非洲猪瘟病毒K196R和A240L蛋白的可溶性表达及酶活力分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0216

摘要/Abstract

摘要：

旨在提供可用候选抗原蛋白,用于非洲猪瘟病毒（African swine fever virus,ASFV）的诊断及免疫预防研究。将ASFV SY18株K196R和A240L蛋白的编码基因进行密码子优化,并在其N端添加促溶标签进行融合表达;TEV酶切后利用亲和层析纯化目的蛋白,并通过Western blot和酶活检测试剂盒对其反应性和体外酶活力进行鉴定。结果显示,K196R和A240L蛋白分别通过添加SUMO和MBP标签实现了可溶性表达,标签去除后蛋白大小分别为22 kD和27 kD左右;Western blot结果显示,K196R和A240L蛋白均能够与ASF阳性血清发生较好的特异性反应;酶活检测结果表明,二者均具有一定的体外酶催化活性。利用大肠杆菌表达的ASFV K196R和A240L可溶性蛋白,具有较好的反应性和一定的体外酶催化活性,可为ASFV的诊断及蛋白的相关功能研究提供一定的依据。

关键词: 非洲猪瘟, K196R蛋白, A240L蛋白, 可溶性表达, Western blot鉴定, 酶活力分析

Abstract:

The objective of this work is to provide effective antigens for the clinical diagnosis and immunoprophylaxis of African swine fever virus（ASFV）. K196R and A240L coding sequences derived from ASFV SY18 were executed codon optimization and co-expression with soluble-tag in their N terminals. The target proteins were purified by affinity chromatography after TEV digestion,and their reactivity and enzymatic activity were evaluated through Western blot and enzyme activity detection kit,respectively. As results,the soluble expressions of K196R and A240L proteins were obtained by severally fusing with SUMO and MBP tag,and the size was about 22 kD and 27 kD after tag removed. Western blot results showed both K196R and A240L proteins well specifically reacted with the ASF positive serum. Enzyme activity detection results demonstrated the K196R and A240L proteins all presented definite enzyme catalytic activity in vitro. In conclusion,the soluble K196R and A240L proteins primarily expressed in Escherichia coli all show better reactivity and certain enzymatic activity in vitro,which will be the basis for the ASFV clinical diagnosis and protein relevant functions investigation.

Key words: African Swine Fever, K196R protein, A240L protein, soluble expression, western blot identification, enzymatic activity analysis

李鹏昊, 梁严予, 王彦伟, 关洋, 逄文强, 田克恭. 非洲猪瘟病毒K196R和A240L蛋白的可溶性表达及酶活力分析[J]. 生物技术通报, 2020, 36(11): 70-75.

LI Peng-hao, LIANG Yan-yu, WANG Yan-wei, GUAN Yang, PANG Wen-qiang, TIAN Ke-gong. Soluble Expression and Enzyme Activity Analysis of African Swine Fever Virus K196R and A240L Proteins[J]. Biotechnology Bulletin, 2020, 36(11): 70-75.

图/表 5

图1 K196R和A240L编码基因的PCR扩增 M：DNA分子质量标准（DL5000）;1：A240L基因PCR扩增产物;2：K196R基因PCR扩增产物;3：阴性对照

图2 目的蛋白表达的SDS-PAGE检测 M：蛋白质分子质量标准;1-2：K196R蛋白表达上清和沉淀;3-4：SUMO-K196R蛋白表达上清和沉淀;5-6：A240L蛋白表达上清和沉淀;7-8：MBP-A240L蛋白表达上清和沉淀

图3 目的蛋白纯化与酶切的SDS-PAGE检测 M：蛋白质分子质量标准;1：SUMO-K196R蛋白纯化样品;2：SUMO-K196R蛋白纯化后酶切;3：K196R蛋白无标签;4：MBP-A240L蛋白纯化样品;5：MBP-A240L蛋白纯化后酶切;6：A240L蛋白无标签

