猪圆环病毒3型四川株Cap蛋白的截短表达及其抗体间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2024.07.030

摘要/Abstract

摘要： 【目的】原核截短表达猪圆环病毒3型(Porcine circovirus type 3,PCV3)四川株Cap蛋白(1－197位氨基酸),并建立其抗体间接ELISA检测方法,为PCV3血清学调查提供材料。【方法】以PCV3四川分离株基因组为模板,PCR扩增获得Cap蛋白编码基因片段,将其克隆至原核表达载体pET-30a(＋)中构建重组质粒pET30a-PCV3-Cap。经酶切和测序鉴定后,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,Ni-NTA树脂亲和层析纯化,获得具有良好抗原性的重组Cap蛋白。基于纯化的重组Cap蛋白建立间接ELISA方法,确定最佳抗原包被浓度、待检血清最佳稀释比例、酶标抗体稀释比例和阴阳性临界值,对间接ELISA方法的特异性、敏感性、重复性、相关性和符合率进行分析,并对2018－2022年采自川东北地区猪场的351份临床猪血清进行检测,分析该区域PCV3流行情况。【结果】试验成功构建pET30a-PCV3-Cap重组表达载体,实现重组Cap蛋白(1－197位氨基酸)的原核截短表达,蛋白大小约为26 ku,可与PCV3阳性猪血清发生特异性反应;确定最佳抗原包被浓度为1 ng/μL,待检血清最佳稀释比例为1∶100,酶标抗体稀释比例为1∶60 000,阴阳性临界值D_{450 nm}为0.684;建立的间接ELISA方法与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、日本乙型脑炎病毒(JEV)和猪细小病毒1型(PPV1)阳性猪血清均不发生交叉反应,敏感度达1∶1 600,批内变异系数和批间变异系数均＜10%,与病毒中和试验(VNT)结果呈正相关,与Western blotting方法的阳性符合率为95.8%;2018－2022年川东北地区的351份猪血清样品阳性率为7.12%。【结论】本研究成功截短表达了PCV3四川株Cap蛋白(1－197位氨基酸),建立了特异性强、灵敏度高、重复性好和符合率高的PCV3间接ELISA方法,为PCV3的血清学调查和检测试剂盒的开发提供了材料。

关键词: 猪圆环病毒3型(PCV3); Cap蛋白; 截短表达; ELISA

Abstract: 【Objective】 This study was aimed to express truncatly Cap protein (1-197 amino acids) of Porcine circovirus type 3 (PCV3) Sichuan strain in prokaryotic expression system,and develop an indirect ELISA (i-ELISA) method for rapid detection of PCV3 antibody,and provide material for serological investigation of PCV3.【Method】 The genome of PCV3 Sichuan strain was used as template.The Cap protein coding gene of PCV3 was obtained by PCR amplification and inserted into the prokaryotic expression vector pET-30a(＋) to construct the recombinant expression plasmid pET30a-PCV3-Cap.The recombined plasmid pET30a-PCV3-Cap was expressed in E.coli BL21 (DE3) competent cells and was induced by IPTG.The recombinant Cap protein with good antigenicity was purified by Ni-NTA,and analyzed by SDS-PAGE and Western blotting.The i-ELISA was developed using the recombinant Cap protein as coating antigen.The optimal concentration of antigen,the optimal dilution ratio of serum to be tested,the dilution ratio of enzyme-labeled antibody and the threshold of negative and positive were determined,and the specificity,sensitivity,repeatability,correlation and coincidence rate of the i-ELISA were analyzed.The established i-ELISA method was used to detect 351 pig serum samples collected from pig farms in Northeast Sichuan from 2018 to 2022 to analyze the prevalence of PCV3 in the region.【Result】 The recombinant expression plasmid pET30a-PCV3-Cap was successfully obtained,and the recombinant Cap protein (1-197 amino acids) was successfully expressed in E.coli BL21(DE3) competent cells could react specifically with positive pig serum of PCV3 with a relative molecular weight of 26 ku.The coating concentration of Cap protein was 1 ng/μL,and the detection positive threshold was D_{450 nm}＞0.684.The dilution concentration of serum and HRP-rabbit anti-pig IgG were 1∶100 and 1∶60 000,respectively.The i-ELISA was not cross reaction with positive sera of CSFV,PRRSV,PCV2,PRV,JEV and PPV1.The sensitivity of i-ELISA was 1∶1 600.The method was of high duplicability with less than 10% variation of intra-and inter-assay coefficients.The result of i-ELISA was positive correlation with that of virus neutralization test,and the coincidence rate with Western blotting method was 95.8%.The positive rate of PCV3 antibody was 7.12% in 351 pig serum samples collected in Northeast Sichuan from 2018 to 2022.【Conclusion】 This experiment successfully truncated expression Cap protein (1-197 amino acids) and established i-ELISA of PCV3 with better specificity,sensitivity,repeatability and coincidence rate,which provided material for serological investigation and test kit development of PCV3.

