铜绿假单胞菌oprD基因DNA疫苗和重组亚单位疫苗的免疫效果研究

Immune efficacy of DNA vaccine and recombinant subunit vaccine base on oprD gene of Pseudomonas aeruginosa

为探究铜绿假单胞菌oprD基因DNA疫苗和重组亚单位疫苗的免疫效果,本研究经PCR扩增铜绿假单胞菌oprD基因,将其克隆于真核表达载体pcaggs-HA中,构建重组质粒pOPRD。同时将oprD基因克隆于原核表达载体pET32a中构建重组质粒,随后将该重组质粒转入大肠杆菌BL21(DE3)感受态细胞中进行蛋白表达并纯化后制备该基因的重组亚单位疫苗。分别以pOPRD质粒和重组亚单位疫苗免疫BALB/c小鼠,同时设置pOPRD初免-重组亚单位疫苗加强免疫组、灭活疫苗(本研究室制备)免疫组、铜绿假单胞菌外膜蛋白(本研究室制备)免疫组以及pcaggs-HA空载体对照组和PBS对照组。免疫后采用间接ELISA测定小鼠血清抗体水平,MTT法检测小鼠脾淋巴细胞增殖水平,双抗夹心ELISA测定脾淋巴细胞分泌的IFN-γ和IL-4的水平。结果显示各疫苗均可诱导免疫小鼠产生较高水平的血清抗体,初免-加强免疫组小鼠脾细胞的增殖指数(SI)值及小鼠脾淋巴细胞分泌的IFN-γ和IL-4含量显著高于DNA疫苗组和重组亚单位疫苗组(P<0.05)。三免两周后以铜绿假单胞菌强毒株CAV0792对各组小鼠进行攻毒试验,测定攻毒后各组小鼠的保护率,结果显示DNA疫苗组、重组亚单位疫苗组、初免-加强免疫组、外膜蛋白疫苗组和灭活疫苗组的保护率分别为53.3%、60%、73.3%、80%和93.3%。本研究首次证实以铜绿假单胞菌oprD基因制备的DNA疫苗和重组亚单位疫苗均可诱导小鼠产生免疫应答,并为其提供对铜绿假单胞菌感染的免疫保护,且两者以初免-加强免疫的方式诱导的免疫应答水平更高、保护效果更好。本研究为铜绿假单胞菌DNA疫苗和重组亚单位疫苗的研究奠定了基础。

To evaluate the immune efficacy of DNA vaccine of oprD gene from Pseudomonas aeruginosa,oprD gene fragment amplified by PCR from Pseudomonas aeruginosa was cloned into the eukaryotic expression vector pcaggs-HA to construct the recombinant plasmid pOPRD,namely DNA vaccine of oprD gene.At the same time,the gene fragment was cloned into the prokaryotic expression vector pET32a and expressed in Escherichia coli BL21(DE3).Then the recombinant protein was purified and the recombinant subunit vaccine was prepared.BALB/c mice were divided into seven groups,namely DNA vaccine group,recombinant subunit vaccine group,pOPRD primary immunization-recombinant subunit vaccine booster group(prime-boost group),inactive vaccine group,outer membrane protein(OMP)vaccine group,pcaggs-HA group and PBS group,and vaccinated with corresponding vaccines.Then the levels of serum antibody,lymphocyte proliferation assays,interferon-γ(IFN-γ)and interleukin-4(IL-4)concentrations were detected by indirect ELISA,MTT and double antibody sandwich ELISA,respectively.After challenging with virulent Pseudomonas aeruginosa,protective efficacy was evaluated.The results showed that each vaccine can induce immunized mice to produce high levels of serum antibodies.The stimulation index(SI)values,IFN-γand IL-4 concentrations in the prime-boost group were higher than those in the DNA vaccine group and recombinant subunit vaccine group(P<0.05).The protective efficacy rate of DNA vaccine group,recombinant subunit vaccine group,prime-boost group,OMP vaccine group and inactive vaccine group were 53.3%,60%,73.3%,80%and 93.3%,respectively.The results indicated that both the DNA vaccine and recombinant subunit vaccine based on the oprD gene of Pseudomonas aeruginosa could induce a certain immune response and provide certain protection for the vaccinated mice,especially when the two vaccines are applied with the strategy of primary-boost.The goal of this study was to lay the foundation for the development of DNA vaccine and recombinant subunit vaccine against the Pseudomonas aeruginosa.