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

doi:10.11843/j.issn.0366-6964.2024.02.021

摘要/Abstract

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4 108 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5 μg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50 μg蛋白、20 μg 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∶12 800;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。

关键词: 牛冠状病毒, S蛋白, 杆状病毒表达系统, 免疫原性

Abstract: 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 5 μg 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 4 108 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 50 μg protein, 20 μg 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 group (P<0.001), and the highest antibody titer was 1∶12 800. The results of neutralization test showed that the highest neutralization titer of mouse serum was 1∶224, and the average neutralization titer of experimental group was higher than that of inactivated BCoV group, but the difference was not statistically significant (P>0.05). In conclusion, the S protein of BCoV was expressed by baculovirus expression system, and the immune protective effect of S protein was evaluated, which provided a theoretical foundation for the subsequent research of bovine coronavirus vaccine.

Key words: bovine coronavirus, S protein, baculovirus expression system, immunogenicity

中图分类号:

S852.659.6

喻琦胜, 朱庆, 周群, 宋鑫, 张家祺, 陈涛云, 徐林, 张朝辉, 张斌. 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[J]. 畜牧兽医学报, 2024, 55(2): 640-648.

YU Qisheng, ZHU Qing, ZHOU Qun, SONG Xin, ZHANG Jiaqi, CHEN Taoyun, XU Lin, ZHANG Chaohui, ZHANG Bin. Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 640-648.