Key words: Porcine circovirus type 3 (PCV3); Cap protein; truncated expression; ELISA

中图分类号:

S852.65⁺1

欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 翟佳佳, 张杰. 猪圆环病毒3型四川株Cap蛋白的截短表达及其抗体间接ELISA方法的建立[J]. 中国畜牧兽医, 2024, 51(7): 3056-3066.

OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHAI Jiajia, ZHANG Jie. Truncated Expression of Cap Protein of Porcine Circovirus Type 3 and Establishment of Indirect ELISA Method for Its Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 3056-3066.

参考文献

[1] 殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997. YIN Z,LIU J H.Animal Virology[M].2nd ed.Beijing:Science Press,1997.(in Chinese)
[2] 韦平,秦爱建.重要动物病毒分子生物学[M].北京:科学出版社,2008. WEI P,QIN A J.Molecular Biology of Important Animal Viruses[M].Beijing:Science Press,2008.(in Chinese)
[3] HA Z,LI J,XIE C,et al.Prevalence,pathogenesis,and evolution of Porcine circovirus type 3 in China from 2016 to 2019[J].Veterinary Microbiology,2020,247:108756.
[4] JIA Y,ZHU Q,XU T,et al.Detection and genetic characteristics of Porcine circovirus type 2 and 3 in Henan province of China[J].Molecular and Cellular Probes,2022,61:101790.
[5] SOMASHEKARA S,RAVISHANKAR C,RAVINDRAN R,et al.Molecular characterization and phylogenetic analysis of Porcine circovirus 2 from Kerala,India[J].Virus Disease,2023,34(2):331-338.
[6] QI S,SU M,GUO D,et al.Molecular detection and phylogenetic analysis of Porcine circovirus type 3 in 21 provinces of China during 2015-2017[J].Transboundary and Emerging Diseases,2019,66:1004-1015.
[7] 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PPV7Cap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 genetic variation analysis for PPV7Cap gene in Fujian and Guangdong[J].China Animal Husbandry&Veterinary Medicine,2022,49(6):2291-2297.(in Chinese)
[8] DEKA D,BARMAN N N,DEKA N,et al.Sero-epidemiology of Porcine parvovirus,Circovirus,and Classical swine fever virus infections in India[J].Tropical Animal Health and Production,2021,53(1):180.
[9] PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novel Circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virology Journal,2016,13(1):184.
[10] ASSAO V S,SANTOS M R,PEREIRA C E R,et al.Porcine circovirus 3 in North and South America:Epidemiology and genetic diversity[J].Transboundary and Emerging Diseases,2021,68(6):2949-2956.
[11] SAVIC B,MILICEVIC V,RADANOVIC O,et al.Identification and genetic characterization of Porcine circovirus 3 on pig farms in Serbia[J].Archives of Virology,2020,165(1):193-199.
[12] PARTHIBAN S,RAMESH A,RAJ G D,et al.Molecular evidence of Porcine circovirus 3 infection in swine:First report in Southern India[J].Virus Disease,2022,33(3):284-290.
[13] SANTO A C D,CEZARIO K C,BENNEMANN P E,et al.Full-genome sequences of Porcine circovirus 3(PCV3) and high prevalence in mummified fetuses from commercial farms in Brazil[J].Microbial Pathogenesis,2020,141:104027.
[14] CHEN D J,HUANG Y,GUO Y T,et al.Prevalence and evolution analysis of Porcine circovirus 3 in China from 2018 to 2022[J].Animals,2022,12:1588.
[15] WANG M,YU Y,WU J,et al.Genetic and in vitro characteristics of a Porcine circovirus type 3 isolate from Northeast China[J].Veterinary Science,2023,10:517.
[16] JIANG M,GUO J,ZHANG G,et al.Fine mapping of linear B cell epitopes on capsid protein of Porcine circovirus 3[J].Applied Microbiology and Biotechnology,2020,104(14):6223-6234.
