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

doi:10.16431/j.cnki.1671-7236.2022.01.039

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (1): 368-374.doi: 10.16431/j.cnki.1671-7236.2022.01.039

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

刘莉¹, 顾玲玲¹, 张硕², 谢军¹, 朱善元¹, 王安平¹

1. 江苏农牧科技职业学院, 江苏省兽用生物制药高技术研究重点实验室, 泰州 225300;
2. 江苏海洋大学食品科学与工程学院, 连云港 222005

收稿日期:2021-05-28 出版日期:2022-01-05 发布日期:2021-12-29
通讯作者: 王安平 E-mail:wap4017@163.com
作者简介:刘莉,E-mail:vetlily@163.com;顾玲玲,E-mail:546374954@qq.com。
基金资助:
江苏省农业科技自主创新项目（CX（18）1004）；江苏省现代农业产业技术体系集成创新中心（JATS[2020]345）；江苏高校"青蓝工程"苏教师函[2020]10号；江苏省高等学校自然科学研究重大项目（18KJA230001）

Prokaryotic Expression of ORF2 Protein of Goose Astrovirus 1 and Preparation and Identification of Its Polyclonal Antibody

LIU Li¹, GU Lingling¹, ZHANG Shuo², XIE Jun¹, ZHU Shanyuan¹, WANG Anping¹

1. Jiangsu Key Laboratory of Veterinary Bio-pharmaceutical High Technology Research, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China;
2. School of Food Science and Engineering, Jiangsu Ocean University, Lianyungang 222005, China

Received:2021-05-28 Online:2022-01-05 Published:2021-12-29

摘要/Abstract

摘要： [目的] 利用原核表达系统表达1型鹅星状病毒（Goose astrovirus 1，GAstV-1）结构蛋白ORF2，并制备其多克隆抗体。[方法] 根据GAstV-1 TZ03株基因序列，对ORF2基因序列进行大肠杆菌密码子偏爱性优化和合成，将其克隆至原核表达载体pET-30a（+），构建重组质粒pET-ORF2，转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导表达重组蛋白，通过SDS-PAGE和Western blotting鉴定重组蛋白；将鉴定正确的重组蛋白纯化后免疫家兔制备多克隆抗体，以间接免疫荧光试验鉴定多克隆抗体的特异性。[结果] 酶切和测序结果显示，成功获得重组质粒pET-ORF2；SDS-PAGE结果显示，表达的重组蛋白分子质量约为70 ku，主要以包涵体形式存在；Western blotting结果表明，该蛋白能与抗His标签鼠单克隆抗体发生特异性反应。间接ELISA检测制备的兔抗ORF2蛋白多克隆抗体效价达1：128 000；间接免疫荧光试验结果显示，制备的多克隆抗体能与GAstV-1发生特异性反应。[结论] 原核表达的GAstV-1 ORF2重组蛋白具有良好的免疫原性，制备的兔源多克隆抗体具有较高的效价和特异性，为研制GAstV-1相关诊断试剂奠定了基础。

关键词: 1型鹅星状病毒(GAstV-1); ORF2基因; 原核表达; 多克隆抗体

Abstract: [Objective] The purpose of this study was to express the structural protein ORF2 of Goose astrovirus 1 (GAstV-1) by prokaryotic expression system and prepare its polyclonal antibody. [Method] According to the gene sequence of GAstV-1 TZ03 strain, the ORF2 gene sequence was optimized and synthesized. It was cloned into prokaryotic expression vector pET-30a(+). The recombinant plasmid pET-ORF2 was constructed and transformed into E. coli BL21(DE3) competent cells. The recombinant protein was induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting. The polyclonal antibody was prepared by immunizing rabbits with the purified recombinant protein, and the specificity of the polyclonal antibody was identified by indirect immunofluorescence assay (IFA). [Result] The results of enzyme digestion and sequencing showed that the recombinant plasmid pET-ORF2 was successfully obtained. SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 70 ku, mainly in the form of inclusion body. Western blotting showed that the protein could react specifically with mouse monoclonal antibody against His tag. The titer of rabbit polyclonal antibody against ORF2 protein detected by indirect ELISA was 1:128 000. IFA results showed that the polyclonal antibody could react specifically with GAstV-1. [Conclusion] The prokaryotic expression of GAstV-1 ORF2 recombinant protein had good immunogenicity, and the rabbit polyclonal antibody had high titer and specificity, which laid a foundation for the development of GAstV-1 related diagnostic reagents.

Key words: Goose astrovirus 1 (GAstV-1); ORF2 gene; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺7

刘莉, 顾玲玲, 张硕, 谢军, 朱善元, 王安平. 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 Li, GU Lingling, ZHANG Shuo, XIE Jun, ZHU Shanyuan, WANG Anping. Prokaryotic Expression of ORF2 Protein of Goose Astrovirus 1 and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 368-374.

