ADAM28基因原核表达载体的构建和在大肠杆菌中的融合表达

Construction of ADAM28 gene prokaryotic expression vector and fusion expression in E.coli

目的:利用消减杂交技术克隆出牙齿发育相关基因ADAM28。方法:本研究为构建ADAM28基因的原核表达载体,在大肠杆菌中诱导其表达并纯化出ADAM28蛋白;采用反转录-多聚酶链式反应(RT-PCR)扩增出ADAM28基因的蛋白编码序列,克隆到中间载体pMD18-TVector中产生pMD18-T-ADAM28,再经双酶切得到ADAM28片段定向克隆到pGEX-4T-1载体中,构建融合表达载体pGEX-4T-ADAM28;在大肠杆菌中利用IPTG进行诱导表达,得到GST-ADAM28融合蛋白,并经SDS-PAGE电泳初步纯化。结果:①成功构建融合表达载体pGEX-4T-ADAM28,并经酶切鉴定、PCR鉴定和测序验证显示插入的ADAM28序列正确,阅读框架完整,未发现突变。②得到初步纯化的GST-ADAM28融合蛋白。经IPTG诱导后出现一条约35.3×103的新蛋白带。结论:ADAM28基因编码蛋白质能够在原核细胞中表达并纯化,为进一步研究该蛋白质的功能打下基础。

AIM:To clone the tooth growth related gene ADAM28 by subtractive hybridization technique.METHODS:Our research is to construct high-level prokaryotic expression vector of ADAM28,induce its expression in E.coli,and purify ADAM28 protein.The protein coding region of ADAM28 was amplified by RT-PCR and cloned into pMD18-T vector to produce the new construction,pMD18-T-ADAM28.The cloned ADAM28 segment was cut out with two restriction enzymes and was cloned into the prokaryotic expression vector,pGEX-4T-1,to produce the expression vector pGEX-4T-ADAM28.The recombinant plasmid was transformed into E.coli DH5α.GST- ADAM28 fusion protein was obtained after induction of IPTG.The fusion protein was extracted and purified by SDS-PAGE.RESULTS:①The expression vector pGEX-4T-ADAM28 was constructed successfully.The coding region sequence of ADAM28 was correctly inserted into the expression vector by endonucleases digestion,PCR identification and DNA sequencing analysis. Its open reading frame was integra.Mutation was not detected.②GST-ADAM28 fusion protein was obtained by initial purification.A new protein band of 35.3 kDa appeared on SDS-PAGE after induction of IPTG.CONCLUSION:ADAM28 protein could be expressed in E.coli expression system and purified initially.which may be the foundation to further study of the functions of ADAM28 protein.