重组人普兰林肽原核表达载体的构建及表达  

Construction of Recombinant Human Pramlintide Expression Vector and Its Expression in E. coli

作　　者：杨薇[1,2] 吴红梅 李佳楠[1] YANG Wei;WU Hongmei;LI Jianan(School of Life Sciences,Jianghan University,Wuhan 430056,Hubei,China;Technique Center of Wuhan Hiteck Biological Pharma Co.,Ltd.,Wuhan 430056,Hubei,China)

机构地区：[1]江汉大学生命科学学院,湖北武汉430056 [2]武汉海特生物制药股份有限公司,湖北武汉430056

出　　处：《江汉大学学报（自然科学版）》2017年第1期77-82,共6页Journal of Jianghan University：Natural Science Edition

基　　金：江汉大学研究生科研创新基金项目(301004210001)

摘　　要：目的构建人普兰林肽基因原核表达载体,并诱导其在大肠杆菌中大量高效表达。方法将胰淀素25位的丙氨酸、28位的丝氨酸和29位的丝氨酸用脯氨酸代替,选用大肠杆菌偏爱的密码子对天然胰淀素碱基序列进行修饰,通过化学合成的方式合成了普兰林肽基因片段,经Kpn I、Hind III双酶切后插入p ET-39b(+)载体,构建p ET-39b+[Pramlintide]重组质粒,转化BL21(λDE3)菌株,筛选重组子,并经IPTG诱导重组蛋白表达。结果构建的普兰林肽基因插入载体位置正确,且序列测定结果与预期一致;在37℃条件下,经0.1 mmol/L IPTG诱导后3 h,重组蛋白表达量最高,且重组蛋白主要存在于胞质蛋白中。结论成功构建重组人普兰林肽原核表达载体,并诱导其在大肠杆菌得以表达,为进一步工业化制备普兰林肽奠定了基础。Objective To construct the recombination human pramlintide prokaryotic expression vectorand induce the expression of pramlintide in E.coli.Methods The human nature pramlintide gene whosegenetic codon mutated to prokaryotic preference codon were chemically synthesized.The gene wasinserted into pET-39b+expression vector with double enzyme(KpnI、HindIII)digest to construct therecombination plasmid pET-39b+[Pramlintide],and then,the recombination plasmid was transferredinto E.coli BL21to construct high expression genetic engineering bacteria of pramlintide which wasinducted with isopropylβ-D-thiogalactoside(IPTG).Results Pramlintide was inserted in plasmid atcorrect site,and gene sequencing indicated that the sequence of pramlintide was the same as exspected.Upon induction with0.1mmol/L IPTG for3hours,the recombination protein was expressed in E.coliBL21cells at37℃,The expression of pramlintide reached the highest level and large amounts ofrecombination protein accumulated in cytoplasm.Conclusion The expression vector pET-39b+[Pramlintide]was successfully constructed.Upon induction with IPTG,large amounts of recombinationprotein accumulated in E.coli BL21cells.It will establish a foundation for producing large amounts ofpramlintide in the future.

关 键 词：普兰林肽 密码子突变 原核表达 诱导表达 

分 类 号：Q81[生物学—生物工程]