禽多杀性巴氏杆菌ompa DNA疫苗在小鼠体内免疫效果试验

Immune efficacy of ompa DNA vaccine from avian Pasteurella multocida in mice

应用PCR技术扩增获得禽多杀性巴氏杆菌的ompa基因片段,克隆到pUCm-T载体,再亚克隆到真核表达质粒载体pCDNA3.1(+)上,构建重组质粒pcA,体外转染Vero细胞,RT-PCR和间接免疫荧光试验检测其转录表达情况。动物免疫分为3组:pCDNA3.1(+)组、PBS对照组和pcA组,每组16只BALB/c小鼠,pCDNA3.1(+)组和pcA组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2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2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。结果显示,间接免疫荧光试验和RT-PCR检测结果均表明pcA可在体外培养的Vero细胞中表达目的蛋白。动物免疫后,pcA组免疫小鼠血清抗体水平持续上升,与pCDNA3.1(+)组和PBS组相比差异极为显著(P<0.01)。经提取的禽多杀性巴氏杆菌总外膜蛋白(Omps)刺激后,pcA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)。脾细胞产生的IFN-γ极显著高于两对照组(P<0.01)。强毒攻击后pcA组保护率明显高于两对照组,可达60%。结果表明,成功构建了禽多杀性巴氏杆菌ompa 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一定的保护效果。

The ompa gene from avian Pasteurella multocida fragment,which was amplified by PCR was cloned into pUCm-T,and subcloned into the eukaryotic expression vector pCDNA3.1（＋）,the recombinant plasmid pcA was obtained.Then the recombinant plasmid was transfected into Vero cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR and indirect immunofluorescence.Three groups of BALB/c mice（n=16） named pCDNA3.1（＋）,PBS and pcA were intramuscularly vaccinated with control vector,PBS and the recombinant plasmid respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation（SLP） and the secreted IFN-γ 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.RT-PCR and indirect immunofluorescence showed the ompa gene could be transfected into Vero cells in vitro and expressed the target protein.Indirect ELISA showed the levels of antibodies in pcA group was significantly higher than the other groups（P0.01）.SLP experiments indicated the SI value induced with avian Pasteurella multocida Omps in pcA group was higher than pCDNA3.1（＋） and PBS groups（P0.05）.The IFN-γ experiments showed the levels of IFN-γ induced with Omps in the group of pcA was higher than the other control groups apperent（P0.01）.The protection rate of pcA（60%） was better than the other groups.The ompa DNA vaccine against avian Pasteurella multocida has been constructed successfully,which could enhance the immunity level and the protective effect of the vaccinated mice.