非洲猪瘟病毒pS273R蛋白的原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.11.025

Abstract

Abstract: To provide the material for the functional study of ASFV pS273R, ASFV pS273R protein was expressed and polyclonal antibody against pS273R protein was prepared in present study. The recombinant pS273R protein expressed in E. coli expression system was purified by Ni-affinity column and molecular sieve, respectively. Specific polyclonal antibodies against the pS273R protein were obtained by the immunized BALB/c mice, and the anti-pS273R polyclonal antibody (pAb) was purified from the mouse serum by using a Protein G affinity chromatography column. The reactivity and specificity of polyclonal antibodies were evaluated by Western blotting, IFA and IP. The results showed recombinant pS273R protein was efficiently expressed in E. coli as when induced with 16 ℃ and the concentration of IPTG at 0.1 mmol/L. The Flag-tagged pS273R protein in HEK293T cells and the ASFV-encoded pS273R in porcine alveolar macrophage (PAMs) were detected by Western blotting, IFA and IP using the mouse anti-pS273R pAb. In summary, ASFV pS273R was successfully expressed in E. coli, preparation and purification of mouse anti-S273R pAb had good specificity and reactivity. The availability of anti-pS273R pAb laid an important foundation for further research studying the biological function of pS273R protein.

Key words: African swine fever virus (ASFV); pS273R protein; polyclonal antibody

CLC Number:

S855.3

ZHAO Gaihong, LI Tingting, ZHANG Taoqing, CHEN Xin, WANG Xiao, LI Changyao, ZHANG Zhaoxia, ZHENG Jun, WENG Changjiang. Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4154-4161.

References

[1] KARGER A, PÉREZ-NU'EZ D, URQUIZA J, et al. An update on African swine fever virology[J]. Viruses, 2019, 11(9):864.
[2] DIXON L, ISLAM M, NASH R, et al. African swine fever virus evasion of host defences[J]. Virus Research, 2019, 266:25-33.
[3] DIXON L K, SUN H, ROBERTS H.African swine fever[J]. Antiviral Research, 2019, 165:34-41.
[4] LUBISI B A, BASTOS A D, DWARKA R M, et al. Molecular epidemiology of African swine fever in East Africa[J]. Archives of Virology, 2005, 150(12):2439-2452.
[5] BASTOS A D, PENRITH M L, MACOME F, et al. Co-circulation of two genetically distinct viruses in an outbreak of African swine fever in Mozambique:No evidence for individual co-infection[J]. Veterinary Microbiology, 2004, 103(3-4):169-182.
[6] ZHAO D, LIU R, ZHANG X, et al. Replication and virulence in pigs of the first African swine fever virus isolated in China[J]. Emerging microbes & Infections, 2019, 8(1):438-477.
[7] GE S, LI J, FAN X, et al. Molecular Characterization of African swine fever virus, China, 2018[J]. Emerging Infectious Diseases, 2018, 24(11):2131-2133.
[8] ARIAS M, DE LA TORRE A, DIXON L, et al. Approaches and perspectives for development of African swine fever virus vaccines[J]. Vaccines, 2017, 5(4):35.
[9] 欧云文, 阎传忠, 张杰, 等.非洲猪瘟病毒的分子病原学及致病机理研究进展[J]. 中国畜牧兽医, 2017, 44(7):2139-2146. OU Y W, YAN C Z, ZHANG J et al. Research progress on molecular etiology and pathogenesis for African swine fever virus[J]. China Animal Husbandy & Veterinary Medicine, 2017, 44(7):2139-2146.(in Chinese)
[10] ROJO G, GARCÍA-BEATO R, VIUELA E, et al. Replication of African swine fever virus DNA in infected cells[J]. Virology, 1999, 257(2):524-536.
[11] ALEJO A, MATAMOROS T, GUERRA M, et al. A proteomic atlas of the African swine fever virus particle[J]. Journal of Virology, 2018, 92(23):e01293-18.
[12] DIXON L K, CHAPMAN D A, NETHERTON C L, et al. African swine fever virus replication and genomics[J]. Virus Research, 2013, 173(1):3-14.
[13] XIA N, WANG H, LIU X, et al. African swine fever virus structural protein p17 inhibits cell proliferation through ER stress-ROS mediated cell cycle arrest[J]. Viruses, 2020, 13(1):21.
[14] GARCÍA-BELMONTE R, PÉREZ-NÚÑEZ D, PITTAU M, et al. African swine fever virus armenia/07 virulent strain controls interferon beta production through the cGAS-STING pathway[J]. Journal of Virology, 2019, 93(12):e02298-18.
[15] REIS A, NETHERTON C, DIXON L.Unraveling the armor of a killer:Evasion of host defenses by African swine fever virus[J]. Journal of Virolog, 2017, 91(6):e02338-16.
[16] BANJARA S, CARIA S, DIXON L K, et al. Structural insight into African swine fever virus A179L-mediated inhibition of apoptosis[J]. Journal of Virology, 2017, 91(6):e02228-16.
[17] ANDRÉS G, ALEJO A, SALAS J, et al. African swine fever virus polyproteins pp220 and pp62 assemble into the core shell[J]. Journal of Virology, 2002, 76(24):12473-12482.
[18] ANDRÉS G, ALEJO A, SIMÓN-MATEO C, et al. African swine fever virus protease, a new viral member of the SUMO-1-specific protease family[J]. The Journal of Biological Chemistry, 2001, 276(1):780-787.
[19] LI G, LIU X, YANG M, et al. Crystal structure of African swine fever virus pS273R protease and implications for inhibitor design[J]. Journal of Virology, 2020, 94(10):e02125-19.
[20] ALEJO A, ANDRÉS G, SALAS M L.African swine fever virus proteinase is essential for core maturation and infectivity[J]. Journal of Virology, 2003, 77(10):5571-5577.
[21] WEN X, HE X, ZHANG X, et al. Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018[J]. Emerging Microbes & Infections, 2019, 8(1):303-306.
[22] 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 Science, 2018, 38(9):1831-1832.(in Chinese)
[23] CHEN W, ZHAO D, HE X, et al. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs[J]. Science China Life Sciences, 2020, 63(5):623-634.
[24] 杨思成, 高瞻, 邵军军, 等.非洲猪瘟新型疫苗研究进展[J]. 中国动物检疫, 2020, 331(12):87-91. YANG S C, GAO Z, SHAO J J, et al. Research progress of novel vaccines against African swine fever virus[J]. China Animal Health Inspection, 2020, 331(37):87-91.(in Chinese)
[25] LI C, CHAI Y, SONG H, et al. Crystal structure of African swine fever virus dUTPase reveals a potential drug target[J]. mBio, 2019, 10(5):e02483-19.
[26] 张萌, 刘英楠, 陈鸿军.非洲猪瘟病毒pS273R蛋白水解酶单克隆抗体制备[J]. 畜牧兽医学报, 2021, 52(3):839-844. ZHANG M, LIU Y N, CHEN H J.Preparation of monoclonal antibodies against African swine fever virus pS273R protease[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3):839-844.(in Chinese)

Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody

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