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为了提高流感病毒血凝素(HA)基因的表达量及其免疫原性,按照哺乳动物偏爱密码子将A/Goose/HLJ/QFY/04(H5N1)流感病毒的血凝素基因进行优化改造,然后插入到真核表达载体pCI-neo中,构建了真核表达质粒pCI-opti-HA.将此质粒和含野生HA基因的真核表达质粒pCI-wt-HA分别转染Vero或293T细胞,通过免疫过氧化物酶单层细胞试验(IPMA)和蛋白免疫印迹(WB)实验比较HA蛋白的表达量.将两种重组质粒免疫Balb/c小鼠,比较两者诱导的抗体产生水平.三次免疫后两周用强毒攻击,通过体温和体重变化评价两种重组质粒的免疫保护效果.IP-MA和WB实验结果表明:密码子优化后,HA蛋白在哺乳动物细胞中的表达水平显著提高.酶联免疫吸附实验(ELISA)结果表明:小鼠免疫后密码子优化的重组质粒诱导的特异性抗体效价较高.攻毒试验结果表明:两种重组质粒均能够提供较好的保护.
In order to improve the gene expression and immunogenicity of the influenza virus hemagglutinin (HA) gene, the hemagglutinin gene of A / Goose / HLJ / QFY / 04 (H5N1) influenza virus was optimized and modified according to mammalian preference codons, And then inserted into the eukaryotic expression vector pCI-neo to construct the eukaryotic expression plasmid pCI-opti-HA. The plasmid and pCI-wt-HA containing the wild-type HA gene were transfected into Vero or 293T cells respectively, The expression of HA protein was compared by immunoperoxidase monolayer assay (IPMA) and western blot (WB) assay.The two recombinant plasmids were immunized with Balb / c mice to compare the level of antibody production induced by the two Two weeks after the challenge, virulent challenge was used to evaluate the protective effect of two recombinant plasmids on the basis of body temperature and body weight.The results of IP-MA and WB showed that the expression of HA protein was significantly increased in mammalian cells after codon optimization. The results of ELISA showed that the specific antibody titer induced by the codon-optimized recombinant plasmids was higher than that of the control, and the results of the challenge test showed that both recombinant plasmids could provide better protection.