密码子优化的甲型H1N1流感病毒HA基因DNA疫苗在小鼠体内免疫保护效力研究

为了提高甲型H1N1流感病毒HA基因DNA疫苗的免疫原性,本研究将流感病毒rPan09(HA和NA来自A/California/04/09,其余6个片段来自PR8的重组病毒)的HA基因通过人工合成的方法将其密码子优化为哺乳动物体内偏嗜性密码子opti-HA,同未优化的rPan09的HA基因分别与真核表达载体pCAGGS连接构成重组质粒pCA-optiHA和pCA-HA转染293T细胞,48 h后采用间接免疫荧光的方法检测HA基因的体外表达情况,结果显示重组质粒pCA-optiHA的蛋白表达量明显高于pCA-HA。为评价这两种重组质粒的免疫及保护效力,选取6~8周龄雌性BALB/c小鼠,将质粒pCA-HA、pCA-optiHA以100μg/只的剂量,进行后腿肌肉多点注射,同时设立空载体pCAGGS对照,共免疫3次,每次间隔2周。结果表明DNA疫苗pCA-optiHA可显著提高小鼠的体液免疫和细胞免疫应答水平。三免2周后用107.45EID50的rPan09采用滴鼻方式进行攻毒,用Real-time PCR及制作肺组织石蜡切片检测DNA疫苗的保护效力。结果表明,pCA-optiHA免疫组的保护效力明显高于pCA-HA免疫组。本研究为进一步研究和设计有效的甲型流感病毒DNA疫苗奠定了基础。

EFFICACY OF DNA VACCINE CONTAINING CODON-OPTIMIZED HA GENE OF PANDEMIC H1N1 INFLUENZA VIRUS IN MICE

In order to enhance the immunogenicity of DNA vaccine,the HA gene of recombinant influenza virus rPan09(HA and NA genes from A/California/04/09 and remaining six genes from PR8) was optimized according to the mammalian biased codon usages(optiHA).The optiHA and original HA gene were respectively inserted into the pCAGGS vector,and two recombinant plasmids PCA-optiHA and pCA-HA were generated.The HA protein was expressed in 293T cell as detected at 48 h post transfection in indirect immunofluorescence.The results showed the recombinant plasmid pCA-optiHA significantly increased HA expression.In order to evaluate the immunogenicity of these two plasmids,each of six to eight-old BALB/c mice was injected at multiple sites with 100 μg of pCA-optiHA or pCA-HA plasmid.The control group was injected with pCAGGS vector alone.The injection was repeated two times at interval of two weeks.The immune responses of the two DNA vaccines were evaluated for HA / HI antibody,neutralization,cytokines and ELISPOT.The results showed that the pCA-optiHA vaccine significantly enhanced humoral and cellular immune responses.All mice were intranasally challenged with 107.45EID50 of rPan09(H1N1) after two weeks of the third injection.The virus replication was detected in lungs in Real-time PCR and paraffin sections.There were no viruses detected in lungs of mice immunized with pCA-optiHA vaccine.The information is useful for future study and design of an effective DNA vaccine against pandemic H1N1 influenza virus.