Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody

doi:10.15933/j.cnki.1004-3268.2021.08.018

Journal of Henan Agricultural Sciences ›› 2021, Vol. 50 ›› Issue (8): 154-159.DOI: 10.15933/j.cnki.1004-3268.2021.08.018

Special Issue: 非洲猪瘟专题

• Animal Science Veterinary Medicine • Previous Articles Next Articles

Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody

GENG Xiaolin^1,2，SUN Jie³，WANG Yanwei^1,2，HUANG Tian³，LIU Peng^1,2，CAO Hongmei³，PANG Wenqiang^1,2，HAO Liying³，DENG Junhua³，HUANG Yuxin^1,2，TIAN Kegong^1,2

（1.National Research Center for Veterinary Medicine，Luoyang 471000，China；2.Publike Bioengineering Co.，Ltd.，Luoyang 471000，China；3.Luoyang Putai Biotechnology Co.，Ltd.，Luoyang 471000，China）

Received:2021-01-21 Published:2021-08-15 Online:2021-09-17

非洲猪瘟病毒K145R 蛋白的原核表达及其单克隆抗体制备

耿笑林^1,2，孙杰³，王彦伟^1,2，黄甜³，刘鹏^1,2，曹红梅³，逄文强^1,2，郝丽影³，邓均华³，黄玉欣^1,2，田克恭^1,2,3

（1.国家兽用药品工程技术研究中心，河南洛阳471000；2.普莱柯生物工程股份有限公司，河南洛阳471000；3.洛阳普泰生物技术有限公司，河南洛阳471000）

通讯作者: 黄玉欣（1974-），女，河南鲁山人，兽医师，主要从事微生物发酵技术研究。E-mail：nvchyx@163.com 田克恭（1964-），男，山西万荣人，教授，博士，主要从事动物疫病诊断与防控技术研究。E-mail：vetvac@126.com
作者简介:耿笑林（1985-），男，河南洛阳人，兽医师，硕士，主要从事微生物发酵工艺研究。E-mail：gengxiaolin1017@163.com 孙杰为同等贡献作者
基金资助:
洛阳市重大科技专项（1901029A）

Abstract

Abstract: In order to develop an immunodiagnostic reagent for African swine fever virus（ASFV），the recombinant ASFV K145R protein was expressed by E. coli system，and the monoclonal antibody against K145R protein was prepared and identified. The results showed that the pET28a‑K145R expression vector was successfully constructed，and the soluble recombinant protein of K145R with the size of about 17 ku was obtained. Western blot analysis confirmed that recombinant K145R protein had a good
reactivity with ASFV positive serum. A monoclonal antibody 1D4 was obtained by cell fusion and screening. The titer was 1∶5 120 000 by ELISA. The heavy chain was identified as IgG2a and the light chain as κ. Western blot analysis showed that it could specifically recognize recombinant K145R protein.IFA test showed that it could react with ASFV. In summary，the soluble expression of K145R protein in E.coli and the preparation of monoclonal antibody were achieved.

Key words: African swine fever virus, K145R protein, Prokaryotic expression, Western blot identification, Monoclonal antibody

摘要： 为研发非洲猪瘟病毒（ASFV）的免疫诊断学试剂，利用大肠杆菌系统表达ASFV重组K145R蛋白，制备并鉴定K145R蛋白单克隆抗体。结果显示，成功构建pET28a-K145R表达载体，表达得到大小约为17 ku可溶性的重组K145R蛋白。Western blot分析证实，重组K145R蛋白与ASFV阳性血清有良好的反应性。经细胞融合、筛选，得到单克隆抗体1D4；ELISA检测其效价为1∶5 120 000；亚类鉴定其重链为IgG2a，轻链为κ；Western blot鉴定其能特异性识别重组K145R蛋白；IFA检测其能与ASFV反应。综上，实现K145R蛋白在大肠杆菌中的可溶性表达并制备单克隆抗体。

关键词: 非洲猪瘟病毒, K145R蛋白, 原核表达, Western blot鉴定, 单克隆抗体

CLC Number:

S855.3

GENG Xiaolin, SUN Jie, WANG Yanwei, HUANG Tian, LIU Peng, CAO Hongmei, PANG Wenqiang, HAO Liying, DENG Junhua, HUANG Yuxin, TIAN Kegong. Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody[J]. Journal of Henan Agricultural Sciences, 2021, 50(8): 154-159.

