禽多杀性巴氏杆菌外膜蛋白单价DNA疫苗及融合DNA疫苗的免疫效果分析

为探讨禽多杀性巴氏杆菌omph和ompa基因单价DNA疫苗及融合DNA疫苗的免疫效果,利用PCR技术扩增了禽多杀性巴氏杆菌omph和ompa基因片段,并克隆入pcDNA3.1(+),构建了DNA疫苗pcDNA-OMPH(pOMPH)、pcDNA-OMPA(pOMPA)和pcDNA-OMPH/OMPA(pOMPHA),将其体外转染SP2/0细胞,用间接免疫荧光试验检测其表达情况。同时以弱毒活疫苗作为阳性对照,pcDNA3.1(+)和PBS作为阴性对照,分别免疫4周龄鸡,检测血清特异性抗体水平、外周血淋巴细胞增殖情况和IFN-γ分泌情况,经强毒攻击后计算各疫苗的保护率。结果显示,融合DNA疫苗组血清抗体水平与弱毒活疫苗相当,明显高于单价DNA疫苗(P<0.05);pOMPHA组的刺激值(SI值)和IFN-γ分泌水平显著高于其他各组(P<0.05),单价DNA疫苗组略高于弱毒活疫苗组;强毒攻击后各DNA疫苗均可为动物提供一定的保护力,其中融合DNA疫苗的保护率最高,可达86.7%,其次为弱毒活疫苗,为73.3%,而单价DNA疫苗则低于弱毒活疫苗,仅为60%和66.7%。结果表明,DNA疫苗尤其是融合DNA疫苗在预防禽多杀性巴氏杆菌病(禽霍乱)方面具有较好的应用前景。

Immune efficacy of single DNA vaccine and double fusion DNA vaccine encoding for outer membrane proteins against Pasteurella multocida

The objective of the study is to evaluate the protective immunity of single DNA vaccine and double fusion DNA vaccine against avian Pasteurella multocida.The DNA fragments of omph and ompa were amplified by PCR from avian P.multocida.These fragments were inserted into pcDNA3.1(+) vector to construct three DNA vaccines as follows:pcDNA-OMPH(pOMPH),pcDNA-OMPA(pOMPA) and pcDNA-OMPH/OMPA(pOMPHA).The three plasmids were then transfected into SP2 /0 cell in vitro with the expression of target genes analyzed by indirect immunofluorescence.Four-weeks-old chickens were vaccinated with these DNA vaccines.The chickens injected with attenuated vaccine were used as positive control and the chickens injected with pcDNA3.1(+) and PBS were used as negative control.The serum antibodies,peripheral blood lymphocyte proliferation(PBLP) and secreted IFN-γ of PBL were tested.Immunized chickens were challenged with virulent of avian P.multocida,with the protection rate counted.In result,ELISA showed the levels of antibodies in fusion DNA group were equivalent to group of attenuated vaccine,and significantly higher than the two single DNA groups(P<0.05).The SI values and IFN-γ secreted by PBL in pOMPHA group were significantly higher than the other groups(P<0.05),and the index in single DNA group slightly higher than attenuated vaccine group.All the DNA vaccines could provide partial protection for chickens after the challenge.The protection rate of fusion DNA vaccine was higher than the attenuated vaccine,and the latter was higher than the two single DNA vaccines. In conclusion,DNA vaccines especially the fusion DNA vaccine provide a promising approach for the prevention of fowl cholera.