副猪嗜血杆菌OMP5抗原免疫刺激复合物的制备及其对小鼠免疫功能的影响  

Study on the preparation of immunostimulatory complexes of Glaesserella parasuis OMP5 antigen and its effects on immune function in mice

作　　者：刘栋 薛少华 龙凡利 梁蓉娟 吴剑梅 郑新添[1,3] 李晓华 LIU Dong;XUE Shaohua;LONG Fanli;LIANG Rongjuan;WU Jianmei;ZHENG Xintian;LI Xiaohua(College of Life Science,Longyan University,Longyan 364012,China;College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou 350000,China;Fujian Provincial Key Laboratory of Prevention and Control of Infectious Diseases and Biotechnology,Longyan 364012,China;Fujian Provincial University Engineering Research Center for Animal-Derived Zoonoses,Longyan 364012,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364012 [2]福建农林大学动物科学学院,福州350000 [3]福建省家畜传染病防治与生物技术重点实验室,福建龙岩364012 [4]动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩364012

出　　处：《黑龙江畜牧兽医》2023年第9期67-72,79,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：福建省科技厅高校产学合作科技项目(2018N5012);动物源性人兽共患病防控福建省高校工程研究中心开放基金项目(2021ZN003);大学生创新创业训练计划项目(202011312005)。

摘　　要：为探究副猪嗜血杆菌(Haemophilus parasuis,HPS)外膜蛋白(outer membrane prtein,OMP)免疫刺激复合物(immune stimulating complexs,ISCOM)的抗原性,试验采用PCR方法扩增HPS的OMP5基因,将其克隆至原核表达载体pET-28a(+)中,获得重组表达质粒pET-28a-OMP5;进一步以QuilA、胆固醇和磷脂酰胆碱为原料,用透析法制备HPS OMP5抗原免疫刺激复合物(ISCOM-OMP5),用其免疫ICR小鼠后检测HPS抗体水平、细胞因子基因相对表达量及淋巴细胞增殖情况,以评价ISCOM-OMP5的免疫增强效果。结果表明:构建的重组表达质粒能够成功表达目的蛋白,大小约为40 ku;制备的ISCOM-OMP5具有ISCOM的典型蜂窝状结构,直径为30~40 nm,ISCOM-OMP5蛋白的包封率达85.8%;用ISCOM-OMP5对小鼠进行免疫,二免后14天即可在小鼠血清中检测到较高水平HPS抗体;ISCOM-OMP5能极显著提高白细胞介素-10(IL-10)和γ干扰素(IFN-γ)基因的相对表达量(P<0.01),显著提高T淋巴细胞的增殖能力(P<0.05),极显著提高B淋巴细胞的增殖能力(P<0.01)。说明ISCOM-OMP5可有效诱导小鼠产生针对HPS的免疫应答,能显著增强免疫效果。In order to ivestigate the antigenicity of Glaesserella parasuis(HPS) outer membrane prtein(OMP) immune stimulating complexes(ISCOMs), in this experiment, PCR method was used to amplify OMP5 gene of HPS, which was cloned into pET-28a(+) vector;the recombinant expression plasmid pET-28a-OMP5 was obtained. Furthermore, using QuilA, cholesterol and phospholipids as raw materials, dialysis method was used to prepare the HPS OMP5 antigen immune stimulating complexes(ISCOM-OMP5), which was immunized to ICR mice for the determination of the HPS-specific IgG antibody levels, cytokines and lymphocyte proliferation effect to evaluate the immune enhancing effect of ISCOM-OMP5. The results showed that the recombinant expression plasmid was able to successfully express the target protein with a band size of about 40.0 ku;the prepared ISCOM-OMP5 had a typical honeycomb structure of ISCOM with a diameter of 30-40 nm. The encapsulation rate of OMP5 protein reached 85.8%. ISCOM-OMP5 was used for the immunological evaluation results of mice. High levels of HPS-specific IgG antibodies were detected in the serum of mice 14 days after secondary immunization. ISCOM-OMP5 could significantly increase the relative expression of interleukin 10(IL-10) and gamma interferon(IFN-γ) genes(P<0.01);ISCOM-OMP5 could significantly increase the proliferative capacity of T lymphocytes(P<0.05), and very significantly increase the proliferative capacity of T lymphocytes(P<0.01). The results suggested that ISCOM-OMP5 could effectively induce the immune response against HPS in mice, and could significantly enhance the immune effect.

关 键 词：副猪嗜血杆菌 免疫刺激复合物 OMP5 小鼠 细胞因子 

分 类 号：S852.4[农业科学—基础兽医学]