表达禽流感病毒NP和M2融合基因重组乳酸菌的构建及鉴定

Construction and Identification of recombinant Lactobacillus plantarum NC8 expressing NP fused with M2 proteins of avian influenza virus

关键词:  禽流感病毒  NP基因  M2基因  原核表达  重组乳酸菌

Key words:  avian influenza virus  NP gene  M2 gene  Prokaryotic expression  Recombinant bacteria

摘要:

为了在乳酸菌中表达H9N2亚型禽流感病毒（AIV）NP和M2基因并检测其反应原性，先以pMD19-T-M2质粒作为模板，采用PCR方法克隆M2全长基因，然后将M2基因亚克隆入pSIP409-pgsA′-NP质粒中，构建重组质粒pSIP409-pgsA′-NP-M2。并将重组质粒pSIP409-pgsA′-NP-M2通过电转化的方法转入植物乳杆菌Lb.plantarum NC8中，制备重组菌NC8-pSIP409-pgsA′-NP-M2。诱导表达NC8-pSIP409-pgsA′-NP-M2重组乳酸菌，并通过流式细胞术、免疫荧光和免疫印迹技术验证。结果表明，重组菌NC8-pSIP409-pgsA′-NP-M2能够经诱导表达NP-M2融合蛋白，并且与兔源NP单克隆抗体和抗M2的小鼠血清具有反应原性。这为后续研究禽流感广谱口服疫苗奠定基础。

Abstract:

To express the M2-NP fusion gene of the H9N2 subtype avian influenza virus and to study the reactivity of its prokaryotic expression product in Lb.plantarum NC8, pMD19-T-M2 plasmid supplied from the laboratory serves as the template, the M2 gene was then amplified by PCR. The M2 gene was subcloned in the pSIP409-pgsA''-NP vector to construct a recombinant plasmid pSIP409-pgsA''-NP-M2. The recombinant plasmid pSIP409-pgsA''-NP-M2 was transformed into Lb. plantarum NC8 by electroporation to prepare recombinant bacteria NC8-pSIP409-pgsA''-NP-M2. The NC8-pSIP409-pgsA′-NP-M2 was induced, and reactogenicity of antigens were detected using flow cytometry, immunofluorescence and Western-blot. The results showed that NP-M2 fusion protein was able to express on NC8-pSIP409-pgsA''-NP-M2. Antigens of recombinant bacteria were also recognized with anti-NP monoclonal antibody and anti-M2 mouse source serum, which play an important role on developing universal oral vaccine against avian influenza viruses.