共表达牛病毒性腹泻病毒E0基因和牛传染性鼻气管炎病毒gD基因的重组质粒DNA构建及其对小鼠的免疫效应

doi:10.11843/j.issn.0366-6964.2021.016

摘要/Abstract

摘要： 本研究计划构建能同时表达牛病毒性腹泻病毒（BVDV）和牛传染性鼻气管炎病毒（IBRV）优势抗原基因的重组质粒并研究其免疫效果，旨在为BVD-IBR二联核酸疫苗的研制提供参考。采用PCR扩增目的基因E0及gD并将其依次连接至真核表达载体pVAX1-IRES中，构建pVAX1-E0-IRES-gD重组真核表达载体；将pVAX1-E0-IRES-gD转染至293T细胞中，间接免疫荧光法检测目的基因在细胞中的表达情况；将pVAX1-E0-IRES-gD以不同剂量采用肌内注射的方式接种小鼠，通过抗体水平和细胞因子检测以及脾淋巴细胞增殖试验对该重组质粒的免疫效果进行评价。结果表明，成功构建pVAX1-E0-IRES-gD重组质粒，目的基因在293T细胞内成功表达。在抗体和细胞因子水平以及脾淋巴细胞增殖能力这3个指标，重组质粒组均极显著高于空载体和阴性对照组，高剂量免疫组优于中、低剂量组；另外，高剂量重组质粒免疫组与二联灭活疫苗组相比无显著性差异。结果显示，成功构建了共表达BVDV E0和IBRV gD基因的重组质粒，体外表达检测证明目的蛋白具有良好的反应原性，动物免疫试验证明其能刺激机体产生良好免疫应答。

关键词: 牛病毒性腹泻病毒, E0, 牛传染性鼻气管炎病毒, gD, 重组质粒, 共表达, 免疫效果

Abstract: This study aimed to construct a recombinant plasmid that can simultaneously express the dominant antigen genes of bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV), and study its immune effect, so as to provide reliable data and theoretical support for the development of BVD-IBR bivalent DNA vaccine. In our experiment, PCR was used to amplify the target genes, E0 and gD, which were sequentially connected to the eukaryotic expression vector pVAX1-IRES to construct the pVAX1-E0-IRES-gD recombinant eukaryotic expression vector. The recombinant plasmid pVAX1-E0-IRES-gD was transfected into 293T cells and the expression of the target genes in foreign cells were detected by indirect immunofluorescence assay. Following, immunization test with the pVAX1-E0-IRES-gD, pVAX1-IRES and BVD-IBR combined inactivated vaccine was inoculated with SPF mice at different doses by intramuscular injection, subsequently, immune effects have been evaluated through antibody level and cytokine detection and splenic lymphocyte proliferation assay. The results showed that the pVAX1-E0-IRES-gD recombinant plasmid was successfully constructed and both antigen genes obtained a high level of expression in 293T cells. In terms of antibody and cytokine levels and the proliferation of spleen lymphocytes, the recombinant plasmid group was significantly higher than that of the empty vector and the negative control group, and the differences were extremely significant, the high-dose immunization group was better than the medium and low dose groups. Also, the high-dose recombinant plasmid immunization group was not significantly different from the BVD-IBR inactivated vaccine group. The results showed that the co-expression recombinant plasmid which contains the BVDV E0 gene and IBRV gD gene was constructed successfully. The expression test in vitro proved that the target protein has good reactionogenicity, and animal immunity tests proved that it can stimulate the body to produce a good immune response.

Key words: bovine viral diarrhea virus, E0, infectious bovine rhinotracheities virus, gD, recombinant plasmid, co-expression, immune effect

中图分类号:

S858.235.3

马小静, 李梦莹, 张淮瑜, 宋阿北, 许立华, 吴心华, 张巧娥, 李继东. 共表达牛病毒性腹泻病毒E0基因和牛传染性鼻气管炎病毒gD基因的重组质粒DNA构建及其对小鼠的免疫效应[J]. 畜牧兽医学报, 2021, 52(1): 154-165.

MA Xiaojing, LI Mengying, ZHANG Huaiyu, SONG Abei, XU Lihua, WU Xinhua, ZHANG Qiaoe, LI Jidong. Construction of a Recombinant Plasmid Co-expressing Bovine Viral Diarrhea Virus E0 Gene and Infectious Bovine Rhinotracheitis Virus gD Gene and Consider Its Immune Effect on Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 154-165.