耿笑林, 孙杰, 王彦伟, 黄甜, 刘鹏, 曹红梅, 逄文强, 郝丽影, 邓均华, 黄玉欣, 田克恭. 非洲猪瘟病毒K145R 蛋白的原核表达及其单克隆抗体制备[J]. 河南农业科学, 2021, 50(8): 154-159.

Figures/Tables 5

References

［1］ COSTARD S，MUR L，LUBEOTH J，et al.Epidemiology of African swine fever virus［J］.Virus Res，2013，173（1）：191‑197.
［2］贾云飞，赵福杰，朱静静，等.非洲猪瘟病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立［J］.河南农业大学学报，2020，54（1）：69‑73.
JIA Y F，ZHAO F J，ZHU J J，et al.Development of a SYBR Green Ⅰ based real‑time quantitative PCR method for the detection of African swine fever virus［J］.Journal of Henan Agricultural University，2020，54（1）：69‑73.
［3］ DI A V，NETHERTON C L，DIXON L K，et al.African swine fever virus strain Georgia 2007/1 in ornithodoros erraticus ticks［J］.Emerg Infect Dis，2012，18（6）：1026‑1028.
［4］王清华，任炜杰，包静月，等.我国首例非洲猪瘟的确诊［J］.中国动物检疫，2018，35（9）：1‑4.
WANG Q H，REN W J，BAO J Y，et al.The first outbreak of African swine fever was confirmed in China［J］.China Journal of Animal Quarantine，2018，35（9）：1‑4.
［5］陈腾，张守峰，周鑫韬，等.我国首次非洲猪瘟疫情的发现和流行分析［J］.中国兽医学报，2018，38（9）：1831‑1832.
CHEN T，ZHANG S F，ZHOU X T，et al.The discovery and epidemic analysis of the first African swine fever epidemic in China［J］.Chinese Journal of Veterinary Medicine，2018，38（9）：1831‑1832.
［6］ SALAS M L，ANDRES G.African swine fever virus morphogenesis［J］.Virus Res，2013，173（1）：29‑41.
［7］ DIXON L K，CHAPMAN D，NETHERTON C L，et al.African swine fever virus replication and genomics［J］.Virus Res，2013，173（1）：3‑14.
［8］ DIXON L K，TWIGG S R，BAVLIS S A，et al.Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate （Malawi LIL20/1）［J］.J Gen Virol，1994，75（7）：1655‑1684.
［9］ YANEZ R J，RODRIGUEZ J M，NOGAL M L，et al.Analysis of the complete nucleotide sequence of African swine fever virus［J］.Virology，1995，208（1）：249‑278.
［10］ ZSAK L，LU Z，KUTISH G F，et al.An African swine fever virus virulence‑associated gene NL-S with similarity to the herpes simplex virus ICP34. 5 gene［J］.J Virol，1996，70（12）：8865‑8871.
［11］ ZASK L，CALER E，LU Z，et al.A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine［J］.J Virol，1998，72（2）：1028‑1035.
［12］ CARRASCOSA A L，VAL M D，SANTAREN J F，et al.Purification and properties of African swine fever virus［J］.J Virol，1985，54（2）：337‑344.
［13］ ESTEVES A， MARQUES M I， COSTA J V.Two‑dimensional analysis of African swine fever virus proteins and proteins induced in infected cells［J］.Virology，1986，152（1）：192‑206.
