MTB线粒体翻译控制28蛋白在昆虫细胞中的表达和纯化

摘要

To clone the Mycobacterium tuberculosis (MTB) mitochondrial translation control (MTC) gene, construct the Bacmid-MTC28 eukaryotic expression plasmid and express MTC28 protein in insect cells.MTC28 gene fragment was amplified from the genome of the MTB H37Rv standard strain by PCR.Then the PCR products and plasmid pFastbac1was digested with EcoRI and XhoI, linked with T4 ligase, the recombinant plasmid pFastbac1-MTC28 was constructed.The pFastbac1-MTC28 was transfected into DH5α competent cells, positive clone was screened by Ampicillin L-B medium and biological amplified by LB liquid medium.The plasmid (pFastbac1-MTC28) extracted from positive clone was identified by PCR and transfected into DH10 Bac competent cells to construct Bacmid-MTC28.The competent cells was amplified with liquid LB medium containing gentamicin, kanamycin and tetracycline, positive clone was screened by blue-white technique.The positive bacteria was amplified by LB liquid medium, extracted the plasmid (Bacmid-MTC28) and identified the target genes by PCR.By liposome transfection, the recombinant plasmid Bacmid-MTC28 was transfected into Sf9 insect cells in logarithmic growth phase, packaged recombinant baculovirus and expressed protein.We successfully constructed the eukaryotic expression plasmid of Bacmid-MTC28, established a baculovirus expression vector and expressed the corresponding proteins.High purity and homogenized MTC28 protein was obtained by purification.%对MTB线粒体翻译控制(mitochondrial translation control,MTC) 蛋白MTC28基因进行克隆, 构建真核表达质粒并在昆虫细胞中表达MTC28蛋白.采用PCR技术从MTB H37Rv 标准株基因组中扩增MTC28基因片断,MTC28基因片断和质粒pFastbac1经EcoRI和XhoI双酶切,T4 DNH连接酶连接,构建重组质粒pFastbac1-MTC28,转染DH5α感受态细菌,在含有氨苄青霉素溶菌肉汤(lysogeny broth,LB)固体培养基中筛选阳性克隆,挑选生长的菌落进而在含有氨苄青霉素的液体LB培养基中进行细菌克隆.PCR鉴定目的基因插入质粒后,阳性克隆细菌进行质粒(pFastbac1-MTC28)的提取.pFastbac1-MTC28质粒转染DH10 Bac感受态细菌构建重组杆状质粒(Bacmid-MTC28),用含有庆大霉素、卡那霉素和四环素的液体LB培养基进行细菌克隆,采取蓝白斑技术筛选阳性菌落.PCR鉴定为阳性的细菌进行杆状重组质粒(Bacmid-MTC28)的提取.利用脂质介导的转染技术,将杆状重组质粒(Bacmid-MTC28)转染sf9昆虫细胞中,包装重组杆状病毒,并表达蛋白质.结果成功构建了MTC28真核表达质粒,建立了杆状病毒表达MTC28蛋白的表达体系,并表达MTC28蛋白,通过纯化获得了高纯度且均一化的MTC28蛋白.