表达甲型H1N1流感病毒血凝素抗原的DNA疫苗构建及其免疫原性评价

Construction and immunogenic evaluation of DNA vaccine expressing influenza A （H1N1） virushemagglutinin antigen

目的构建表达甲型HIN1流感病毒血凝素（hemagglutinin，HA）抗原的DNA疫苗，并在小鼠中测试其免疫原性。方法运用人密码子优化技术合成甲型HIN1流感病毒HA序列，并转移至DNA疫苗载体，用Westernblotting检测其表达效率。采用单纯随机抽样方法把BALB／c小鼠分成DNA疫苗组和对照组。用DNA疫苗免疫疫苗组小鼠，免疫后取小鼠血清和脾细胞，分别用ELISA方法和酶联免疫斑点试验（ELISPOT）方法检测血清中的特异性抗体应答和脾细胞中的特异性T细胞应答，最后用血凝抑制（hemagglutination inhibition，HI）试验和假病毒中和试验分别检测血清中的HI抗体和中和抗体应答。采用t检验作两组之间比较。结果所构建的DNA疫苗能够高效表达HA蛋白。免疫小鼠后能有效诱导T细胞免疫应答[429．0±113．4斑点形成细胞（spot—forming cell，SFC）／10。脾细胞]，血清结合抗体滴度为16127．0±2698．0，HI抗体滴度为100．8±16．9，所有数据均显著高于载体对照（分别为t=3．863，P=0．0042；t=5．734，P=0．0002；t=6．018，P=0．0001）。DNA疫苗能活化产生高水平的针对同源H1N1毒株的中和抗体，但不能活化产生针对异源H1N1毒株的中和抗体。结论本研究构建的DNA疫苗有较好的免疫原性，并能诱导一定水平的保护性抗体产生。

Objective To construct DNA vaccine expressing influenza A （ HI N1 ） virus hemagglutinin （HA） antigen, and to evaluate its immunogenicity in mice. Methods The condon-optimized influenza A （ H1N1 ） virus HA sequence was synthesized and transferred to DNA vaccine vector, and its expression efficacy was proved by Western blotting in vitro. BALB/c mice were divided into DNA vaccine group and control group by simple random sampling for the evaluation of DNA vaccine immunogenicity. After the final inoculation, spleens and sera were collected from the immunized mice. HA-specific antibody responses in sera and T cell responses in splenocytes were quantified by ELISA and enzyme-linked immunospot assay （ELISPOT）, respectively. In addition, hemagglutination inhibition （HI） and neutralizing antibodies were determined by HI assay and pseudovirus based neutralization assay, respectively. The t test was adopted in the comparison between the two groups. Results The constructed DNA vaccine could efficiently express HA protein in vitro. The inoculation of DNA vaccine in mice was capable of inducing vigorous T cell responses [429.0±113.4 spot-forming cell （SFC）/IO6 splenocytes] , the binding antibody titers in sera reached 16127.0±2698.0, and HI antibody titers were 100.8±16.9. All these data were significantly higher than those in vector control group （t =3. 863, P =0. 0042; t =5. 734, P =0. 0002; t =6. 018, P =0. 0001, respectively）. Furthermore,DNA vaccine could elicit high levels of neutralizing antibodies against homogenous H1N1 virus, but not against H1N1 heterogenous ones. Conclusions The constructed DNA vaccine is highly immunogenic and could induce high level of protective immunity.