禽巴氏杆菌OmpH外膜蛋白DNA疫苗的免疫效果

The immune efficacy of OmpH DNA vaccine from avian Pasteurella multocida

目的:研究禽多杀性巴氏杆菌omph DNA疫苗在小鼠体内诱导的免疫保护效果。方法:利用PCR技术扩增出禽多杀性巴氏杆菌的omph基因片段,克隆到pMD18-T上,再亚克隆到真核载体pcDNA3.1(+)上,构建重组质粒pOMPH,体外转染SP2/0细胞,RT-PCR、间接免疫荧光试验、Western blot检测其转录及表达情况。然后进行动物免疫,实验动物分为三组即:pOMPH组、pCDNA3.1(+)空载体组和PBS对照组,每组16只BALB/c小鼠,pOMPH组和pCDNA3.1(+)组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μl1×PBS,各组均进行了三次免疫,每次间隔两周。间接ELISA检测免疫后小鼠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,三免两周后检测脾淋巴细胞IFN-γ分泌情况。强毒攻击,计算小鼠存活数目及保护率。结果:间接免疫荧光试验、Western blot和RT-PCR检测结果均表明pOMPH可在体外培养的SP2/0细胞中表达目的蛋白。动物免疫后,pOMPH组免疫小鼠血清抗体水平持续上升,与pCDNA3.1(+)组和PBS组相比差异极显著(P<0.01)。经提取的外膜蛋白(Omps)刺激后,pOMPH组的刺激值(SI值)与pCDNA3.1(+)组及PBS免疫组相比均差异显著(P<0.05)。免疫小鼠脾细胞产生的IFN-γ极显著高于两对照组产生的IFN-γ(P<0.01)。攻毒后pOMPH组保护率明显高于两对照组,可达70%。结论:成功构建了禽多杀性巴氏杆菌omph DNA疫苗,该疫苗可诱导免疫小鼠产生较强的体液和细胞免疫应答及较好的保护效果。

Objective： To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice. Methotis： The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T. Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1 （ ＋ ）, and the recombinant plasmid pOMPH was obtained. Then the recombinant plasmid was transfected into SP2/0 cells in vitro .The transcription and expression of target gene were analyzed by RT-PCR, Western blot analysis and indirect immunotluorescence.Three groups of BALB/c mice（ n = 16） named pOMPH,pCDNA3.1（ ＋ ） and PBS were intramuscularly vaccinated with the recombinant plasmid, control vector and PBS respectively. The serum antibodies were detected by indirect ELISA. The spleen lymphocyte proliferation （SLP） and secreted IFN-7 of spleen were tested by MTT. The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization, the protection rate were counted. Results： RT-PCR, Western blot analysis and indirect showed that the omph gene could be transfected into SP2/0 cells in vitro and expressed the target protein. Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups（ P 〈 0.01 ）. Spleen lymphocyte proliferation by MTF assay indicated that the SI value induced with avian Pasteurella multocida Omps in pOMPH group was higher than those in pCDNA3.1 （ ＋ ） and PBS groups （P 〈 0.05）. The IFN-γ experiments（Double-antibodies-sandwich-ELISA） showed that the levels of IFN-γ induced with Omps in the group of pOMPH was mostly higher than in the other control groups apperent（ P 〈 0.01 ） .The protection rate of pOMPH（70% ） was better than in the other groups. Conclusion：The omph DNA vaccine against avian Pasteurella multocida had been constructed successfully.The DNA vaccine could enhance the immunity level and the protective effect of the vaccinated mice. Present study may be useful for the development of avian Pasteurella multocida vaccine.