小鼠肺炎链球菌psaA核酸疫苗的制备及其初免-蛋白加强免疫策略的研究

Improvement of immunogenicity of Streptococcus pneumoniae psaA DNA vaccine by prime and boost strategy

目的制备肺炎链球菌核酸疫苗，初步探讨初免一蛋白加强免疫策略能否增强疫苗在动物体内的免疫原性。方法从肺炎链球菌基因组中克隆出肺炎球菌表面黏附素A（pneumococcalsurfaceadhesinA，PsaA）基因，将其分别插入到真核表达载体pVAX1和原核表达载体pET28a中，分别构建出表达PsaA的核酸疫苗及其基因工程表达菌株。pVAX1-psaA核酸疫苗体外转染293T细胞，并通过反转录-聚合酶链反应（RT—PCR）加以鉴定。实验组5只BALB／c小鼠经核酸疫苗免疫3次后，PsaA纯化蛋白加强免疫1次，对照组5只小鼠经pVAXl空载体免疫3次后，生理盐水加强免疫1次，分别采集血清测定两组抗PsaA抗体水平。结果成功克隆出psaA基因并实现其亚克隆；所构建的psaA核酸疫苗序列正确，同时实现了PsaA蛋白的一步亲和纯化；psaA核酸疫苗可在293T真核细胞中成功表达；核酸疫苗初免后蛋白加强组的抗PsaA抗体滴度高于对照组（t=87．518，P〈0．05）。结论成功构建出psaA核酸疫苗，采用核酸疫苗初免一蛋白加强免疫策略能增强肺炎链球菌psaA核酸疫苗的免疫原性。

Objective To study on the preparation of Streptococcus pneumoniae psaA DNA vaccine and to analyse the immunogenicity by the prime-boost strategy. Methods The psaA gene was amplified from the genome of Streptococcus pneumoniae by PCR, and then was inserted into plasmid pVAX1 and pET28a to construct recombinant expression vectors respectively. 293T cells were transiently transfected with pVAX1- psaA,and RT-PCR analysis of total cell RNA extracts showed successful expression of psaA. BALB/c mices （n = 5） were intramuscularly injected with 100 μg psaA DNA vaccine for three times, and then boosted with 50 μg recombinant PsaA protein. The antibody response against PsaA was measured by ELISA. Results The psaA gene was amplified and subcloned successfully. The constructed psaA DNA vaccine was confirmed by DNA sequencing, and the recombinant PsaA protein was purified by the one-step Ni2＋affinity chromatography. Expression of the PsaA was observed in cells transfected with pVAXl-psaA. The animal experiment resuhs showed that the anti-PsaA level of the DNA prime-protein boosting mice was higher significantly than the other groups （ t = 87. 518, P 〈 0. 05 ）. Conclusion The psaA DNA vaccine was prepared successfully, and the immunogenicity of Streptococcus pneumoniae psaA DNA vaccine could be improved significantaly by the DNA prime and protein boost strategy.