TOPO克隆构建SARS-CoV M蛋白基因裂殖酵母表达载体及其稳定性

目的 利用TOPO克隆法构建SARS-CoV M蛋白基因的裂殖酵母重组表达载体，并验证重组载体在宿主细胞中的稳定性。方法 运用逆转录-聚合酶链反应（RT-PCR）技术从SARS-CoV RNA中扩增出M蛋白基因,AT克隆构建出序列正确的pMD18-T-M重组载体。设计含Kozak序列的引物从pMD18-T-M载体上亚克隆出M蛋白基因，与裂殖酵母表达载体pNMT1-TOPO进行TOPO克隆,构建出重组表达载体pNMT1-M,转化TOP10感受态细胞，菌落PCR鉴定阳性转化子后进行测序鉴定。将序列正确的pNMT1-M重组载体电转化入裂殖酵母TCP1菌株中,在EMM培养基中诱导表达,连续传代100代，在EMM+T培养基中验证其稳定性。结果 RT-PCR获得666 bp的片段,pMD18-T-M重组载体经测序验证序列正确；重组表达载体pNMT1-M经菌落PCR和测序鉴定均正确;重组裂殖酵母菌经诱导后，SDS-PAGE检测出了表达条带;重组表达载体连续传代后,未发现丢失现象。结论 成功地构建出了SARS-CoV M蛋白基因的裂殖酵母表达载体，验证了其在裂殖酵母中能稳定地进行遗传,为下一步的表达优化、活性和功能研究垫定了基础.

Construction and Stability of Fission Yeast Expression Vector of the M Gene of SARS CoV by TOPO Cloning

Objective To construct Fission Yeast expression vector of the M gene of SARS coronavirus(SARS-CoV) and to testify its stability. Methods M gene was amplified by RT-PCR from the total RNA of SARS-CoV,and then was inserted into pMD18-T vector by AT cloning.Primers including Kozak sequence were designed to amplify M gene from pMD18-T-M,and then the purified M gene was inserted into the fission yeast' expression vector pNMT1-TOPO by TOPO-cloning to construct recombinant expression vector pNMT1-M.The vector pNMT1-M was transformed into fission yeast TCP1 by electroshock to construct genetic strains.The positive clones screened by EMM T plates were induced in EMM by removal of thiamine.SDS-PAGE was used to analyze expression of M protein in fission yeast.Recombinant fission yeast cells were cultured on YPD culture and EMM culture after many passages to testify the stability of pNMT1-M. Results It was found that the 666 bp gene fragment was amplified by RT-PCR and the 797 bp fragment was also amplified from the recombinant vector pNMT1-M with the upstream primer of the M gene and the downstream sequencing primer of the pNMT1-TOPO vector.The sequence of the M gene in pNMT1-M was correct.A specific band of 28.2KD was gained after induction.After over 100 passages,recombinant strains still kept stable plasmid inheritance and consistence. Conclusion The recombinant expression vector of M gene of SARS-CoV was successively constructed and it could be kept stably in fission yeast,which would be the basis of further study.