitor (Arresten) gene, optimize the expression conditions and explore the inhibition of angiogenesis activity of the expression protein. Methods: Arresten gene was searched from GenBank library and its gene sequence and protein sequence were obtained. Primers were designed by DNAman software. The Arresten gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from placental tissue of healthy puerpera. The Arresten gene was purified by gel electrophoresis with low melting point agarose gel electrophoresis. The purified product was ligated with plasmid pBV220, and transformed into E.coli JM109 DH5 伪 -BL21 BL21 (DE3) cell. Positive colonies were screened in ampicillin positive plate. The recombinant plasmids were identified by PCR and restriction endonuclease BamH 鈪，
本文编号：2155838
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