论文部分内容阅读
目的:构建禽流感病毒M2基因胞外区(M2e)多拷贝片段的原核表达载体,并对表达、纯化的重组蛋白进行免疫原性的分析。方法:以含有全长M2基因的质粒19T-M2为模板PCR扩增获得M2e,通过PCR及同尾酶连接的技术构建一系列的M2e多拷贝中间载体,得到顺次连接的9个拷贝M2e(9c),将其克隆到原核表达载体pET-32a中,经酶切和DNA测序鉴定正确后转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导,SDS-PAGE和Western blot分析蛋白表达情况。用纯化后的融合蛋白免疫BALB/c小鼠,取动物血清作ELISA检测M2e特异性抗体IgG并取脾脏细胞作ELISpot检测细胞分泌IFN-γ的情况。结果:结果显示,重组菌能够表达出一条相对分子质量(Mr)约为45 000的可溶性融合蛋白(M2e-9c),该蛋白表达量约占菌体表达量的37.2%。纯化蛋白免疫小鼠后,M2e特异性IgG滴度可达到1×104,M2e肽段能够刺激免疫组小鼠脾细胞分泌IFN-γ且具有一定的浓度依赖性。结论:M2e多拷贝重组蛋白可以诱导小鼠产生抗M2e特异性的抗体反应和细胞免疫反应,说明该蛋白具有良好的免疫原性,为流感病毒广谱疫苗的进一步研发打下了基础。
OBJECTIVE: To construct a prokaryotic expression vector of M2e extracellular region (M2e) of avian influenza virus and to analyze the immunogenicity of expressed and purified recombinant protein. Methods: M2e was amplified by PCR using plasmid 19T-M2 containing the full-length M2 gene as a template. A series of multi-copy M2e intermediate vectors were constructed by PCR and ligation by homologous recombination, resulting in 9 copies of sequentially linked M2e 9c), and cloned into prokaryotic expression vector pET-32a. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, induced by IPTG and analyzed by SDS-PAGE and Western blot. The purified fusion protein was used to immunize BALB / c mice. The serum of mice was used to detect the M2e specific IgG by ELISA and the spleen cells were taken as ELISpot to detect the secretion of IFN-γ. Results: The results showed that the recombinant fusion protein could express a soluble fusion protein (Mre) with a relative molecular mass of 45 000 (M2e-9c), which accounted for 37.2% of the total bacterial expression. The M2e-specific IgG titer can reach 1 × 104 after immunization with the purified protein, and the M2e peptide can stimulate the secretion of IFN-γ by the spleen cells of the immunized mice in a concentration-dependent manner. CONCLUSION: M2e multi-copy recombinant protein can induce specific anti-M2e antibody response and cellular immune response in mice, indicating that the protein has good immunogenicity, laying a foundation for further research and development of a broad-spectrum influenza virus vaccine.