H1亚型猪流感病毒HA基因密码子优化的DNA疫苗免疫保护效力研究

DNA疫苗的免疫效果与抗原基因的表达量及表达抗原的免疫原性有直接关系。为了提高猪流感病毒（Swine influenza virus，SIV）HA基因DNA疫苗的表达量，增强其免疫效果，本研究通过人工合成的方法将H1亚型猪流感病毒A／Swine／Guangdong／1／01（H1N1）的HA基因密码子优化为猪体内偏嗜性密码子optiHA，同野生型A／Swine／Guangdong／1／01（H1N1）的HA基因分别与真核表达载体PCAGGS连接构成重组质粒PCAGGS—optiHA和PCAGGS—HA，然后分别转染293T细胞，48h后采用间接免疫荧光的方法检测Ⅲ基因的瞬时表达蛋白情况。将质粒PCAGGS—HA、PCAGGS—optiHA以100μg／只的剂量，采用后腿肌肉多点注射的方式，免疫6-8周龄雌性BALB／c小鼠，同时设立空载体PCAGGS对照。共免疫3次，每次间隔2周，三免2周后对每组以10^3.87 EID50的A／Swine／Guangdong／1／01（H1N1）进行攻毒。采用ELISA、血凝抑制试验、细胞因子检测和肺组织病毒含量测定等实验评价这两种DNA疫苗的免疫效果。结果表明，HA基因密码子优化的DNA疫苗可显著提高体液免疫和细胞免疫的应答水平，攻毒后免疫组PCAGGS—optiHA的保护效力明显高于免疫组PCAGGS—HA。这一结果为进一步研究和设计有效的SIVDNA疫苗奠定了基础。

PROTECTIVE EFFICACY OF DNA VACCINE ENCODING CODON- OPTIMIZED HA GENE OF H1 SUBTYPE SWINE INFLUENZA VIRUS.

The immunogenicity of DNA vaccine directly related to antigen gene expression and immunogenicity of antigen expression. In order to enhance the immunogenicity of DNA vaccine, the HA gene of H1 subtype Swine influenza virus was optimized according to the swine bias codon usages （optiHA）. The optiHA and the wild-type HA gene were inserted into the PCAGGS vector, respectively, and then the two recombinant plasmids PCAGGS-optiHA and PCAGGS-HA were constructed. The HA protein transiently expressed in 293T cell was detected by indirect immunofluorescence at 48h post transfection. Each of six to eight week-old BALB/c mouse was inoculated by multi-point injection with 100 μg of plasmids PCAGGS-optiHA and PCAGGS-HA. The control group was injected with PCAGGS vector alone. A total of immunity were three times interval of two weeks. All mice were challenged with 10387EID50 of A/Swine/Guangdong/1/01（H1N1） after two weeks of the third immunity. The immunogenicity of the two vaccines were evaluated by HA specific antibody production, HI antibody production, cytokines production and so on. The result showed that the DNA vaccine with encoding codon-optimized HA gene can significantly enhanced the expression of HA gene and improved the response level of humoral and cellular immune compared to the DNA vaccine with wild-type HA gene. In addition, the virus replication were inhibitated completely in the lungs of mice in the PCAGGS-optiHA vaccine group . These results might lay the foundation for the further study and design of the effective DNA vaccine of Swine influenza virus.