［14］ ALEJO A，MATAMOROS T，GUERRA M，et al.Aproteomic atlas of the African swine fever virus particle［J］.J Virol，2018，12，92（23）：1293‑1310.
［15］ KEIL G M，GIESOW K，PORTIGAL R.A novel bromodeoxyuridine‑resistantwild boar lung cell line facilitates generation of African swine fever virus recombinants ［J］.Arch Virol， 2014， 159 （9）：2421‑2428.
［16］ SANCHEZ E G，RIERA E，NOGAL M，et al.Phenotyping and susceptibility of established porcine cells lines to African swine fever virus infection and viral production［J］.Sci Rep，2017，7（1）：10369.
［17］ KEBLER C，FORTH J H，KEIL G M，et al.The intracellular proteome of African swine fever virus［J］.Sci Rep，2018，8（1）：14714.
［18］ JAING C，ROWLAND R，ALLEN J E，et al.Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses［J］.Sci Rep，2017，7（1）：10115.
［19］张素玲，吴芃，岳亚男，等.非洲猪瘟病毒MGF505-5R蛋白表达与纯化［J］.河南农业科学，2020，49（10）：130‑136.
ZHANG S L，WU P，YUE Y N，et al.Expression and purification of MGF505‑5R protein of African swine fever virus［J］.Journal of Henan Agricultural Sciences，2020，49（10）：130‑136.
［20］郝丽影，王彦伟，孙玉洁，等.非洲猪瘟病毒P30蛋白单克隆抗体的制备与应用［J］.河南农业科学，2020，49（10）：124‑129.
HAO L Y，WANG Y W，SUN Y J，et al.Preparation and application of monoclonal antibodies against P30 protein
of African swine fever virus［J］.Journal of Henan Agricultural Sciences，2020，49（10）：124‑129.
［21］白晶晶，宋欢欢，白晨雨，等.非洲猪瘟病毒p17蛋白单克隆抗体的制备与鉴定［J］.河南农业科学，2020，49（12）：137‑143.
BAI J J，SONG H H，BAI C Y，et al.Preparation and identification of monoclonal antibodies against African swine fever virus p17 protein［J］.Journal of Henan Agricultural Sciences，2020，49（12）：137‑143.
［22］ ALEXANDAR H，KELER C，PANNHORST K，et al.Identification and characterization of the 285L and K145R proteins of African swine fever virus［J］.J Gen Virol，2019，100（9）：1303‑1314.
［23］ RODRIGUEZ J M，MORENO L T，ALEJO A，et al.Genome sequence of African swine fever virus BA71，the virulent parental strain of the nonpathogenic and tissue‑culture adapted BA71V［J］.PLoS One，2015，10（11）：e0142889.
［24］ NATALIA M P，MAREKW，MALGORZATA J，et al.The spillover of African swine fever in western Poland revealed its estimated origin on the basis of O174L，K145R，MGF 505-5R and IGR I73R/I329L genomic sequences［J］.Viruses，2020，12（10）：1094.

