發(fā)布時間：2018-01-21 15:44

  本文關(guān)鍵詞： 增殖抑制基因 載體 基因重組 生物信息學(xué)　出處：《寧夏醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的(1)在前期構(gòu)建pExpress-1-rHSG重組質(zhì)粒載體基礎(chǔ)上,構(gòu)建大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)pAdtrack-cmv-rHSG重組穿梭質(zhì)粒并鑒定,為進一步構(gòu)建腺病毒載體及腦血管痙攣的基因治療提供物質(zhì)基礎(chǔ);(2)對測序報告結(jié)果中rHSG基因序列進行序列與結(jié)構(gòu)相關(guān)的生物信息分析,為認識該基因結(jié)構(gòu)與功能提供借鑒。方法(1)目的基因的提取:復(fù)蘇本室保存的含pExpress-1-rHSG質(zhì)粒的Jm109菌,用質(zhì)粒抽取試劑盒抽取質(zhì)粒pExpress-1-rHSG,并在PCR儀中擴增出目的基因rHSG cDNA。(2)穿梭質(zhì)粒的構(gòu)建及鑒定:①復(fù)蘇本室保存的含腺病毒穿梭質(zhì)粒pAdtrack-cmv的DH5α菌種,用質(zhì)粒抽取試劑盒抽取質(zhì)粒pAdtrack-cmv;②將PCR后rHSG cDNA連接到pGM-T質(zhì)粒上,構(gòu)建重組質(zhì)粒pGM-T-rHSG,并對重組質(zhì)粒進行限制性內(nèi)切酶酶切鑒定;③將pGM-T-rHSG重組質(zhì)粒在LB固體瓊脂培養(yǎng)基上轉(zhuǎn)化到大腸桿菌DH5α,提取pGM-T-rHSG質(zhì)粒;④用BglⅡ、Eco RV對pGM-T-rHSG重組質(zhì)粒進行雙酶切,提取目的基因并鑒定;⑤用BglⅡ和Eco RV雙酶切pAdtrack-cmv質(zhì)粒得到線性化的pAdTrack-CMV片斷,在T4DNA連接酶下與目的基因連接,構(gòu)建出重組pAdtrack-cmv-rHSG穿梭質(zhì)粒,并對重組穿梭質(zhì)粒pAdtrack-cmv-rHSG進行酶切鑒定;⑥將重組穿梭質(zhì)粒送測序公司進行DNA序列分析。(3)rHSG基因生物信息分析:根據(jù)測序報告結(jié)果中rHSG基因序列,用生物信息學(xué)方法對其進行同源性、分子量、蛋白跨膜區(qū)、等電點、親疏水性、蛋白二級結(jié)構(gòu)等項的分析和預(yù)測。結(jié)果(1)從質(zhì)粒pExpress-1-rHSG中成功提取出了rHSG cDNA基因。(2)重組pAdtrack-cmv- rHSG穿梭質(zhì)粒經(jīng)BglⅡ和Eco RV雙酶切后,進一步將重組體進行基因測序分析,驗證了克隆的rHSG cDNA序列與GenBank[AF036536]公布的rHSG序列吻合。這一結(jié)果表明重組體pAdtrack-cmv-rHSG中插入的目的基因是正向、單倍插入。(3)rHSG生物信息分析結(jié)果顯示,rHSG全長4151bp,編碼一757氨基酸殘基的蛋白,可能為一跨膜蛋白,經(jīng)基因Genebank查詢發(fā)現(xiàn)與人HSG基因序列有95.2%同源性。結(jié)論(1)成功構(gòu)建了大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)真核表達載體pAdtrack-cmv- rHSG,為深入研究rHSG功能和rHSG基因治療腦血管痙攣奠定了物質(zhì)基礎(chǔ);(2)分析rHSG基因序列及蛋白結(jié)構(gòu),有助于進一步研究該基因的分子和生物學(xué)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 偽 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 偽 on LB solid Agar medium and pGM-T-rHSG plasmid was extracted. 4Recombinant pGM-T-rHSG plasmid was digested with Bgl 鈪，

本文編號：1451883