参考文献 30

[1]	刘占悝,刘泽余,李智杰,等.牛传染性鼻气管炎病毒和牛病毒性腹泻病毒双重Nano-PCR检测方法的建立与应用[J].中国兽医科学,2019,49(12):1499-1504.LIU Z L,LIU Z Y,LI Z J,et al. Establishment and application of dual Nano-PCR detection method for infectious bovine rhinotracheitis virus and bovine viral diarrhea virus[J]. Chinese Veterinary Science,2019,49(12):1499-1504. (in Chinese)
[2]	谢春芳,于瑞嵩,李震,等.规模化奶牛场牛传染性鼻气管炎与牛病毒性腹泻的流行病学调查[J].中国奶牛,2016, 312(4):38-41.XIE C F,YU R S,LI Z,et al. Epidemiological investigation of bovine viral diarrhea and infectious bovine rhinotracheitis in dairy herds[J]. China Dairy Cattle, 2016, 312(4):38-41. (in Chinese)
[3]	季新成.牛传染性鼻气管炎病毒和牛病毒性腹泻病毒分子生物学检测技术研究[D].杨凌:西北农林科技大学,2010.JI X C. Study on molecular biology detection technology of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus[D]. Yangling:Northwest A&F University,2010. (in Chinese)
[4]	阮东. 某牛场牛BVD-MD的诊断与BVD-MD/IBR二联苗免疫效果评价[D]. 石河子:石河子大学,2017.RUAN D. Evaluation of BVD-MD diagnosis and BVD-MD/IBR combined vaccine in a cattle farm[D]. Shihezi:Shihezi University,2017. (in Chinese)
[5]	何小丽,李凡飞,张凯,等. 国内外牛传染性鼻气管炎的流行现状及防控措施的研究进展[J]. 现代畜牧兽医,2018(6):53-57.HE X L,LI F F,ZHANG K,et al. Epidemiology and control of infectious bovine rhinotracheitis in the domestic and overseas[J]. Modern Journal of Animal Husbandry and Veterinary Medicine,2018(6):53-57. (in Chinese)
[6]	刘瑞宁,邓明亮,刘玉辉,等. 牛传染性鼻气管炎疫苗研究进展[J]. 动物医学进展,2016,37(2):85-90.LIU R N,DENG M L,LIU Y H,et al. Advance in vaccines of infectious bovine rhinotracheitis[J]. Progress in Veterinary Medicine, 2016, 37(2):85-90. (in Chinese)
[7]	KELLING C L. Evolution of bovine viral diarrhea virus vaccines[J]. Vet Clin North Am Food Anim Pract, 2003, 20(1):115-129.
[8]	冷雪,郭利,张淑琴,等. 牛传染性鼻气管炎活疫苗安全性和免疫保护效果研究[J]. 中国畜牧兽医. 2011, 38(10):181-184.LENG X,GUO L,ZHANG S Q,et al. Safety and efficacy of an attenuated infectious bovine rhinotracheitis virus vaccine[J].China Animal Husbandry & Veterinary Medicine, 2011, 38(10):181-184. (in Chinese)
[9]	VAN OIRSCHOT J T,BRUSCHKE C J M,VAN RIJIN P A. Vaccination of cattle against bovine viral diarrhea[J]. Vet Microbiol, 1999, 64(2-3):169.
[10]	王利丽,王云峰,孙惠玲,等.传染性喉气管炎病毒基因疫苗免疫效应[J].畜牧兽医学报,2007,38(2):175-183.WANG L L, WANG Y F, SUN H L, et al. Immune efficacy of DNA vaccine of avian infectious laryngotracheitis virus[J]. Acta Veterinaria et Zootechnica Sinica,2007,38(2):175-183. (in Chinese)
[11]	BELÁKOVÁ J,HORYNOVÁ M,KRUPKA M, et al. DNA vaccines:are they still just a powerful tool for the future?[J].Arch Immunol Ther Exp (Warsz),2007, 55(6):387-398.
[12]	LI L,PETROVSKY N. Molecular adjuvants for DNA vaccines[J]. Curr Issues Mol Biol, 2017, 22:17-40.
[13]	FAUREZ F, DORY D, LE MOIGNE V, et al. Biosafety of DNA vaccines:New generation of DNA vectors and current knowledge on the fate of plasmids after injection[J].Vaccine, 2010,28(23):3888-3895.
[14]	石明.牛病毒性腹泻/粘膜病病毒E0、E2基因免疫原性的比较[D]. 呼和浩特:新疆农业大学,2014.SHI M. The difference of immunogenicity between E0 and E2 gene in Bovine viral diarrhea/mucosal disease virus[D]. Urumqi:Xinjiang Agricultural University,2014. (in Chinese)
[15]	DESHPANDE M S, AMBAGALA T C, HEGDE N R, et al. Induction of cytotoxic T-lymphocytes specific for bovine herpesvirus-1 by DNA immunization[J]. Vaccine, 2002, 20(31-32):3744-3751.
[16]	张帆,刘行波,倪宏波.牛传染性鼻气管炎病毒gE蛋白的真核表达及其多克隆抗体的制备[J]. 中国生物制品学杂志, 2020,33(5):507-509,515.ZHANG F,LIU X B,NI H B. Eukaryotic expression of gE protein of infectious bovine rhinotracheitis virus and preparation of polyclonal antibody[J]. Chinese Journal of Biologicals, 2020,33(5):507-509, 515. (in Chinese)
