pAdtrack-cmv-rHSG重组穿梭质粒载体的构建、序列分析及rHSG生物信息分析

发布时间：2018-01-21 15:44

  本文关键词： 增殖抑制基因 载体 基因重组 生物信息学　出处：《宁夏医科大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的(1)在前期构建pExpress-1-rHSG重组质粒载体基础上,构建大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)pAdtrack-cmv-rHSG重组穿梭质粒并鉴定,为进一步构建腺病毒载体及脑血管痉挛的基因治疗提供物质基础;(2)对测序报告结果中rHSG基因序列进行序列与结构相关的生物信息分析,为认识该基因结构与功能提供借鉴。方法(1)目的基因的提取:复苏本室保存的含pExpress-1-rHSG质粒的Jm109菌,用质粒抽取试剂盒抽取质粒pExpress-1-rHSG,并在PCR仪中扩增出目的基因rHSG cDNA。(2)穿梭质粒的构建及鉴定:①复苏本室保存的含腺病毒穿梭质粒pAdtrack-cmv的DH5α菌种,用质粒抽取试剂盒抽取质粒pAdtrack-cmv;②将PCR后rHSG cDNA连接到pGM-T质粒上,构建重组质粒pGM-T-rHSG,并对重组质粒进行限制性内切酶酶切鉴定;③将pGM-T-rHSG重组质粒在LB固体琼脂培养基上转化到大肠杆菌DH5α,提取pGM-T-rHSG质粒;④用BglⅡ、Eco RV对pGM-T-rHSG重组质粒进行双酶切,提取目的基因并鉴定;⑤用BglⅡ和Eco RV双酶切pAdtrack-cmv质粒得到线性化的pAdTrack-CMV片断,在T4DNA连接酶下与目的基因连接,构建出重组pAdtrack-cmv-rHSG穿梭质粒,并对重组穿梭质粒pAdtrack-cmv-rHSG进行酶切鉴定;⑥将重组穿梭质粒送测序公司进行DNA序列分析。(3)rHSG基因生物信息分析:根据测序报告结果中rHSG基因序列,用生物信息学方法对其进行同源性、分子量、蛋白跨膜区、等电点、亲疏水性、蛋白二级结构等项的分析和预测。结果(1)从质粒pExpress-1-rHSG中成功提取出了rHSG cDNA基因。(2)重组pAdtrack-cmv- rHSG穿梭质粒经BglⅡ和Eco RV双酶切后,进一步将重组体进行基因测序分析,验证了克隆的rHSG cDNA序列与GenBank[AF036536]公布的rHSG序列吻合。这一结果表明重组体pAdtrack-cmv-rHSG中插入的目的基因是正向、单倍插入。(3)rHSG生物信息分析结果显示,rHSG全长4151bp,编码一757氨基酸残基的蛋白,可能为一跨膜蛋白,经基因Genebank查询发现与人HSG基因序列有95.2%同源性。结论(1)成功构建了大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)真核表达载体pAdtrack-cmv- rHSG,为深入研究rHSG功能和rHSG基因治疗脑血管痉挛奠定了物质基础;(2)分析rHSG基因序列及蛋白结构,有助于进一步研究该基因的分子和生物学功能。
[Abstract]:Objective 1) to construct pExpress-1-rHSG recombinant plasmid vector. Rat hyperplasia suppressor gene was constructed. RHSG)pAdtrack-cmv-rHSG recombinant shuttle plasmid was identified to provide material basis for further construction of adenovirus vector and gene therapy of cerebral vasospasm. (2) the sequence of rHSG gene in the sequencing report was analyzed by structure-related biological information analysis. Methods 1) extraction of the target gene: resuscitation of Jm109 bacteria containing pExpress-1-rHSG plasmid. Plasmid pExpress-1-rHSG was extracted with plasmid extraction kit. The target gene rHSG cDNA2 was amplified by PCR. Construction and identification of shuttle plasmid DH5 伪 containing adenovirus shuttle plasmid pAdtrack-cmv in resuscitation room. Plasmid pAdtrack-cmv was extracted by plasmid extraction kit. (2) the recombinant plasmid pGM-T-rHSG was constructed by ligating rHSG cDNA after PCR to pGM-T plasmid, and the recombinant plasmid was identified by restriction endonuclease digestion. 3Recombinant pGM-T-rHSG plasmid was transformed into Escherichia coli DH5 伪 on LB solid Agar medium and pGM-T-rHSG plasmid was extracted. 4Recombinant pGM-T-rHSG plasmid was digested with Bgl 鈪，

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