猪繁殖与呼吸综合征病毒ORF5基因重组DNA疫苗的构建及其免疫效果评价  

Construction of recombinant DNA vaccines expressing the ORF5 gene of PRRSV and evaluation of their immune responses in mice

作　　者：魏春华[1,2,3] 黄夏玲 杨圆 何乐 林志锋 徐叶 黄文琳 柳开隆 戴爱玲[1,2,3] 杨小燕[1,2,3] 刘建奎[1,2,3] WEI Chunhua;HUANG Xialing;YANG Yuan;HE Le;LIN Zhifeng;XU Ye;HUANG Wenlin;LIU Kailong;DAI Ailing;YANG Xiaoyan;LIU Jiankui(College of Life Sciences,Longyan University,Longyan,Fujian 364000,China;Fujian Provincial Key Laboratory of Preventive Veterinary Medicine and Veterinary Biotechnology,Longyan University,Longyan,Fujian 364000,China;Fujian Provincial Key Laboratory of Preventive Veterinary Medicine and Biotechnology,Longyan University,Longyan,Fujian 364000,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364000 [2]龙岩学院福建省家畜传染病防治与生物技术重点实验室,福建龙岩364000 [3]龙岩学院预防兽医学与生物技术福建省高校重点实验室,福建龙岩364000 [4]福建农林大学动物科技学院,福建福州350002

出　　处：《西北农林科技大学学报（自然科学版）》2022年第7期17-25,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：中央引导地方科技发展专项(2019L3011,2021L3028);福建省科技重大专项项目(2019NZ09005);福建省科技厅引导性项目(2018N0022);国家级大学生创新创业训练计划资助项目(202011312003);奇迈基金项目(2020SHQM07,XLQM007);龙岩市科技计划项目(2019LYF1001)。

摘　　要：【目的】利用DNA改组技术(DNA shuffling)对不同谱系的猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因进行体外重组,构建具有良好交叉保护作用的DNA疫苗,为研制新型PRRSV疫苗提供参考。【方法】利用DNA改组技术对PRRSV谱系1、3、5.1、8.7毒株的ORF5基因进行体外突变重组,筛选去除信号肽的重组突变体。将重组突变体与真核表达载体pCAGGS-HA连接,构建重组表达质粒并进行酶切和测序鉴定,经脂质体转染MARC-145细胞,利用间接免疫荧光试验(IFA)和Western blotting试验检测目的蛋白的表达情况。将重组质粒pCA-ΔORF5-8和pCA-ΔORF5-46(ORF5基因重组的DNA疫苗)免疫小鼠,试验同时设pCAGGS-HA空载体组、PBS空白对照组、弱毒疫苗组(经典弱毒疫苗Ingelvac PRRS MLV,MLV组)和pCA-ORF5-MLV组(ORF5基因来自Ingelvac PRRS MLV),评估其免疫效果。【结果】筛选到2个去除信号肽的重组突变体(ΔORF5-8和ΔORF5-46)。成功构建重组表达质粒pCA-ΔORF5-8和pCA-ΔORF5-46,其可在MARC-145细胞中表达GP5蛋白。与pCAGGS-HA空载体组和PBS空白对照组相比,pCA-ΔORF5-8和pCA-ΔORF5-46组免疫小鼠血清白介素4(IL-4)和干扰素γ(INF-γ)含量均极显著提高(P<0.01),脾淋巴细胞增殖能力均极显著增强(P<0.01),均能产生针对GP5蛋白的抗体,血清中和试验表明,免疫组小鼠血清能对异源毒株产生交叉中和抗体(抗体平均滴度为10~16)。【结论】重组DNA疫苗pCA-ΔORF5-8和pCA-ΔORF5-46均能有效刺激并诱导机体产生细胞及体液免疫应答,具有良好的免疫原性。【Objective】This study developed DNA vaccines with good heterologous cross-neutralizing ability against heterologous virus using DNA shuffling technology to provide basis for further development of new vaccines against PRRS.【Method】Recombine PRRSV ORF5 genes of four different lineages were constructed by DNA shuffling technology,and two mutants(ΔORF5-8 andΔORF5-46)were selected by sequencing and analysis.Recombinant DNA plasmid pCA-ΔORF5-8/pCA-ΔORF5-46 was transfected into MARC-145 cells,and the expression of target proteins was confirmed by indirect immunofluorescent assay(IFA)and Western blotting(WB).To evaluate the immune effect,the recombinant plasmids pCA-ΔORF5-8 and pCA-ΔORF5-46 were used to immunize mice.The experiment also set up pCAGGS-HA empty vector control group,PBS blank control group,attenuated vaccine group(classic attenuated vaccine Ingelvac PRRS MLV,MLV group),and pCA-ORF5-MLV(ORF5 gene from Ingelvac PRRS MLV).【Result】Compared with the pCAGGS-HA and PBS groups,the mice from pCA-ΔORF5-8 and pCA-ΔORF5-46 groups showed significantly higher IL-4 and INF-γlevels(P<0.01)and the splenic lymphocyte proliferation abi-lity was significantly enhanced(P<0.01).Both pCA-ΔORF5-8 and pCA-ΔORF5-46 induced GP5 neutralizing antibodies with mean neutralizing antibody titers of 10-16 for different lineage viruses.Serum neutralization experiments showed that the sera of mice in the immunized group could produce cross-neutra-lization ability against heterologous strains.【Conclusion】Two recombinant DNA vaccines of pCA-ΔORF5-8 and pCA-ΔORF5-46 could effectively induce cellular and immune responses.

关 键 词：猪繁殖与呼吸综合征病毒 ORF5基因 DNA改组技术 重组DNA疫苗 交叉保护 

分 类 号：S852.651[农业科学—基础兽医学]