参考文献

[1] SAIF L J. Bovine respiratory coronavirus[J]. Vet Clin North Am Food Anim Pract, 2010, 26(2):349-364.
[2] GENG H L, MENG X Z, YAN W L, et al. Prevalence of bovine coronavirus in cattle in China:A systematic review and meta-analysis[J]. Microb Pathog, 2023, 176:106009.
[3] 苗艳, 朱庆贺, 陈亮, 等. 牛冠状病毒流行病学和疫苗研究进展[J]. 中国兽药杂志, 2022, 56(7):89-94.
MIAO Y, ZHU Q H, CHEN L, et al. Progress on epidemiology and vaccine of bovine coronavirus[J]. Chinese Journal of Veterinary Drug, 2022, 56(7):89-94.(in Chinese)
[4] 孙飞雁, 叶京飞, 魏宇, 等. 吉林省肉牛冠状病毒感染状况调查及分析[J]. 畜牧兽医学报, 2023, 54(2):673-682.
SUN F Y, YE J F, WEI Y, et al. Epidemiological investigation and analysis of bovine coronavirus in beef cattle in Jilin province[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2):673-682.(in Chinese)
[5] ZHANG X M, HERBST W, KOUSOULAS K G, et al. Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child[J]. J Med Virol, 1994, 44(2):152-161.
[6] AMER H M. Bovine-like coronaviruses in domestic and wild ruminants[J]. Anim Health Res Rev, 2018, 19(2):113-124.
[7] VLASOVA A N, SAIF L J. Bovine coronavirus and the associated diseases[J]. Front Vet Sci, 2021, 8:643220.
[8] 王苗, 杜万年, 史秋梅, 等. 牛冠状病毒的研究进展[J]. 黑龙江畜牧兽医, 2021(7):48-51.
WANG M, DU W N, SHI Q M, et al. Progress in bovine coronavirus research[J]. Heilongjiang Animal Science and Veterinary Medicine, 2021(7):48-51.(in Chinese)
[9] PRÉVOST J, GASSER R, BEAUDOIN-BUSSIÈRES G, et al. Cross-sectional evaluation of humoral responses against SARS-CoV-2 spike[J]. Cell Rep Med, 2020, 1(7):10126.
[10] PARKER M D, YOO D, COX G J, et al. Primary structure of the S peplomer gene of bovine coronavirus and surface expression in insect cells[J]. J Gen Virol, 1990, 71(2):263-70.
[11] 周可悦, 冯生, 范泽昌, 等. 表达新型冠状病毒奥密克戎BA.1变异株S1基因重组DNA疫苗的构建及免疫原性[J]. 中国兽医学报, 2023, 43(5):964-970.
ZHOU K Y, FENG S, FAN Z C, et al. Construction and immunogenicity of recombinant DNA vaccine expressing S1 protein of a novel coronavirus Omicron BA. 1 variant[J]. Chinese Journal of Veterinary Science, 2023, 43(5):964-970.(in Chinese)
[12] SADOFF J, GRAY G, VANDEBOSCH A, et al. Safety and efficacy of single-dose Ad26. COV2. S vaccine against Covid-19[J]. N Engl J Med, 2021, 384(23):2187-2201.
[13] 周晶晶. 新型冠状病毒RBD重组蛋白疫苗及多肽疫苗的设计和免疫原性研究[D]. 杭州:浙江大学, 2021.
ZHOU J J. The design and immunogenicity of SARS-CoV-2 RBD recombinant protein vaccine and peptide vaccine[D]. Hangzhou:Zhejiang University, 2021.(in Chinese)
[14] LONG G, PAN X Y, KORMELINK R, et al. Functional entry of baculovirus into insect and mammalian cells is dependent on clathrin-mediated endocytosis[J]. J Virol, 2006, 80(17):8830-8833.
[15] FELBERBAUM R S. The baculovirus expression vector system:A commercial manufacturing platform for viral vaccines and gene therapy vectors[J]. Biotechnol J, 2015, 10(5):702-714.
[16] 刘拂晓, 柳增善, 王志亮. 杆状病毒表达系统——有效的VLP构建工具[J]. 生物技术通报, 2012(6):25-31.
LIU F X, LIU Z S, WANG Z L. Baculovirus expression system-effective tool of VLP production[J]. Biotechnology Bulletin, 2012(6):25-31.(in Chinese)
[17] 李利山, 刘玉香, 韩斌. 猪瘟疫苗的研究进展[J]. 中国动物保健, 2023, 25(3):116-118.
LI L S, LIU Y X, HAN B. Research progress of swine fever vaccine[J]. China Animal Health, 2023, 25(3):116-118.(in Chinese)
[18] YANG J Y, WANG W, CHEN Z M, et al. Publisher correction:a vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity[J]. Nature, 2021, 590(7844):E23.
[19] 高国强, 王梦心, 刘明明, 等. 牛冠状病毒S基因的序列分析及原核表达[J]. 中国畜牧兽医, 2018, 45(7):1740-1749.
GAO G Q, WANG M X, LIU M M, et al. Sequence analysis and prokaryotic expression of bovine coronavirus S gene[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(7):1740-1749.(in Chinese)
[20] 程凯慧, 沈付娆, 邹积振, 等. 牛冠状病毒S蛋白抗原表位基因的串联表达及抗血清制备[J]. 中国兽医杂志, 2018, 54(10):59-61, 66.
CHENG K H, SHEN F R, ZOU J Z, et al. Preparation of antisera and tandem expression of the antigen epitopes of bovine coronavirus S protein[J]. Chinese Journal of Veterinary Medicine, 2018, 54(10):59-61, 66.(in Chinese)
[21] MEBUS C A, STAIR E L, RHODES M B, et al. Pathology of neonatal calf diarrhea induced by a coronavirus-like agent[J]. Vet Pathol, 1973, 10(1):45-64.
[22] ZHU Q H, LI B, SUN D B. Advances in bovine coronavirus epidemiology[J]. Viruses, 2022, 14(5):1109.
[23] 沈付娆. 牛冠状病毒实时荧光定量PCR检测方法的建立及应用[D]. 济南:山东师范大学, 2015.
SHEN F R. Development and application of a SYBR Green Ⅰ real-time PCR assay for detection of bovine coronavirus[D]. Ji'nan:Shandong Normal University, 2015.(in Chinese)
[24] KANNO T, KAMIYOSHI T, ISHIHARA R, et al. Phylogenetic studies of bovine coronaviruses isolated in Japan[J]. J Vet Med Sci, 2009, 71(1):83-86.
[25] BIDOKHTI MR M, TRÅVÉN M, KRISHNA N K, et al. Evolutionary dynamics of bovine coronaviruses:natural selection pattern of the spike gene implies adaptive evolution of the strains[J]. J Gen Virol, 2013, 94(9):2036-2049.
[26] 郭晓旭, 顾文源, 赵云环, 等. 五种分子伴侣促进牛冠状病毒病毒样颗粒可溶性表达的研究[J/OL]. 中国动物传染病学报, 2023:1-15[2023-10-08]. https://doi.org/10.19958/j.cnki.cn31-2031/s.20230206.005.
GUO X X, GU W Y, ZHAO Y H, et al. Five molecular chaperones to promote soluble expression of bovine coronavirus virus-like particles[J/OL]. Chinese Journal of Animal Infectious Diseases, 2023:1-15[2023-10-08]. https://doi.org/10.19958/j.cnki.cn31-2031/s.20230206.005.(in Chinese)
[27] COHEN J. New Chinese vaccine could bolster global arsenal[J]. Science, 2021, 374(6563):12-13.
[28] HE C, YANG J Y, HONG W Q, et al. A self-assembled trimeric protein vaccine induces protective immunity against Omicron variant[J]. Nat Commun, 2022, 13(1):5459.
[29] 郝建伟, 薛春宜, 曹永长. 应用杆状病毒表达系统有效表达PEDV纤突蛋白[J]. 山西农业科学, 2022, 50(5):714-719.
HAO J W, XUE C Y, CAO Y C. Effective expression of spike protein of PEDV with expression system of Bac-to-Bac[J]. Journal of Shanxi Agricultural Sciences, 2022, 50(5):714-719.(in Chinese)
[30] O'HAGAN D T, OTT G S, DE GREGORIO E, et al. The mechanism of action of MF59-an innately attractive adjuvant formulation[J]. Vaccine, 2012, 30(29):4341-4348.
[31] 徐莉, 李敏. CpG ODN佐剂的研究进展及其药学研究的思考[J]. 中国生物制品学杂志, 2021, 34(12):1524-1528.
XU L, LI M. Research progress of CpG ODN adjuvant and reflections on pharmaceutical research[J]. Chinese Journal of Biologicals, 2021, 34(12):1524-1528.(in Chinese)