摘要
为获得副猪格拉瑟菌(GPS)亚单位疫苗的高效保护性抗原,本研究利用原核表达系统对GPS重要保护性抗原Hps06257与HbpB及其C端片段(HbpB2)进行了融合表达,获得4个重组蛋白rHps06257-HbpB、rHbpBHps06257、rHps06257-HbpB2和rHbpB2-Hps06257。采用His标签蛋白纯化试剂盒纯化该4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。采用超微量核酸蛋白浓度测定仪测定这4个串联重组蛋白以及实验室前期保存的两个重组蛋白(rHps06257和rHbpB)的浓度后,分别与ISA201佐剂混合制成亚单位疫苗,对小鼠经皮下注射方式免疫2次,分别在小鼠免疫前、首免后21 d和二免后14 d采血,采用间接ELISA方法检测各组小鼠的抗体水平;二免后15 d分别以5 LD50(8.40×10^(8)cfu)和10 LD50(1.68×10^(9)cfu)的GPS 5型H5L3株菌液对小鼠经腹腔攻毒,统计各重组蛋白疫苗对小鼠的攻毒保护率。SDS-PAGE检测结果显示,4个重组蛋白分别在约92.4 ku、92.5 ku、63.6 ku和63.6 ku处出现目的条带,均以包涵体形式表达,其表达量占菌体总蛋白的11.2%~28.3%。4个重组蛋白的纯化效果均较好,浓度介于1.2 mg/mL~3.0 mg/mL。Western blot结果显示,4个重组蛋白均能够与GPS阳性血清发生特异性反应。ELISA结果显示,各重组蛋白疫苗免疫组小鼠血清中IgG抗体水平随免疫次数的增加而升高,且均与PBS对照组差异极显著(P<0.01),其中rHps06257-HbpB组小鼠血清中的IgG抗体水平最高。攻毒试验结果显示,采用5 LD50H5L3株攻毒后,重组蛋白疫苗rHps06257-HbpB、rHbpB-Hps06257、rHps06257-HbpB2和rHbpB2-Hps06257对小鼠的免疫保护率均为100%(6/6),重组蛋白疫苗rHps06257和rHbpB对小鼠的免疫保护率分别为67%(4/6)和50%(3/6)(对照组存活率为0);采用10 LD50H5L3株攻毒后,6个重组蛋白疫苗对小鼠的免疫保护率分别为83%(5/6)、0、67%(4/6)、33%(3/6)、0和0。本研究对GPS重要保护性抗原Hps06257与HbpB及其C端片段(HbpB2)进行了4种方式的串联表达,并对4个重组蛋白疫苗的免疫保护效力进行了评价,证实重组蛋白rHps06257-HbpB和rHps06257-HbpB2具备成为GPS亚单位疫苗候选靶抗原的潜力,为GPS亚单位疫苗的研发提供了重要参考依据。
To obtain highly effective protective antigen for Glaesserella parasuis(GPS)subunit vaccines,the important protective antigen Hps06257 of GPS was fused with HbpB and its C-terminal fragment(HbpB2)respectively,and four recombinant proteins rHps06257-HbpB,rHbpB-Hps06257,rHps06257-HbpB2 and rHbpB2-Hps06257 were expressed in prokaryotic expression system,following by purification using His-tagged protein purification kit.The expression and purification were monitored by SDS-PAGE.The concentrations of these four tandem recombinant proteins and previously stored two recombinant proteins(rHps06257 and rHbpB)were measured by ultra-micro nucleic acid protein concentration analyzer,and then each protein was respectively mixed with ISA201 adjuvant to make a subunit vaccine candidate.Subsequently,the mice were immunized twice by subcutaneous injection,and blood was collected before immunization,21 days after the first immunization and 14 days after the second immunization.The antibody levels of mice in each group were detected by indirect ELISA.On the 15th day after the second immunization,mice were challenged intraperitoneally with GPS type 5 H5L3 strains of 5LD50(8.40×10^(8)cfu)and 10LD50(1.68×10^(9)cfu),and the protection rate of each vaccine candidate in mice was calculated.The results of SDS-PAGE showed that the target bands appeared at about 92.4ku,92.5ku,63.6ku and 63.6ku,respectively,and were expressed in the form of inclusion bodies,which accounted for 11.2%to 28.3%of the total bacterial protein.The four recombinant proteins were purified effectively,and the concentrations ranged from 1.2mg/mL to 3.0mg/mL.The results of western blot showed that the four recombinant proteins all reacted specifically with GPS-positive serum.ELISA results indicated that the IgG antibody levels in the sera of mice immunized with the corresponding vaccine candidate increased with the number of immunizations,and significantly higher than that of the PBS control group(P<0.01).Among the vaccines,rHps06257-HbpB elicited the highest IgG antibody levels.The challenge experiment results demonstrated that following exposure to 5LD50 of the H5L3 strain,the protective rates of tandem expressed proteins(rHps06257-HbpB,rHbpB-Hps06257,rHps06257-HbpB2 and rHbpB2-Hps06257)were all 100%(6/6),while they were 67%(4/6)and 50%(3/6)for rHps06257 and rHbpB protein,respectively(the survival rate of control group was 0).After being challenged with 10LD50 of the H5L3 strain,the protective rates of the six recombinant protein vaccines were 83%(5/6),0,67%(4/6),33%(2/6),0 and 0,respectively.In this study,the important GPS protective antigen Hps06257 was expressed in tandem with HbpB and its C-terminal fragment(HbpB2)in four ways,and evaluation of the immunoprotective efficacy of the four fusion proteins revealed that recombinant proteins rHps06257-HbpB and rHps06257-HbpB2 have the potential to be the target antigens of subunit vaccines,which provides an important reference for the research and development of GPS subunit vaccines.
作者
杨金钱
董玉岗
丁略烜
史琳琪
杨梦娟
张俊峰
关丽君
司丽芳
薛云
赵战勤
YANG Jin-qian;DONG Yu-gang;DING Lue-xuan;SHI Lin-qi;YANG Meng-juan;ZHANG Jun-feng;GUAN Li-jun;SI Li-fang;XUE Yun;ZHAO Zhan-qin(Luoyang Key Laboratory of Prevention and Control Technology of Zoonotic Bacterial Infectious Diseases,College ofAnimal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China;Molecular Diagnostic Laboratory,College of Medical Technology and Engineering,Henan University of Science and Techology,Luoyang 471000,China)
出处
《中国预防兽医学报》
CSCD
北大核心
2024年第11期1166-1173,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31672530、U1704117)。
关键词
副猪格拉瑟菌
串联表达
小鼠
保护效力
亚单位疫苗
Glaesserella parasuis
tandem expression
mice
protective efficacy
subunit vaccine
作者简介
杨金钱(1994-),男,河南周口人,硕士研究生,主要从事家畜传染病及其疫苗研究;通信作者:张俊峰,E-mail:jfzhang018@163.com;通信作者:赵战勤,E-mail:zhaozhanqin@126.com。