参考文献

[1] MADELEY C R,COSGROVE B P.Letter:Viruses in infantile gastroenteritis[J].Lancet,1975,2(7925):124.
[2] APPLETON H,HIGGINS P G.Letter:Viruses and gastroenteritis in infants[J].Lancet,1975,1(7919):1297.
[3] ZHANG Y X,WANG F M,LIU N,et al.Complete genome sequence of a novel Avastrovirus in goose[J].Archives of Virology,2017,162(7):2135-2139.
[4] BENEDICTIS P D,SCHULTZ-CHERRY S,BURNHAM A,et al.Astrovirus infections in humans and animals-molecular biology,genetic diversity,and interspecies transmissions[J].Infection Genetics and Evolution,2011,11(7):1529-1544.
[5] CORTEZ V,MELIOPOULOS V A,KARLSSON E A,et al.Astrovirus biology and pathogenesis[J].Annual Review of Virology,2017,4(1):327-348.
[6] ZHANG X Y,REN D,LI T F,et al.An emerging novel Goose astrovirus associated with gosling gout disease,China[J].Emergingicrobes and Infections,2018,7(1):152.
[7] YANG J,TIAN J J,TANG Y,et al.Isolation and genomic characterization of goslinggout caused by a novel Goose astrovirus[J].Transboundary and Emerging Diseases,2018,65(6):1689-1696.
[8] LIU N,JIANG M,DONG Y,et al.Genetic characterization of a novel group of Avastroviruses in geese[J].Transboundary and Emerging Diseases,2018,65(4):927-932.
[9] ZHANG Q S,CAO Y X,WANG J,et al.Isolation and characterization of an Astrovirus causing fatal visceral gout in domestic goslings[J].Emerging Microbes and Infections,2018,7(1):71
[10] WAN C H,CHEN C T,CHENG L F,et al.A novel group of Avian astrovirus in domestic geese,China[J].Journal of Veterinary Medical Science,2018,80(5):798-801.
[11] CHEN Q X,XU X,YU Z L,et al.Characterization and genomic analysis of emerging Astroviruses causing fatal gout in goslings[J].Transboundary and Emerging Diseases,2019,67(2):865-876.
[12] WANG A P,ZHANG S,XIE J,et al.Isolation and characterization of a Goose astrovirus 1 strain causing fatal gout in goslings,China[J].Poultry Science,2021,100(11):101432.
[13] 张玉杰,孙宁,刘东,等.鹅星状病毒的分离鉴定及全基因组序列分析[J].畜牧兽医学报,2020,51(11):2765-2777. ZHANG Y J,SUN N,LIU D,et al.Isolation,identification and complete genomic sequence analysis of Goose astrovirus[J].Acta Veterinaria et Zootechnica Sinica,2020,51(11):2765-2777.(in Chinese)
[14] 张玉霞,杨金兴,孟凯,等.鹅星状病毒LAMP快速检测方法的建立与应用[J].中国预防兽医学报,2019,41(11):1137-1140. ZHANG Y X,YANG J X,MENG K,et al.Establishment and application of LAMP method for rapid detection of Goose astrovirus[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(11):1137-1140.(in Chinese)
[15] YUAN X Y,MENG K,ZHANG Y X,et al.Establishment and application of rapid diagnosis for reverse transcription-quantitative PCR of newly emerging goose origin nephrotic Astrovirus in China[J].mSphere,2018,3(6):e00380-18.
[16] 吕炫,张淼,胡增金,等.鹅星状病毒衣壳纤突蛋白VP27多克隆抗体的制备和鉴定[J].江苏农业学报,2021,37(2):412-417. LYU X,ZHANG M,HU Z J,et al.Preparation and identification of polyclonal antibody against the capsid spike protein VP27 of Goose astrovirus[J].Jiangsu Journal of Agriculture Science,2021,37(2):412-417.(in Chinese)
[17] 刘雪婷,王召阳,鑫婷,等.非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定[J].中国畜牧兽医,2021,48(3):991-1000. LIU X T,WANG Z Y,XIN T,et al.Prokaryotic expression and polyclonal antibody preparation and identification of African swine fever virus B438L protein[J].China Animal Husbandry & Veterinary Medicine,2021,48(3):991-1000.(in Chinese)
[18] WU W K,XU R,LV Y J,et al.Goose astrovirus infection affects uric acid production and excretion in goslings[J].Poultry Science,2020,99(4):1967-1974.
[19] XU D W,LI C F,LIU G Q,et al.Generation and evaluation of a recombinant goose origin Newcastle disease virus expressing Cap protein of goose origin Avastrovirus as a bivalent vaccine in goslings[J].Poultry Science,2019,98(10):4426-4432.
[20] ARIAS C F,DUBOIS R M.The Astrovirus capsid:A review[J].Viruses,2017,9(1):15
[21] BIDOKHTI M R M,ULLMAN K,HAMMER A S,et al.Immunogenicity and efficacy evaluation of subunit Astrovirus vaccines[J].Vaccines,2019,7(3):79.
[22] 蒲路莎,苏世博,陈肖韩,等.鹅源星状病毒ORF2基因原核表达及遗传进化分析[J].浙江农业学报,2020,32(5):789-796. PU L S,SU S B,CHEN X H,et al.Prokaryotic expression and phylogenetic analysis of ORF2 gene of Goose astrovirus[J].Acta Agriculture Zhejiangensis,2020,32(5):789-796.(in Chinese)
[23] 孙佳卉,刘志艺,黄聪,等.抗鹅星状病毒Cap蛋白单克隆抗体的制备及鉴定[J].中国动物传染病学报,2021,29(1):58-63. SUN J H,LIU Z Y,HUANG C,et al.Preparation and identification of monoclonal antibodies against Cap protein of Goose astrovirus[J].Chinese Journal of Animal Infectious Diseases,2021,29(1):58-63.(in Chinese)

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

Prokaryotic Expression of ORF2 Protein of Goose Astrovirus 1 and Preparation and Identification of Its Polyclonal Antibody

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