机构地区:[1]河南科技大学动物科技学院/洛阳市动物细菌性传染病防控技术重点实验室,河南洛阳471000 [2]河南科技大学医学技术与工程学院/分子诊断实验室,河南洛阳471000
出 处:《中国预防兽医学报》2025年第1期63-70,共8页Chinese Journal of Preventive Veterinary Medicine
基 金:国家自然科学基金项目(32072899、U1704117)。
摘 要:为筛选多杀性巴氏杆菌(PM)的优势保护性抗原,本研究利用原核表达系统表达了PM 5个重组蛋白rTorA、rPlpE、rThiB、rPGAM和rPrX,采用His标签蛋白纯化试剂盒纯化该5个重组蛋白后,通过SDS-PAGE检测各重组蛋白的表达及纯化效果;采用western blot检测各重组蛋白的反应原性。SDS-PAGE结果显示,重组蛋白rTorA主要以包涵体的形式表达,重组蛋白rPlpE以可溶性和包涵体两种形式表达,重组蛋白rThiB、rPGAM和rPrX主要以可溶性形式表达。纯化后分别在91 ku、45 ku、41 ku、32 ku和25 ku处出现单一目的条带,纯化效果较好;western blot结果显示,5个重组蛋白均可与猪PM阳性血清发生特异性反应,表明5个重组蛋白的反应原性均较强;采用超微量核酸蛋白浓度测定仪测定这5个重组蛋白的浓度分别0.7 mg/mL、2.0 mg/mL、1.0 mg/mL、1.5 mg/mL和0.5 mg/mL。将这5个重组蛋白分别与ISA201佐剂混合制成疫苗后以皮下注射方式免疫小鼠2次,间隔21 d。利用间接ELISA方法检测各组小鼠免疫后不同时间的抗体水平。二免后15 d,分别从各组随机取一半小鼠,以约4 LD50(210 cfu)A型PM A7强毒株第一次经腹腔注射攻毒;7 d后,将各组剩余的小鼠再以相同剂量的PM A7株攻毒。间接ELISA检测结果显示,5个重组蛋白疫苗均能诱导免疫小鼠产生较高水平的IgG抗体。攻毒结果显示,对照组小鼠全部死亡,各蛋白疫苗组(除rPlpE组外)也出现不同程度的发病死亡情况,死亡小鼠剖检均呈典型的败血症性病理变化,从死亡小鼠的心、肺、肝等组织脏器中均能分离到PM;在第一次攻毒试验中,rTorA、rPlpE、rThiB、rPGAM和rPrX疫苗对小鼠提供的免疫保护率分别为60%(6/10)、100%(10/10)、90%(9/10)、70%(7/10)、20%(2/10);在第二次重复攻毒试验中rTorA、rPlpE、rThiB、rPGAM和rPrX疫苗分别对小鼠提供60%(6/10)、100%(10/10)、80%(8/10)、60%(6/10)、40%(4/10)的免疫保护率。两次攻毒试验结果基本一致。In order to screen the dominant protective antigen of Pasteurella multocida(PM),the recombinant proteins of five antigens,PM rTorA,rPlpE,rThiB,rPGAM and rPrX,were expressed by prokaryotic expression system in this study.After the five recombinant proteins were purified using a His-tagged protein purification kit,SDS-PAGE were used to detect the expression and purification effect of each recombinant protein.Western blot was used to detect the reactivity of each recombinant protein.The results showed that the recombinant protein rTorA expressed mainly in the form of inclusion body,the recombinant protein rPlpE expressed in the form of soluble and the recombinant protein rThiB,rPGAM and rPrX expressed mainly in soluble form.SDS-PAGE results showed that the target bands appeared at 91ku,45ku,41ku,32ku,and 25ku,respectively,and the purification effect was better.Western blot results showed that the five recombinant proteins could react specifically with porcine PM-positive serum,indicating that the reactogenicity of the five recombinant proteins was good.The concentration of the five recombinant proteins was determined by using the ultra-micro nucleic acid protein concentration analyzer at 0.7mg/mL,2.0mg/mL,1.0mg/mL,1.5mg/mL and 0.5mg/mL,respectively.These five proteins were mixed with ISA201 adjuvant to prepara vaccines,and then immunize mice twice by subcutaneous injection with a 21 days interval.Indirect ELISA was used to detect the antibody levels of mice in each group at different times after immunization.15 days after the second immunization,half of the mice were randomly selected from each group and challenged by intraperitoneal injection of about 4LD50(210cfu)PM A7 virulent strain.After 7 days,the remaining mice in each group were challenged again with the same dose of PM A7 strain.Indirect ELISA results showed that the five recombinant protein vaccines could induce higher levels of IgG antibodies in immunized mice.The challenge test results showed that all mice in the control group died,and the protein vaccin
分 类 号:S852.61[农业科学—基础兽医学]
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