猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.04.023

摘要/Abstract

摘要： 【目的】研究旨在对猫细小病毒(Feline panleukopenia virus,FPV)洛阳分离株VP2蛋白进行原核表达和鼠源多克隆抗体制备。【方法】根据已公布的FPV VP2基因序列(GenBank登录号:EU018143.1)设计1对特异性引物,以洛阳地区7例疑似FPV感染猫粪便为样本提取DNA,对VP2基因进行PCR鉴定及测序,利用Mega 11.0软件对VP2基因进行遗传进化分析;应用在线软件预测分离株VP2蛋白结构特征并找出优势抗原表位,克隆VP2优势抗原(sVP2)并连接pET-32a (+)载体,构建pET-32a-sVP2重组质粒,转化大肠杆菌Rosetta (DE3)感受态细胞后进行诱导表达,对重组蛋白表达条件进行优化,并使用SDS-PAGE与Western blotting方法进行鉴定;将pET-32a-sVP2用镍柱亲和层析蛋白纯化后免疫BABL/c小鼠,制备多克隆抗体,采用ELISA法检测抗体效价。【结果】分离得到1株FPV,命名为LY-1。遗传进化分析发现,该毒株属于FPV-G2亚群,与北京株(GenBank登录号:MT270581)亲缘关系最近。生物信息学分析显示,VP2属于稳定膜内蛋白,无信号肽与跨膜区,在N-端具有丰富的B细胞抗原表位(1―800 bp,sVP2)。试验成功构建pET-32a-sVP2重组质粒并表达,SDS-PAGE显示重组蛋白分子质量约50 ku,主要以包涵体形式存在,在30 ℃、1 mmol/L IPTG诱导6 h蛋白表达量最佳;Western blotting结果表明,重组蛋白能与鼠抗His单克隆抗体发生特异性反应;ELISA结果表明,该重组蛋白抗体效价为1∶128 000,能够较好地识别免疫原sVP2和原核表达VP2蛋白并发生特异性反应。【结论】本试验克隆获得FPV sVP2片段,并制备多克隆抗体,为进一步研究VP2在FPV复制和致病过程中的作用及建立诊断FPV方法提供参考。

关键词: 猫细小病毒(FPV); 洛阳分离株; VP2基因; 原核表达; 多克隆抗体

Abstract: 【Objective】 The aim of this study was to prokaryotically express VP2 protein of Luoyang Feline panleukopenia virus (FPV) isolates and prepare polyclonal antibodies from mice.【Method】 A pair of specific primers were designed according to the published FPV VP2 gene sequence (GenBank accession No.:EU018143.1),DNA was extracted from the feces of 7 cats suspected of FPV infection in Luoyang area,and VP2 gene was identified by PCR and sequenced.The genetic evolution of VP2 gene was analyzed by Mega 11.0 software.The online software was used to predict the structure characteristics of VP2 protein and find the dominant epitopes.The VP2 dominant antigen (sVP2) was cloned into pET-32a(+) vector,the recombinant plasmid pET-32a-sVP2 was constructed and transformed into E.coli Rosetta (DE3) competent cell for induction and optimal expression.The recombinant protein was identified by SDS-PAGE and Western blotting.BABL/c mice were immunized with pET-32a-sVP2 purified by nickel column affinity chromatography to prepare polyclonal antibody,and the titer of the antibody was detected by ELISA method.【Result】 A FPV strain was isolated and named LY-1.Genetic evolution analysis showed that this strain belonged to FPV-G2 subgroup,which was the closest relative to Beijing strain (GenBank accession No.:MT270581).Bioinformatics analysis showed that VP2 was a stable intramembrane protein with no signal peptide and transmembrane region,and there were abundant B cell epitopes (1-800 bp,sVP2) at the N-terminus.The recombinant plasmid pET-32a-sVP2 was successfully constructed and expressed.SDS-PAGE showed that the molecular mass of the recombinant protein was about 50 ku,mainly in the form of inclusion bodies,the optimal protein expression was induced by 1 mmol/L IPTG at 30 ℃ for 6 h;Western blotting results showed that the recombinant protein could react specifically with mouse anti-His monoclonal antibody.ELISA results showed that the antibody titer of the recombinant protein was 1∶128 000.The antibody could recognize the specific reaction between sVP2 and prokaryotic expression of VP2 protein.【Conclusion】 FPV sVP2 fragment was cloned and polyclonal antibody was prepared,which provided a reference for further study of the role of VP2 in FPV replication and pathogenesis and establishing a diagnostic method for FPV.

Key words: Feline panleukopenia virus (FPV); Luoyang isolate; VP2 gene; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺9.2

王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.

WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.

