副猪革拉瑟菌影递送猪圆环病毒2型DNA二联疫苗的制备及小鼠免疫效果评价  

Preparation of Glaesserella parasuis Ghosts Vaccine Delivering Porcine Circovirus Type 2 DNA Vaccine and Evaluation of Its Immunoprotective Effect in Mice

作　　者：王康 刘格言 王宇 杨振 唐欣巍 曹三杰 黄小波 颜其贵 伍锐 赵勤 杜森焱 文心田 文翼平 WANG Kang;LIU Geyan;WANG Yu;YANG Zhen;TANG Xinwei;CAO Sanjie;HUANG Xiaobo;YAN Qigui;WU Rui;ZHAO Qin;DU Senyan;WEN Xintian;WEN Yiping(Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130

出　　处：《畜牧兽医学报》2024年第3期1179-1191,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：成都市科技项目(2022-YF06-00177)。

摘　　要：本研究旨在研制猪圆环病毒2型(PCV2)-副猪革拉瑟菌(GPS)二联菌影疫苗,并评价其在小鼠上的免疫保护效果。选择GPS 5型菌株SH0165作为疫苗株,利用氢氧化钠最低抑菌浓度法将其制备成菌影,再将PCV2的ORF2基因克隆至pEGFP-N1真核表达质粒,将GPS菌影作为PCV2 DNA疫苗的载体,构建PCV2-GPS二联菌影疫苗,设计了商品化疫苗组、PCV2-GPS二联菌影疫苗组、菌影组、质粒组、PBS组、空白组,对4周龄BALB/c小鼠进行免疫,测定免疫后PCV2和GPS的IgG抗体效价、PCV2中和抗体、脏器中PCV2病毒载量、血清杀菌率、GPS攻毒保护率以及血清中细胞因子(IFN-γ、IL-12)水平。结果显示:二联菌影疫苗组小鼠血清中PCV2和GPS的IgG抗体水平显著增高,PCV2中和抗体滴度提高,PCV2攻毒后的小鼠淋巴结和肺脏中病毒载量下降,血清杀菌率显著提高,GPS攻毒后免疫保护率达70%(7/10),小鼠血清中IFN-γ含量显著上调。综上表明,PCV2-GPS二联菌影疫苗显著激活了小鼠对PCV2和GPS的体液免疫反应和细胞免疫反应,且能够抑制PCV2在小鼠体内的增殖,对感染GPS的小鼠达到较好的保护效果,为新型安全高效的PCV2、GPS联合疫苗的研制提供了生物材料和实验数据。The aim of this study was to develop a combined vaccine against porcine circovirus type 2(PCV2)and Glaesserella parasuis(GPS),and to evaluate its immune protective effect in mice.We selected SH0165,a type 5 strain of GPS,as the vaccine strain.The G.parasuis ghosts were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration(MIC)of sodium hydroxide(NaOH).Then,the ORF2 gene of PCV2 was cloned into the pEGFP-N1 vector.We developed a PCV2-GPS combined vaccine by using G.parasuis ghosts as vehicle for pEGFP-ORF2.Furthermore,four-week-old BALB/c mice were divided into commercial vaccine group,PCV2-GPS combined vaccine group,G.parasuis vaccine group,pEGFP-ORF2 group,PBS group,and blank group.The IgG antibody level against PCV2 and GPS,neutralization antibody against PCV2,PCV2 viral load of organs in mice,serum bactericidal activities,challenge protection efficacy,and level of cytokines(IFN-γ,IL-12)in serum were measured after immunization.The results showed that PCV2-GPS combined vaccine elicited significantly higher levels of IgG antibody response of PCV2 and GPS,neutralization antibody titer of PCV2,bactericidal activities and higher levels of IFN-γthan those of the control group.After PCV2 challenge,PCV2-GPS combined vaccine-immunized mice showed lower viral load in lymph nodes and lungs than the control group,and after challenge infection with GPS,immune protection efficacy of mice immunized was 70%(7/10).In conclusion,the PCV2-GPS combined vaccine significantly activated both humoral and cell-mediated immune responses,and inhibited the proliferation of PCV2 in mice,achieving a better protective effect in mice infected with GPS.The results provide biological materials and experimental data for the development of new,safe and efficient combined vaccines against porcine circovirus type 2 and Glaesserella parasuis.

关 键 词：猪圆环病毒2型 ORF2基因 副猪革拉瑟菌 菌影 小鼠 免疫保护 

分 类 号：S855[农业科学—临床兽医学]