猪多杀性巴氏杆菌4个重组蛋白的小鼠免疫保护效力比较  

Comparative analysis of protective efficacy of four antigenic proteins of Pasteurella multocida

作　　者：孙鑫妍 王怡涤 关丽君 史琳琪 薛云[1] 司丽芳[1,2] 赵战勤[1,2] SUN Xin-yan;WANG Yi-di;GUAN Li-jun;SHI Lin-qi;XUE Yun;SI Li-fang;ZHAO Zhan-qin(l.Luoyang Key Laboratory of Prevention and Control Technology of Zoonotic Bacterious Diseases,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China;Key Disciplines Laboratory of Safety of Environment and Animal Product,Collegt of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China)

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]河南科技大学动物科技学院河南省高等学校环境与畜产品安全重点学科开放实验室,河南洛阳471000

出　　处：《中国预防兽医学报》2023年第11期1172-1178,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31672530、U1704117)。

摘　　要：为筛选多杀性巴氏杆菌(Pasteurella multocida,Pm)亚单位疫苗的优势保护性抗原,本研究根据GenBank中登录的Pm基因序列,设计相应引物,经PCR扩增Pm torA、prX、gpmA和thiB基因片段,分别克隆于原核表达载体pET28a中,构建各重组表达质粒,经测序鉴定后转化大肠杆菌BL21感受态细胞,利用IPTG诱导表达,采用His6标签蛋白纯化试剂盒纯化获得4个重组蛋白rTorA、rPrX、rPGAM和rThiB,同时利用透析膜制备rTorA的粗提蛋白(rTorA-C)。SDS-PAGE检测结果显示,分别在91 ku、25 ku、32 ku及41 ku处出现目的条带,表明4个重组蛋白均正确表达,其中rTorA主要以包涵体形式表达,少量表达在上清中;rPrX、rPGAM和rThiB主要以可溶性形式表达;各重组蛋白的表达量占菌体总蛋白的7.44%~61.46%,各重组蛋白纯化后的纯度为76%~98.15%,浓度为0.15 mg/mL~4.56 mg/mL。Western blot检测结果显示,分别在上述4个位置处出现特异性条带。将4个纯化蛋白和rTorA-C分别与MontanideTM ISA 201 VG佐剂混合制成亚单位疫苗,免疫小鼠后分别于首免前、首免后14 d、二免后14 d(首免后28 d)采血,分离血清,通过间接ELISA方法检测各组小鼠抗体水平;二免后15 d采用4倍半数致死剂量(LD50)A型Pm强毒A5株菌液进行腹腔注射攻毒,观察并记录小鼠死亡及发病情况至21 d。ELISA检测结果显示,各重组蛋白免疫组小鼠的IgG抗体水平均随免疫次数的增加而升高,与PBS对照组相比差异均极显著(P<0.01)。攻毒试验结果显示,PBS对照组小鼠攻毒后24 h内开始发病,10 d内全部死亡(10/10);发病小鼠主要症状为精神萎靡、食欲减退或废绝,被毛凌乱,对外界刺激无明显反应等;对死亡小鼠剖检,各组织器官主要表现为出血为特征的败血症的剖检变化。各重组蛋白免疫组小鼠中,也出现了不同程度的发病或死亡。rTorA、rTorA-C、rPGAM、rThiB和rPrX免疫组小鼠的免疫保护率分别为60%、40%、100%、20%和0。本研究首�In order to screen for the dominant protective antigen of Pasteurella multocida(Pm)subunit vaccine,this study designed primers based on the registered Pm gene sequence in GenBank and amplified the gene fragments of torA,prX,gpmA and thiB by PCR.Then,they were cloned into the prokaryotic expression vector pET28a to construct recombinant expression plasmid,which was transformed into Escherichia coli BL21 after DNA sequencing and induced by IPTG.Four recombinant proteins rTorA,rPrX,rPGAM and rThiB were extracted by His6 label protein purification kit,and the crude protein of rTorA(rTorA-C)was prepared by dialysis membrane.SDS-PAGE results showed that the four recombinant proteins were correctly constructed and rTorA was mainly expressed in the form of inclusion bodies,with a small amount expressed in supernatant;rPrX,rPGAM and rThiB are mainly expressed in soluble form;accounting for 7.44%to 61.46%of the total protein.The purity of the purified recombinant proteins ranged from 76%to 98.15%,and the concentration ranged from 0.15mg/mL to 4.56mg/mL.Western blot results showed that the recombinant expression protein carrying His6-tag could be recognized by His6 monoclonal antibody,the band size was about 91ku,25ku,32ku and 41ku,respectively.Four purified proteins and rTorA crude protein were mixed with MontanideTM ISA 201 VG adjuvant,respectively,to prepare subunit vaccine.Mice were immunized twice,and blood samples were collected before the first immunization,14 days after the first immunization,and 14 days after the second immunization(28 days after the first immunization)to separate serum.The antibody levels of each group were detected by indirect ELISA.At 15 days after the second immunization,the mice were challenged intraperitoneally with 4LD50 of serogroup A Pm virulent strain A5,and the death and morbidity were observed and recorded for 21 days.ELISA results showed that the IgG antibody level of mice in the recombinant protein immunized group increased with the increase of immunization times,and the difference wa

关 键 词：多杀性巴氏杆菌 重组表达 小鼠 免疫保护效力 亚单位疫苗 

分 类 号：S852.61[农业科学—基础兽医学]