参考文献

[1] YI S,LIU S,MENG X,et al.Feline panleukopenia virus with G299E substitution in the VP2 protein first identified from a captive giant panda in China[J].Frontiers in Cellular and Infection Microbiology,2021,11:820144.
[2] 周群,杨苑,宋鑫,等.2017-2021年成都市猫细小病毒的检测及其VP2基因的变异分析[J].畜牧兽医学报,2022,53(4):1303-1309. ZHOU Q,YANG Y,SONG X,et al.Detection of Feline parvovirus and mutation analysis of VP2 gene in Chengdu from 2017 to 2021[J].Acta Veterinaria et Zooechnica Sinica,2022,53(4):1303-1309.(in Chinese)
[3] MAJUMDER K,BOFTSI M,WHITTLE F B,et al.The NS1 protein of the Parvovirus MVM aids in the localization of the viral genome to cellular sites of DNA damage[J].PLoS Pathogens,2020,16(10):e1009002.
[4] XU P,ZHOU Z,XIONG M,et al.Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway[J].PLoS Pathogens,2017,13(3):e1006266.
[5] ZHANG J,FAN J,LI Y,et al.Porcine parvovirus infection causes pig placenta tissue damage involving nonstructural protein 1 (NS1)-induced intrinsic ROS/mitochondria-mediated apoptosis[J].Viruses,2019,11(4):389.
[6] MILLER C L,PINTEL D J.Interaction between Parvovirus NS2 protein and nuclear export factor Crm1 is important for viral egress from the nucleus of murine cells[J].Journal of Virology,2002,76(7):3257-3266.
[7] KANG H,LIU D,TIAN J,et al.Feline panleucopenia virus NS2 suppresses the host IFN-beta induction by disrupting the interaction between TBK1 and STING[J].Viruses,2017,9(1):23.
[8] LABADIE T,GARCIA D,MUTUEL D,et al.Capsid proteins are necessary for replication of a Parvovirus[J].Journal of Virology,2021,95(17):e0052321.
[9] STRECK A F,CANAL C W,TRUYEN U.Viral fitness and antigenic determinants of Porcine parvovirus at the amino acid level of the capsid protein[J].Journal of Virology,2022,96(2):e0119821.
[10] WU H,JIN H,WANG L,et al.Generation and immunogenicity of virus-like particles based on Mink enteritis virus capsid protein VP2 expressed in Sf9 cells[J].Archives of Virology,2020,165(9):2065-2071.
[11] JIAO C,ZHANG H,LIU W,et al.Construction and immunogenicity of virus-like particles of Feline parvovirus from the tiger[J].Viruses,2020,12(3):315.
[12] 张鑫杰,薛少华,吕紫欣,等.闽粤地区PPV7与PCV2的混合感染调查及PPV7 Cap基因的遗传变异分析[J].中国畜牧兽医,2022,49(6):2291-2297. ZHANG X J,XUE S H,LYU Z X,et al.Investigation of co-infection of PPV7 and PCV2 and gene variation analysis for PPV7 Cap gene in Fujian and Guangdong[J].China Animal Husbandry & Veterinary Medicine,2022,49(6):2291-2297.(in Chinese)
[13] 刘莉,顾玲玲,张硕,等.1型鹅星状病毒ORF2蛋白原核表达及其多克隆抗体的制备与鉴定[J].中国畜牧兽医,2022,49(1):368-374. LIU L,GU L L,ZHANG S,et al.Prokaryotic expression of ORF2 protein of Goose astrovirus 1 and preparation and identification of its polyclonal antibody[J].China Animal Husbandry & Veterinary Medicine,2022,49(1):368-374.(in Chinese)
[14] SHAO R,YE C,ZHANG Y,et al.Novel parvovirus in cats,China[J].Virus Research,2021,304:198529.
[15] SHAO L,SHEN W,WANG S,et al.Recent advances in molecular biology of Human bocavirus 1 and its applications[J].Frontiers in Microbiology,2021,12:696604.
[16] SHEN W,WANG Z,NING K,et al.Hairpin transfer-independent Parvovirus DNA replication produces Infectious virus[J].Journal of Virology,2021,95(20):e0110821.
[17] LEE H,CALLAWAY H M,CIFUENTE J O,et al.Transferrin receptor binds virus capsid with dynamic motion[J].Proceedings of the National Academy of Sciences of the United States of America,2019,116(41):20462-20471.
[18] FERNANDES S,BOISVERT M,SZELEI J,et al.Differential replication of two Porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression[J].Journal of General Virology,2014,95(Pt4):910-921.
[19] WANG J,CHEN X,ZHOU Y,et al.Prevalence and characteristics of a Feline parvovirus-like virus in dogs in China[J].Veterinary Microbiology,2022,270:109473.
[20] STRECK A F,CANAL C W,TRUYEN U.Viral fitness and antigenic determinants of Porcine parvovirus at the amino acid level of the capsid protein[J].Journal of Virology,2022,96(2):e0119821.
[21] WANG J,LIU Y,CHEN Y,et al.Capsid assembly is regulated by amino acid residues asparagine 47 and 48 in the VP2 protein of Porcine parvovirus[J].Veterinary Microbiology,2021,253:108974.
[22] 霍娜,姚威,于婉琪,等.大鼠细小病毒VP2原核表达及间接ELISA方法建立[J].中国动物传染病学报,2018,26(6):53-57. HUO N,YAO W,YU W Q,et al.Expression of VP2 gene of Rat parvovirus and development of a recombinant protein-based indirect ELISA method for antibodies detection[J]. Chinese Journal of Animal Infectious Diseases,2018,26(6):53-57.(in Chinese)
[23] 龙丹丹,嵇辛勤,段志强,等.犬细小病毒VP2蛋白的原核表达与纯化[J].中国畜牧兽医,2018,45(3):643-649. LONG D D,JI X Q,DUAN Z Q,et al.Prokaryotic expression and purification of VP2 protein of Canine parvovirus[J]. China Animal Husbandry & Veterinary Medicine,2018,45(3):643-649.(in Chinese)
[24] 王净,王鹏,李刚,等.犬细小病毒VP2截短基因的原核表达及表达蛋白抗原性分析[J].中国兽医学报,2012,32(7):967-970. WANG J,WANG P,LI G,et al.Prokaryotic expression truncated VP2 gene and antigenicity analysis of expressed soluble proteins of Canine parvovirus[J].Chinese Journal of Veterinary Science,2012,32(7):967-970.(in Chinese)