SARS冠状病毒M蛋白的原核表达及其DNA疫苗构建

Expression of SARS Coronavirus Membrane Protein and its DNA Vaccine Construction

为探讨SARS CoV的M蛋白的免疫学特性以及M蛋白作为SARS CoV病毒疫苗组分的可行性和必要性。分别用pET 15b和pET 22b在大肠杆菌中表达SARS CoV的M蛋白,亲和层析纯化后作为抗原应用。同时,将M蛋白的编码基因克隆进分泌型真核表达载体pSecTagB中得到重组质粒pSecM作为DNA疫苗,免疫BALB/c小白鼠、制备SARS CoV M蛋白的抗血清。并用纯化后的M蛋白建立的SARS CoV M抗体ELISA检测技术研究所构建的M DNA疫苗的免疫效果。结果表明:两种重组M蛋白在大肠杆菌中均以可溶性形式得到高效表达,经与华大产的用灭活SARS全病毒制备的SARS CoV抗体ELISA检测试剂盒比较实验,证明该原核表达的重组M蛋白能与SARS确诊病人血清以及M DNA免疫鼠血清发生特异性抗原抗体反应。这两种重组M蛋白有可能作为抗原组分用于临床SARS CoV检测中;所构建的SARS CoV的M基因核酸疫苗能在小鼠体内产生特异性抗体,提示M蛋白在SARS CoV疫苗尤其是组分疫苗的研制中应加以考虑,为DNA疫苗的开发提供了依据。

To study the immunological speciality of M protein of SARS-CoV and analyze the feasibility and necessity of M protein as the component of the SARS vaccine. M protein of SARS-CoV was expressed in E.coli by pET-15b and pET-22b and purified by affinity resin as antigen. M gene was cloned into the eukaryotic secretory expression plasmid pSecTagB and named recombinant plasmid pSecM as DNA vaccine which will be used to immunize BALB/c mice to obtain the antiserum of M protein. The immune effect of the M-DNA vaccine was studied by the ELISA technique established with purified M protein. Both of the recombinant M proteins can be expressed in soluble form. When compared with the SARS-CoV ELISA kit produced by Huada Co.Ltd, the prokaryotic reconbinant M proteins could react specifically with the sera from immunized M-DNA mice and SARS correct diagnosis patients. The recombinant M protein could be used as an antigen to detect SARS, and the SARS-CoV M gene vaccine could induce specifically antibody. M protein could be considered as SARS-CoV vaccine,especially as the vaccine component, which redounds to the development of a potential DNA vaccine.