Gong Q, Qu N, Niu M F, Qin C L
Department of Biotechnology, College of Food and Bioengineering, Henan University of Science and Technology, Henan Engineering Laboratory of Livestock Disease Diagnosing and Food Safety Testing, Luoyang, 471023, China.
Iran J Vet Res. 2016 Spring;17(2):84-88.
Avian is the causative agent of fowl cholera, a disease much affecting the poultry industry. In order to study the efficacy of the recombinant subunit vaccine constructed with A gene of avian , the A gene fragment amplified by PCR from avian was cloned into the prokaryotic expression vector pET32a and the recombinant plasmid pET32a-ptfA was obtained. The pET32a-ptfA was expressed in BL21(DE3) and the target protein rPtfA was purified. The purified protein was then mixed with Freund's adjuvant and the recombinant subunit vaccine was obtained. Three groups of chickens labeled as rPtfA, attenuated live vaccine and PBS were vaccinated with the recombinant subunit vaccine, attenuated live vaccine and PBS, respectively. Serum antibodies, peripheral blood lymphocyte proliferation (PBLP) and interferon-γ (IFN-γ) level secreted by peripheral blood lymphocyte were tested. The immunized chickens were finally challenged with virulent avian and the protection rate was counted. Indirect ELISA showed the levels of antibodies in rPtfA and attenuated vaccine groups were most significantly higher than the other groups (P<0.01), and the former was slightly lower than the latter. Peripheral blood lymphocyte proliferation experiments and IFN-γ experiments indicated that SI value and the levels of IFN-γ induced by ConA in the two vaccine groups were significantly higher than those of the PBS groups (P<0.01), and that the attenuated vaccine group was higher than the rPtfA group. The protection rates of rPtfA and attenuated live vaccines were 45% and 75%, respectively. The results indicated that the PtfA recombinant subunit vaccine was capable of improving the immunity level and inducing a protective effect for the vaccinated chickens, but it was barely satisfactory.
禽巴氏杆菌是禽霍乱的病原体，该疾病对家禽业影响很大。为研究用禽巴氏杆菌A基因构建的重组亚单位疫苗的效果，通过PCR从禽巴氏杆菌扩增得到的A基因片段被克隆到原核表达载体pET32a中，获得重组质粒pET32a-ptfA。pET32a-ptfA在大肠杆菌BL21(DE3)中表达，纯化得到目标蛋白rPtfA。然后将纯化后的蛋白与弗氏佐剂混合，得到重组亚单位疫苗。将三组鸡分别标记为rPtfA组、弱毒活疫苗组和PBS组，分别用重组亚单位疫苗、弱毒活疫苗和PBS进行免疫接种。检测血清抗体、外周血淋巴细胞增殖(PBLP)以及外周血淋巴细胞分泌的干扰素-γ(IFN-γ)水平。最后用强毒禽巴氏杆菌对免疫后的鸡进行攻毒，并计算保护率。间接ELISA结果显示，rPtfA组和弱毒疫苗组的抗体水平显著高于其他组(P<0.01)，且前者略低于后者。外周血淋巴细胞增殖实验和IFN-γ实验表明，两个疫苗组中ConA诱导的SI值和IFN-γ水平均显著高于PBS组(P<0.01)，且弱毒疫苗组高于rPtfA组。rPtfA和弱毒活疫苗的保护率分别为45%和75%。结果表明，PtfA重组亚单位疫苗能够提高免疫水平并对免疫鸡产生保护作用，但效果不尽人意。