[17] 欧云文,代军飞,马炳,等.猪圆环病毒2型Cap蛋白多克隆抗体的制备及鉴定[J].生物技术通报,2019,35(8):226-231. OU Y W,DAI J F,MA B,et al.Preparation and identification of polyclonal antibody against the Cap protein of Porcine circovirus type 2[J].Biotechnology Bulletin,2019,35(8):226-231.(in Chinese)
[18] BI M,LI X,ZHAI W,et al.Structural insight into the type-specific epitope of Porcine circovirus type 3[J].Bioscience Reports,2020,40(6):1109.
[19] 周一媚,董婉玉,王馨雨,等.基于猪流行性腹泻病毒基因Ⅱ型毒株S蛋白中和表位的间接ELISA和病毒中和试验的相关性分析[J].农业生物技术学报,2022,30(12):2464-2472. ZHOU Y M,DONG W Y,WANG X Y,et al.Correlation of indirect ELISA based on the neutralizing epitopes on the S protein of Porcine epidemic diarrhea virus genotype Ⅱ strain with virus neutralization assay[J].Journal of Agricultural Biotechnology,2022,30(12):2464-2472.(in Chinese)
[20] WANG W,SUN W,CAO L,et al.An epidemiological investigation of Porcine circovirus 3 infection in cattle in Shandong province,China[J].BMC Veterinary Research,2019,15:60.
[21] ZHANG J,LIU Z,ZOU Y,et al.First molecular detection of Porcine circovirus type 3 in dogs in China[J].Virus Genes,2017,54(1):140-144.
[22] HA Z,LI J F,XIE C Z,et al.First detection and genomic characterization of Porcine circovirus 3 in mosquitoes from pig farms in China[J].Veterinary Microbiology,2020,240:108522.
[23] 吕志凯,张伟超,陈永林,等.PCV3自然感染仔猪病例的病理学观察及体内分布[J].中国兽医学报,2022,42(3):421-425. LYU Z K,ZHANG W C,CHEN Y L,et al.Pathological observation of naturally infected piglets with PCV3 and in distribution[J].Chinese Journal of Veterinary Science,2022,42(3):421-425.(in Chinese)
[24] 库旭钢,于学祥,孙琪,等.猪圆环病毒3型的分离及致病性分析[J].畜牧兽医学报,2023,54(4):1568-1578. KU X G,YU X X,SUN Q,et al.Isolation and pathogenicity analysis of Porcine circovirus type 3[J].Acta Veterinaria et Zootechnica Sinica,2023,54(4):1568-1578.(in Chinese)
[25] 湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084. ZHAN Y,WANG D L,WANG N D,et al.Survey on detection and analyses of Cap antigenicity prediction of Porcine circovirus type 3 isolated from partial provinces of Southern China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(6):1076-1084.(in Chinese)
[26] 李周勉,邹亚文,蒋一凡,等.猪圆环病毒3型Cap蛋白序列的生物信息学研究[J].经济动物学报,2020,24(1):9-16. LI Z M,ZOU Y W,JIANG Y F,et al.Bioinformatics analysis of Cap protein sequences of Porcine circovirus type 3[J].Journal of Economic Animal,2020,24(1):9-16.(in Chinese)
[27] 邓家华,吕其壮,陈艳,等.猪圆环病毒3型分子生物学研究进展[J].畜牧与兽医,2019,51(9):125-128. DENG J H,LYU Q Z,CHEN Y,et al.Progress in researches on the molecular biology of Porcine circovirus type 3[J].Animal Husbandry&Veterinary Medicine,2019,51(9):125-128.(in Chinese)
[28] 郭钱菊,查云峰,陈永杰,等.基于截短型Cap蛋白的猪圆环病毒3型间接ELISA检测方法的建立[J].中国兽医科学,2021,51(8):970-975. GUO Q J,ZHA Y F,CHEN Y J,et al.Establishment of an indirect ELISA for the detection of Porcine circovirus type 3 based on truncated Cap protein[J].Chinese Veterinary Science,2021,51(8):970-975.(in Chinese)
[29] WANG Y,WANG G,DUAN W D,et al.Self-assembly into virus-like particles of the recombinant capsid protein of Porcine circovirus type 3 and its application on antibodies detection[J].AMB Express,2020,10:3.
[30] 郭影成,付守鹏,高学勇,等.吉林省猪圆环病毒3型的分子流行病学调查及遗传演化分析[J].中国畜牧兽医,2019,46(5):1483-1490. GUO Y C,FU S P,GAO X Y,et al.Molecular epidemiological investigation and genetic evolution analysis of Porcine circovirus type 3 in Jilin province[J].China Animal Husbandry&Veterinary Medicine,2019,46(5):1483-1490.(in Chinese)
[31] 段群棚,赵硕,秦毅斌,等.广西猪圆环病毒3型流行病学调查与分析[J].中国畜牧兽医,2020,47(4):1183-1190. DUAN Q P,ZHAO S,QIN Y B,et al.Epidemiological investigation and analysis of Porcine circovirus type 3 in Guangxi[J].China Animal Husbandry&Veterinary Medicine,2020,47(4):1183-1190.(in Chinese)