铜绿假单胞菌OprL基因DNA疫苗的免疫效果

The immune efficacy of oprL gene DNA vaccine from Pseudomonas aeruginosa

为研究铜绿假单胞菌OprL基因DNA疫苗在小鼠体内的免疫效果,本研究利用PCR技术扩增得到铜绿假单胞菌的OprL基因,将其克隆于真核表达载体pcDNA3.1(+)中构建重组质粒pOPRL。将BALB/c小鼠分为5组,分别免疫pOPRL、油乳剂灭活疫苗、OMP疫苗、pcDNA3.1(+)空载体和PBS。每两周免疫一次,共免疫3次。间接ELISA检测免疫后血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,双抗夹心ELISA检测IFN-γ分泌情况,计算强毒攻击后各组小鼠的存活数及保护率。结果表明,随着免疫次数的增加,pOPRL、灭活疫苗和OMP疫苗免疫组小鼠产生的抗体水平逐渐升高,与两对照组相比差异极显著(p<0.01),且灭活疫苗组和OMP疫苗组小鼠血清抗体水平高于pOPRL组(p<0.05);灭活疫苗组和OMP疫苗组小鼠脾淋巴细胞的刺激值(SI值)和IFN-γ水平也高于pOPRL组(p<0.05);pOPRL组、灭活疫苗组和OMP疫苗组对小鼠的保护率分别为53.3%、86.7%和73.3%,表明pOPRL疫苗虽可为小鼠提供一定的保护,但效果不如传统的灭活疫苗,还需采取措施进一步增强其免疫原性。

To study the immune efficacy of oprL gene DNA vaccine against Pseudomonas aeruginosa in mice, oprL gene fragment amplified by PCR from P.aeruginosa was cloned into the eukaryotic expression vector pcDNA3.1（＋） to construct the recombinant plasmid pOPRL. BALB/c mice were divided into five groups and vaccinated with pOPRL, inactivated vaccine, outer membrane protein（OMP） vaccine, pcDNA3.1（＋） and PBS, respectively. The serum antibodies were detected by indirect ELISA.The levels of splenic lymphocyte proliferation were tested by MTT. The levels of IFN-γ secreted by splenic lymphocytes were detected by double-antibody-sandwich-ELISA. The mice were challenged with P.aeruginosa at 2 weeks post the third immunization, the protection rate were determinated. Indirect ELISA showed the levels of antibodies in pOPRL, inactivated vaccine and OMP vaccine groups were significantly higher than the two control groups（p0.01）, and the antibodies in inactivated vaccine and OMP vaccine groups were higher than that of the pOPRL group（p0.05）. The results of lymphocyte proliferation and IFN-γ tests indicated the SI value and levels of IFN-γ induced with OMP of P.aeruginosa in inactivated vaccine group and OMP vaccine group were significantly higher than the pOPRL group（p0.05）. The protection rate of pOPRL, inactivated vaccine and OMP vaccine were 53.3%, 86.7% and 73.3%, respectively. The results demonstrated the pOPRL vaccine could provide a certain protective effect for the mice, but it was inferior to the inactive vaccine.