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

doi:10.11843/j.issn.0366-6964.2024.03.030

摘要/Abstract

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。

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

Abstract: 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-γ than 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.

Key words: porcine circovirus type 2, ORF2 gene, Glaesserella parasuis, bacterial ghost, mice, immunoprotection

中图分类号:

S855

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. 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[J]. 畜牧兽医学报, 2024, 55(3): 1179-1191.

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. Preparation of Glaesserella parasuis Ghosts Vaccine Delivering Porcine Circovirus Type 2 DNA Vaccine and Evaluation of Its Immunoprotective Effect in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1179-1191.

参考文献

[1] MUKHERJEE P, KARAM A, CHAKRABORTY A K, et al. Identification of a novel cluster of PCV2 isolates from Meghalaya, India indicates possible recombination along with changes in capsid protein[J]. Infect Genet Evol, 2019, 71:7-15.
[2] CHEN S, LI X, ZHANG L Y, et al. Phylogenetic and structural analysis of porcine circovirus type 2 from 2016 to 2021 in Jilin Province, China[J]. Microorganisms, 2023, 11(4):983.
[3] 莫荣君. 猪圆环病毒病的流行病学分析及防控措施[J]. 畜禽业, 2020, 31(8):91-92.
MO R J. Epidemiological analysis and prevention measures of porcine circovirus disease[J]. Livestock and Poultry Industry, 2020, 31(8):91-92. (in Chinese)
[4] 代科. 副猪嗜血杆菌PotD蛋白的毒力相关性及其通过TLR4诱导先天性免疫的通路研究[D]. 雅安:四川农业大学, 2020.
DAI K. Studies on virulence regulation and innate immunity pathway of Glaesserella parasuis polyamine transporter PotD[D]. Yaan:Sichuan Agricultural University, 2020. (in Chinese)
[5] 陈娟. 副猪嗜血杆菌贵州株的分离鉴定及生物学特性研究[D]. 贵阳:贵州大学, 2020.
CHEN J. Isolation, identification and biological characteristics research of Haemophilus parasuis Guizhou strain[D]. Guiyang:Guizhou University, 2020. (in Chinese)
[6] 何绿琴, 闫雪锋, 文心田, 等. 副猪嗜血杆菌qseB、qseC双基因缺失株的构建及生物学特性[D]. 畜牧兽医学报, 2022, 53(2):529-537.
HE L Q, YAN X F, WEN X T, et al. Construction and biological characteristic of Glaesserella parasuis strain with qseB, qseC double gene deletion [D]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2):529-537. (in Chinese)
[7] 吕茂杰. 猪圆环病毒2型-猪肺炎支原体-副猪嗜血杆菌三联灭活疫苗的研制[D]. 长春:吉林大学, 2022.
LV M J. Development of the triple inactivated vaccine against porcine circovirus type 2, Mycoplasma hyopneumoniae and Haemophilus parasuis[D]. Changchun:Jilin University, 2022. (in Chinese)
[8] BATAH A M, AHMAD T A. The development of ghost vaccines trials[J]. Expert Rev Vaccines, 2020, 19(6):549-562.
[9] SALEH N, MAHMOUD H E, ELTAHER H, et al. Prodigiosin-functionalized probiotic ghosts as a bioinspired combination against colorectal cancer cells[J]. Probiotics Antimicrob Proteins, 2023, 15(5):1271-1286.
[10] HAJAM I A, DAR P A, WON G, et al. Bacterial ghosts as adjuvants:mechanisms and potentia[J]. Vet Res, 2017, 48(1):37.
[11] SOLEYMANI S, TAVASSOLI A, HASHEMI TABAR G, et al. Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype[J]. Vet Res, 2020, 51(1):144.
[12] HARISA G I, SHERIF A Y, YOUSSOF A M E, et al. Bacteriosomes as a promising tool in biomedical applications:immunotherapy and drug delivery[J]. AAPS PharmSciTech, 2020, 21(5):168.
[13] WEN J, YANG Y, ZHAO G Y, et al. Salmonella typhi Ty21a bacterial ghost vector augments HIV-1 gp140 DNA vaccine-induced peripheral and mucosal antibody responses via TLR4 pathway[J]. Vaccine, 2012, 30(39):5733-5739.
[14] VINOD N, NOH H B, OH S, et al. A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges[J]. PLoS One, 2017, 12(9):e0185488.
[15] LI S Y, WANG D, GUO C Y, et al. Study on preparation of a Streptococcus suis ghost vaccine[J]. Microb Pathog, 2021, 154:104865.
[16] 王丹丹, 倪艳秀, 祝昊丹, 等. 应用NaOH制备胸膜肺炎放线杆菌菌影方法的建立[J]. 中国兽医科学, 2017, 38(8):999-1004.
WANG D D, NI Y X, ZHU H D, et al. Establishment of a novel preparation method for Actinobacillus pleuropneumoniae ghost by sodium hydroxide[J]. Chinese Veterinary Science, 2017, 38(8):999-1004. (in Chinese)
[17] JI S, MOON E S, NOH H B, et al. Protective immunity against Listeria monocytogenes in rats, provided by HCl- and NaOH-induced Listeria monocytogenes bacterial ghosts (LMGs) as vaccine candidates[J]. Int J Mol Sci, 2022, 23(4):1946.
[18] VINOD N, OH S, PARK H J, et al. Generation of a novel Staphylococcus aureus ghost vaccine and examination of its immunogenicity against virulent challenge in rats[J]. Infect Immun, 2015, 83(7):2957-2965.
[19] PORTER K R, RAVIPRAKASH K. DNA vaccine delivery and improved immunogenicity[J]. Curr Issues Mol Biol, 2017, 22:129-138.
[20] SYLLA S, CONG Y L, SUN Y X, et al. Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice[J]. Microbiol Immunol, 2014, 58(7):398-408.
[21] WU X Y, JU X R, DU L H, et al. Production of bacterial ghosts from gram-positive pathogen Listeria monocytogenes[J]. Foodborne Pathog Dis, 2017, 14(1):1-7.
[22] WANG Z H, ZHAO Q, WEI H L, et al. Prevalence and seroepidemiology of Haemophilus parasuis in Sichuan province, China[J]. PeerJ, 2017, 5:e3379.
[23] FU S L, ZHANG M M, XU J, et al. Immunogenicity and protective efficacy of recombinant Haemophilus parasuis SH0165 putative outer membrane proteins[J]. Vaccine, 2013, 31(2):347-353.
[24] YUAN F Y, FU S L, HU J, et al. Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model[J]. Res Vet Sci, 2012, 93(1):51-56.
[25] 周鹏. 鹦鹉热衣原体MOMP、MIP重组核酸菌影疫苗的免疫保护作用[D]. 衡阳:南华大学, 2018.
ZHOU P. Immunoprotective effects of recombinant E. coli ghosts expressing Chlamydia psittaci MOMP and MIP[D]. Hengyang:University of South China, 2018. (in Chinese)
[26] CHARERNTANTANAKUL W. Adjuvants for swine vaccines:mechanisms of actions and adjuvant effects[J]. Vaccine, 2020, 38(43):6659-6681.
[27] 周伃欣, 何久香, 全纹萱, 等. 登革病毒多价DNA疫苗构建及免疫保护效果验证[J]. 免疫学杂志, 2023, 39(1):70-77.
ZHOU Y X, HE J X, QUAN W X, et al. Construction and immune protection verification of dengue virus multivalent DNA vaccine[J]. Immunological Journal, 2023, 39(1):70-77. (in Chinese)