摘要
为了检测HPV58型不同L1基因的DNA疫苗的免疫原性,以pcDNA3.1为载体分别构建含HPV58型不同L1基因的DNA疫苗,命名为L1h、L1h△c、L1S、L1SM和L1wt。用免疫印迹法检测各DNA疫苗的体外表达情况;各重组质粒与pcDNA3.1-h58L2和pcDNA3.1-GFP共转染293FT细胞,检测其形成假病毒的能力;并将各DNA疫苗肌肉注射免疫小鼠,利用假病毒中和实验检测中和抗体水平,用ELISPOT检测细胞免疫情况。结果显示,本实验成功构建了五种DNA疫苗,L1h△c的体外表达量最高,L1S和L1SM的表达量次之,L1wt没有表达;重组质粒L1S能够形成假病毒,而其他四种重组质粒均不能形成假病毒。L1S和L1h均可在小鼠体内诱导中和抗体,但L1S诱导的中和抗体的平均滴度为1:6400,明显高于L1h诱导的中和抗体水平(平均滴度为1:48),而其他疫苗在小鼠体内未产生中和抗体。对五种疫苗均未检测出特异性的细胞免疫反应。结果提示,体外能够组装成假病毒的DNA疫苗在免疫动物后可诱导高滴度的中和抗体,为今后DNA疫苗的筛选提供参考。
In order to evaluate the immunogenicity of HPV 58 L1 DNA vaccines, five DNA vaccines had been constructed with pcDNA3.1 vector containing different L1 genes of HPV 58, which were designated as L1h,L1h△c, L1S, L1SM and L1wt. The protein expression of DNA vaccines in vitro was tested by Western blot. The ability of forming pseudovirus was evaluated by transfecting DNA vaccine together with pcDNA3.1-h58L2 and pcDNA3.1-GFP into 293FT cells. The neutralizing antibodies and cellular immune response produced in BALB/c mice immunized with the DNA vaccines were detected by using pseudovirusbased neutralization assay and ELISPOT respectively. The results showed that the five DNA vaccines had been successfully constructed; the level of protein expression of L1h△c was the highest and those for L1S and L1SM were of medium, while no expressed target protein of L1wt was detected. Only L1S could form the pseudovirus while the other four vaccines could not. L1S and L1h could induce neutralizing antibody. However, the average titer of neutralizing antibody for L1S （1：6 400） was much higher than that for L1h （1：48）and the other three vaccines could not induce neutralizing antibody. No cellular immune response for all five DNA vaccines was detectable by ELISPOT. The results indicated that DNA vaccine against HPV 58 can form pseudovirus in vitro, also can induce high level of neutralizing antibodies. This provides reference for screening HPV vaccine in future.
出处
《病毒学报》
CAS
CSCD
北大核心
2008年第3期196-201,共6页
Chinese Journal of Virology