[1]	CHEN Tianmeng, CAI Tongxuan, WANG Jialu, TIAN Muye, ZHAO Baohua, LIU Dong. Expression and Bioinformatics Analysis of Aeromonas hydrophila Outer Membrane TolB Protein [J]. Journal of Henan Agricultural Sciences, 2024, 53(2): 136-143.
[2]	XU Jia, SUN Huaichang. Prokaryotic Expression of Vsp Protein and Preparation of Monoclonal Antibody against Mycoplasma bovis [J]. Journal of Henan Agricultural Sciences, 2023, 52(12): 142-148.
[3]	LU Qingjun, LUO Zhuxing, LIU Huawei, WEI Meng, WANG Yuan, ZHANG Zhaofeng, LI Youshan. Cloning，Sequence Analysis and Prokaryotic Expression of Three Spliceosomes of Abd‑A from Silk Gland of Bombyx mori [J]. Journal of Henan Agricultural Sciences, 2022, 51(4): 138-150.
[4]	JIAN Sijie, CHAO Jia, SUN Wei, CHEN Rui, DING Rui, CHEN Chen, LIU Xiang. Prokaryotic Expression，Purification and Immunogenicity Study of Outer Membrane Protein AHA1182 of Aeromonas hydrophila [J]. Journal of Henan Agricultural Sciences, 2022, 51(11): 135-144.
[5]	WANG Xun, ZHANG Yuhang, SUN Yaning, LI Qingmei, LI Ge, WANG Li, GUO Junqing, DENG Ruiguang, ZHANG Gaiping. Expression and Purification of CRISPR/Cas13a and Establishment of Its Collateral RNase Activity Assay [J]. Journal of Henan Agricultural Sciences, 2021, 50(4): 147-153.
[6]	RONG Na, JIAN Sijie, SUN Wei, KANG Chao, CHEN Chen, LIU Xiang. Prokaryotic Expression and Antigenicity Analysis of Outer Membrane Protein TolC of Fish Pathogenic Aeromonas hydrophila [J]. Journal of Henan Agricultural Sciences, 2021, 50(3): 157-164.
[7]	TAN Zhengwei, LI Lei, YU Yongliang, XU Lanjie, YANG Hongqi, DONG Wei, LU Dandan, MA Xinming, LIANG Huizhen. Cloning and Expression Analysis of CtDXS1 Encoding 1-Deoxy-D-Xylulose-5-Phosphate Synthase Gene in Carthamus tinctorius L. [J]. Journal of Henan Agricultural Sciences, 2021, 50(2): 39-49.
[8]	YAN Shijun, HAO Liying, BAI Lulu, ZHENG Dingding, SONG Huanhuan, LI Shengqiang, WANG Tongyan, TAN Feifei, DENG Junhua, TIAN Kegong. Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus [J]. Journal of Henan Agricultural Sciences, 2021, 50(12): 149-154.
[9]	LIU Yanyang, WU Ke, MEI Hongxian, DU Zhenwei, CUI Chengqi, JIANG Xiaolin, ZHENG Yongzhan. Cloning and Prokaryotic Expression of Capsule Indehiscent Gene SiIND1 from Sesame [J]. Journal of Henan Agricultural Sciences, 2020, 49(5): 63-68.
[10]	WANG Yu, WANG Zhaoguang, LIU Zejun, XIA Zhongxi, FAN Yuding, ZHONG Qiwang. Study on Expression and Cell Localization of atrogin-1 Protein in Monopterus albus [J]. Journal of Henan Agricultural Sciences, 2020, 49(5): 154-160.
[11]	BAI Jingjing, SONG Huanhuan, BAI Chenyu, HAO Liying, YAN Shijun, DU Mengmeng, LI Xiangdong, DENG Junhua, TIAN Kegong. Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein [J]. Journal of Henan Agricultural Sciences, 2020, 49(12): 137-143.
[12]	LIAN Kaiqi, ZHOU Lingling, ZHANG Mingliang, ZHANG Man, WANG Yingjie, LIN Zhengdan, SONG Yuwei. Prokaryotic Expression and Purification of Seneca Valley Virus 3ABC Protein and Preparation of Its Polyclonal Antibodies [J]. Journal of Henan Agricultural Sciences, 2020, 49(1): 123-127.
[13]	ZHANG Xian, LI Chaolin, ZHENG Qiaomu, YAN Zicheng, LIAO Hai, ZHOU Jiayu. Cloning and Expression Analysis of Chymotrypsin Gene PrCT1 of Pieris rapae [J]. Journal of Henan Agricultural Sciences, 2019, 48(9): 74-81.

Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody

非洲猪瘟病毒K145R 蛋白的原核表达及其单克隆抗体制备

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References

Related Articles 13

Recommended Articles

Metrics