图4 K196R和A240L蛋白的Western blot鉴定 M：蛋白质分子质量标准;1：K196R蛋白显色条带;2：A240L蛋白显色条带

图5 K196R和A240L蛋白酶活力检测结果

参考文献 20

[1]	何洋, 康桦华, 冯锈华, 等. 非洲猪瘟疫苗研究进展[J]. 传染病信息, 2019,32(1):76-80.
	He Y, Kang HH, Feng XH, et al. Progress in development of African swine fever vaccine[J]. Infectious Disease Information, 2019,32(1):76-80.
[2]	王涛, 孙元, 罗玉子, 等. 非洲猪瘟防控及疫苗研发:挑战与对策[J]. 生物工程学报, 2018,34(12):66-77.
	Wang T, Sun Y, Luo YZ, et al. Prevention, control and vaccine development of African swine fever: challenges and countermeasures[J]. Chinese Journal of Biotechnology, 2018,34(12):66-77.
[3]	Arias M, Jurado C, Gallardo C, et al. Gaps in African swine fever:analysis and priorities[J]. Transboundary and Emerging Diseases, 2018,65(S1):235-247.
[4]	南木甲. 非洲猪瘟病毒分子生物学研究进展[J]. 中国畜牧兽医, 2005,32(9):51-53.
	Nan MJ. The advancement on molecular biology of African swine fever virus[J]. China Animal Husbandry & Veterinary Medicine, 2005,32(9):51-53.
[5]	Gómezpuertas P, Rodríguez F, Oviedo JM, et al. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response[J]. Virology, 1998,243(2):461-471. URL pmid: 9568043
[6]	Dixon LK, Chapman DAG, Netherton CL, et al. African swine fever virus replication and genomics[J]. Virus Research, 2013,173(1):3-14. doi: 10.1016/j.virusres.2012.10.020 URL pmid: 23142553
[7]	Ramirez-Medina E, Vuono E, O’Donnell V, et al. Differential effect of the deletion of African swine fever virus virulence-associated genes in the induction of attenuation of the highly virulent Georgia strain[J]. Viruses, 2019,11(7):599-613.
[8]	崔贝贝, 仇松寅, 梅琳, 等. 非洲猪瘟病毒K196R基因实时荧光定量PCR检测方法的建立[J]. 中国兽医杂志, 2019,55(5):3-8.
	Cui BB, Qiu SY, Mei L, et al. Establishment of real-time quantitative PCR assay for detection of African swine fever virus K196R gene[J]. Chinese Journal of Veterinary Medicine, 2019,55(5):3-8.
[9]	王侃, 刘次全, 曹槐, 等. 大肠杆菌mRNA编码区长度、形成二级结构倾向与密码子偏好性的关系[J]. 微生物学报, 2006,46(6):895-899. pmid: 17302150
	Wang K, Liu CQ, Cao H, et al. Insight into relationship among mRNA coding region length, folding tendency and codon usage bias in Escherichia coli[J]. Acta Microbiologica Sinica, 2006,46(6):895-899. URL pmid: 17302150
[10]	张宇萌, 童梅, 陆小冬, 等. 提高大肠杆菌可溶性重组蛋白表达产率的研究进展[J]. 中国生物工程杂志, 2016,36(5):118-124.
	Zhang YM, Tong M, Lu XD, et al. Advances in promoting soluble expression of recombinant protein in Escherichia coli[J]. China Biotechnology, 2016,36(5):118-124.
[11]	亓振国, 张宽亮, 陈宗梅, 等. 大肠杆菌分子伴侣表达的辅助载体构建及应[J]. 农业生物技术学报, 2011,19(1):171-177.
	Qi ZG, Zhang KL, Chen ZM, et al. The helper vectors construction of chaperones from Escherichia coli and their application[J]. Journal of Agricultural Biotechnology, 2011,19(1):171-177.
[12]	Sereda AD, Balyshev VM. Antigenic diversity of African swine fever viruses[J]. Voprosy Virusologii, 2011,56(4):38-42. URL pmid: 21899069
[13]	Yanez RJ, Rodriguez JM, Rodriguez JF, et al. African swine fever virus thymidylate kinase gene:sequence and transcriptional mapping[J]. Journal of General Virology, 1993,74(8):1633-1638.
[14]	Portugal R, Leitão A, Martins C. Characterization of African swine fever virus IAP homologue expression in porcine macrophages infected with different virulence isolates[J]. Veterinary Microbiology, 2009,139(1):140-146.
[15]	Sanford B, Holinka LG, O’Donnell V, et al. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus[J]. Virus Research, 2016,213(1):165-171.
[16]	Jaing C, Rowland RRR, Allen JE, et al. Gene expression analysis of whole blood RNA from pigs infected with low and high patho-genic African swine fever viruses[J]. Scientific Reports, 2017,7(1):1-14. doi: 10.1038/s41598-016-0028-x URL pmid: 28127051
[17]	Kollnberger SD, Gutierrez-castan eda B, Foster-Cuevas M, et al. Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody[J]. Journal of General Virology, 2002,83(6):1331-1342. doi: 10.1099/0022-1317-83-6-1331 URL
[18]	Moore DM, Zsak L, Neilan JG, et al. The african swine fever Virus thymidine kinase gene is required for efficient replication in swine macrophages and for virulence in swine[J]. Journal of Virology, 1998,72(12):10310-10315. doi: 10.1128/JVI.72.12.10310-10315.1998 URL pmid: 9811782
[19]	Bishop RP, Fleischauer C, de Villiers EP, et al. Comparative analysis of the complete genome sequences of Kenyan African swine fever virus isolates within p72 genotypes IX and X[J]. Virus Genes, 2015,50(2):303-309. URL pmid: 25645905
[20]	Li CY, Chai Y, Song H, et al. Crystal structure of African swine fever virus dUTPase reveals a potential drug target[J]. Molecular Biology and Physiology, 2019,10(5):1-11.