[17]	张帆,刘行波,倪宏波.牛传染性鼻气管炎病毒gC结构蛋白的真核表达及其反应原性分析[J]. 中国生物制品学杂志,2018,31(12):1333-1335,1339.ZHANG F,LIU X B,NI H B. Eukaryotic expression and reactogenicity of infectious bovine rhinotracheitis virus gC structural protein[J]. Chinese Journal of Biologicals, 2018,31(12):1333-1335, 1339. (in Chinese)
[18]	李婷玉,李雪梅,石慧娟,等.牛病毒性腹泻病毒结构蛋白功能相关研究进展[J].黑龙江科技信息,2016(19):29-30.LI T Y,LI X M,SHI H J,et al. Research progress on the functions of bovine viral diarrhea virus structural proteins[J]. Heilongjiang Science and Technology Information,2016(19):29-30. (in Chinese)
[19]	MARSHALL R L,ISRAEL B A,LETCHWORTH G J. Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection[J]. Virology, 1988, 165(2):338-347.
[20]	BABIUK L A,LEWIS P J,COX G, et al. DNA immunization with bovine herpesvirus-1 genes[J]. Ann N Y Acad Sci, 1995, 772:47-63.
[21]	白艳丽,薛慧文.核糖体内部进入位点介导的翻译起始机制研究进展[J].黑龙江畜牧兽医,2017,(21):67-72.BAI Y L,XUE H W. Review on translation initiation mechanism mediated by internal ribosome entry site[J]. Heilongjiang Animal Science and Veterinary Medicine,2017,(21):67-72. (in Chinese)
[22]	段瑞峰,李刚. 细胞内部核糖体进入位点研究进展[J]. 生物技术通讯,2004, 15(5):59-61.DUAN R F,LI G. Advances in the research of cellular internal ribosome entry site[J]. Letters in Biotechnology,2004, 15(5):59-61. (in Chinese)
[23]	阎瑾琦,刘国栋,刘荷中,等.双顺反子真核表达载体pVAX1-IRES的构建与鉴定[J].军事医学科学院院刊,2008,32(2):111-115.YAN J Q,LIU G D,LIU H Z,et al. Construction and identification of bicistronic eukaryotic expression vector pVAX1-IRES for gene coexpression[J]. Bulletin of the Academy of Military Medical Sciences,2008,32(2):111-115. (in Chinese)
[24]	曲云鹏,刘建国,杨德琴. 真核表达质粒pVAX1的应用[J]. 温州医学院学报,2009, 39(2):194-196.QU Y P,LIU J G,YANG D Q. Application of eukaryotic expression plasmid pVAX1[J].Journal of Wenzhou Medical College,2009, 39(2):194-196. (in Chinese)
[25]	魏昭,张超,徐东刚. 基因回路调控元件的研究进展[J]. 军事医学,2017,41(7):623-627.WEI Z,ZHANG C,XU D G. Regulatory elements in gene circuits:research progress[J]. Military Medical Sciences,2017,41(7):623-627. (in Chinese)
[26]	董博. PCV2 ORF2基因真核表达质粒的构建及与猪IL-15联合免疫效果研究[D]. 哈尔滨:东北农业大学,2013.DONG B. Construction of eukaryotic expression plasmid of PCV2 ORF2 gene and immune responses in mice immunized with porcine IL-15[D]. Harbin:Northeast Agricultural University,2013. (in Chinese)
[27]	石娜. 猪IL-15与PRRSV ORF5基因真核表达质粒的构建及其免疫原性分析[D]. 哈尔滨:东北农业大学,2010.SHI N. Construction of eukaryotic expression plasmids of porcine IL-15 and PRRSV ORF5 gene and its immunogenicity analysis[D]. Harbin:Northeast Agricultural University,2010. (in Chinese)
[28]	梁霄勇.牛病毒性腹泻病毒的分离鉴定及其E0核酸疫苗与细胞因子佐剂联合免疫研究[D].乌鲁木齐:新疆农业大学,2016.LIANG X Y. The Research on Combined Immunization with DNA Vaccine against bovine viral diarrhea virus and bovine cytokine genes[D].Urumqi:Xinjiang Agricultural University,2016. (in Chinese)
[29]	TOUSSAINT J F,COEN L,LETELLIER C,et al. Genetic immunisation of cattle against bovine herpesvirus 1:glycoprotein gD confers higher protection than glycoprotein gC or tegument protein VP8[J]. Vet Res,2005,36(4):529-544.
[30]	仝晓丹.牛传染性鼻气管炎病毒多表位嵌合蛋白表达及免疫原性评价[D].大庆:黑龙江八一农垦大学,2019.TONG X D. Expression of recombinant multiple-epitope chimeric protein of IBRV and immunogenic analysis[D]. Daqing:Heilongjiang Bayi Agricultural University,2019. (in Chinese)