副猪格拉瑟菌OppA过表达株的构建及其对小鼠免疫保护性研究  

Construction of the Gleasserella parasuis Strain Overexpressing OppA and Evaluation of Its Immunogenicity and Efficacity in Mice

作　　者：李枝卉 罗金林 谢虹 李明坤 林杨 张宇姣 徐引弟[2] 陈焕春[1] 蔡旭旺[1] 徐晓娟[1] LI Zhihui;LUO Jinlin;XIE Hong;LI Mingkun;LIN Yang;ZHANG Yujiao;XU Yindi;CHEN Huanchun;CAI Xuwang;XU Xiaojuan(National Key Laboratory of Agricultural Microbiology,Key Laboratory of Preventive Veterinary Medicine in Hubei Province,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Institute of Animal Husbandry and Veterinary Research,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]华中农业大学动物医学院,农业微生物资源发掘与利用重点实验室,湖北省预防兽医学重点实验室,武汉430070 [2]河南省农业科学院畜牧兽医研究所,郑州450002

出　　处：《畜牧兽医学报》2025年第2期860-869,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：“十四五”国家重点研发计划(2022YFD1800902);湖北省科技重大专项(2021ABA005)。

摘　　要：本研究旨在增加副猪格拉瑟菌寡肽渗透酶(oligopeptide permease,OppA)表达量,构建可以过表达OppA的副猪格拉瑟菌株,并对其进行小鼠的免疫保护试验以评价其作为格拉瑟病弱毒活疫苗的潜力。以副猪格拉瑟菌CF7066株为亲本株,利用自然转化和Flp-FRT无抗突变系统构建副猪格拉瑟菌OppA过表达株,通过Western blot检测OppA的表达量,通过阿拉伯糖诱导和提高培养温度消除抗性基因和Flp表达质粒。进行小鼠免疫保护试验,通过检测血清中抗体水平及脾淋巴细胞增殖、观察小鼠死亡率。结果显示:构建了3株副猪格拉瑟菌OppA过表达株ΔnanH::oppA^(R),ΔnanH::oppA和ΔnanH::P qseB-oppA。由于ΔnanH::oppA^(R)株中OppA的表达量显著高于CF7066(P<0.001),所以选择该过表达株进行小鼠免疫保护试验。小鼠在免疫后进行rOppA-ELISA的抗体检测,结果表明,ΔnanH::oppA^(R)免疫组的OppA抗体水平比CF7066组、ΔnanH组和PBS组高,但低于灭活疫苗组。用rOppA蛋白刺激脾淋巴细胞,ΔnanH::oppA^(R)和CF7066组小鼠脾淋巴细胞的增殖量显著高于刺激前(P<0.05),而ΔnanH、灭活疫苗组和PBS对照组没有改变。用CF7066菌株进行攻毒,其中灭活疫苗组、ΔnanH::oppA^(R)组、CF7066组对小鼠的保护率均为100%,ΔnanH组为75%。当前研究构建的3株副猪格拉瑟菌重组菌株中,ΔnanH::oppA^(R)的OppA表达水平最高,用该菌株免疫小鼠后产生OppA特异性的体液免疫和细胞免疫,而且可对CF7066攻毒提供有效保护。基于此,副猪格拉瑟菌OppA过表达株ΔnanH::oppA^(R)有望作为副猪格拉瑟菌的减毒活疫苗。The study aims to construct Glaesserella parasuis(G.parasuis)mutants overexpressing oligopeptide permease gene through improvement of its expression capacity,and to evaluate its immunogenicity and efficacity in mice.G.parasuis CF7066 strain being used as the parent strain,the overexpressing mutans were constructed via natural transformation and Flp-FRT markless mutation system.The OppA expression capacity of the overexpression strains was detected by WB,and then strains were induced by arabinose to excise Kan R cassette and were cultured at high temperature to eliminated Flp expression plasmid.The mutant ofΔnanH::oppA^(R) was selected for conductind immunization and challenge experiments in mice.Comprehensive evaluations were conducted on serum antibody levels,spleen lymphocyte proliferation and the murine survival rate.Three overexpressing mutans ofΔnanH::oppA^(R),ΔnanH::oppA andΔnanH::P qseB-oppA were constructed.BecauseΔnanH::oppA^(R) had the highest capacity for expression of OppA,it wase used for murine immunization experiment.The rOppA-ELISA showed that the mice immunized withΔnanH::oppA^(R) strain produced the higher level of OppA antibody than CF7066 andΔnanH,but lower than the commercial inactivated vaccine group.After being stimulated with rOppA,the number of lymphocyte proliferation were significantly larger than before immunization among the mice immunized withΔnanH::oppA^(R) and with CF7066(P<0.05),but not in the mice immunized withΔnanH,the inactivated vaccine and PBS control.The challenge experiment showed that the survival rate were 100%in the mice immunized withΔnanH::oppA^(R) and CF7066,and it was 75%inΔnanH group.Three overexpressing mutants of G.parasuis were constructed,in whichΔnanH::oppA^(R) has the highest expression capacity of OppA.Immunization and challenge experiments showed thatΔnanH::oppA^(R) elicited murine antibody production and splenic lymphocyte proliferation specific for OppA protein,and simultaneously provided substantial protection against the wild strain CF7066.

关 键 词：副猪格拉瑟菌 寡肽渗透酶 过表达 基因工程 弱毒疫苗 

分 类 号：S852.61[农业科学—基础兽医学]