杆状病毒表达系统表达BCoV纤突蛋白及其对小鼠的免疫原性  

Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice

作　　者：喻琦胜 朱庆 周群 宋鑫[1] 张家祺 陈涛云 徐林 张朝辉 张斌[1] YU Qisheng;ZHU Qing;ZHOU Qun;SONG Xin;ZHANG Jiaqi;CHEN Taoyun;XU Lin;ZHANG Chaohui;ZHANG Bin(College of Animal and Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Center for Animal Disease Control and Prevention,Ganzi Tibetan Autonomous Prefecture,Kangding 626000,China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]四川省甘孜藏族自治州动物疫病预防控制中心,康定626000

出　　处：《畜牧兽医学报》2024年第2期640-648,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省转移支付科技计划项目(210015);国家农业产业技术体系四川肉牛创新团队专项(SCCXTD-2020-13);西南民族大学研究生创新型科研项目(ZD2022455)。

摘　　要：旨在为表达牛冠状病毒(bovine coronavirus,BCoV)的纤突蛋白(S蛋白),并评价其作为免疫原免疫小鼠后的免疫效果。本研究以BCoV/SWUN/HXD-4/2021株的全长S基因为模板,针对昆虫细胞进行密码子优化和合成,构建重组质粒pFastBac-Dual-S,与DH10Bac同源重组后转染昆虫细胞收获重组杆状病毒rpFastBac-S,利用基因组PCR、免疫印迹法(Western blot)和间接免疫荧光试验对其进行鉴定,测定杆状病毒滴度。优化和筛选感染剂量对蛋白表达的影响,超速离心纯化蛋白,免疫小鼠和评估其免疫效果。结果显示:优化后全长4108 bp的BCoV S基因CAI(密码子适应指数)从0.39调整为0.87,平均GC含量从35.8%调整为50.6%。经双酶切、PCR验证,重组质粒与重组杆粒均构建成功。用5μg重组杆粒Bacmid-S转染Sf9细胞并传代至第四代后细胞病变趋于稳定,感染杆状病毒约20 h后产生典型病变,收获重组杆状病毒进行鉴定。PCR电泳结果显示重组杆粒携带S目的蛋白基因;间接免疫荧光试验显示,试验组观察到特异性绿色荧光;Western blot结果显示重组杆状病毒rpFastBac-S所表达的蛋白为S目的蛋白,目的条带位于250 ku左右,感染剂量MOI=0.5时蛋白表达量最高。将50μg蛋白、20μg CpG免疫增强剂与等体积MF59佐剂充分混合乳化后,肌肉注射免疫小鼠,间接ELISA试验结果显示,小鼠免疫蛋白后产生了IgG特异性抗体,且抗体水平高于佐剂对照组(P<0.001),最高抗体效价达1∶12800;微量中和试验结果显示,小鼠血清中和效价最高达1∶224,试验组中和效价平均值高于灭活病毒组,但统计学差异不显著(P>0.05)。本研究利用杆状病毒表达系统表达了牛冠状病毒S蛋白,并对S蛋白免疫保护效果做了进一步评价,为后续牛冠状病毒疫苗研究提供了理论依据。This study aimed to express the S protein of Bovine coronavirus(BCoV)and evaluate its immunogenicity in mice.The S protein of BCoV/SWUN/HXD-4/2021 strain was seclected as a template for codon optimization in insect cells.The complete S gene was synthesized and the recombinant plasmid pFastBac-Dual-S and Bacmid-S were constructed.The recombinant baculovirus rpFastBac-S was harvested by transfection of insect cells and identified by PCR,Western blot and indirect immunofluorescence assay.Baculovirus titers were determined.The effects of different infection doses(MOI=0.005,0.05,0.5,1,2)on protein expression were optimized,and the best infection condition was selected to express protein.The protein was purified by ultracentrifugation and immunized mice,and the immune effect was evaluated by indirect ELISA and neutralization test.After optimization,the S gene CAI(codon adaptation index)was adjusted from 0.39 to 0.87,and the average GC content was adjusted from 35.8%to 50.6%.Double enzyme digestion and PCR confirmed that the recombinant plasmid and the recombinant bacmid were successfully constructed.Sf9 cells were transfected with 5μg Bacmid-S and the cytopathic effect tended to be steady after the fourth cell passage.Typical cytopathic effect appeared 20 h after baculovirus infection.The recombinant bacmid-S was harvested and identified.The results of PCR electrophoresis showed that the recombinant bacmid carried the S target protein gene(size 4108 bp).IFA showed that specific green fluorescence was observed in the experimental group.Western blot results showed that the recombinant baculovirus rpFastBac-S protein was the S target protein,and the target size was about 250 ku.When the MOI was 0.5,the protein expression level was the highest.The mice were immunized with 50μg protein,20μg CpG adjuvant and equal volume of MF59 adjuvant by intramuscular injection.Indirect ELISA results showed that the mice immunized with the protein produced IgG specific antibody,and the antibody titer was higher than that of the control

关 键 词：牛冠状病毒 S蛋白 杆状病毒表达系统 免疫原性 

分 类 号：S852.659.6[农业科学—基础兽医学]