猪肺炎支原体解螺旋酶RuvA的原核表达、多克隆抗体制备及活性鉴定

doi:10.11843/j.issn.0366-6964.2024.01.025

Abstract

Abstract: Homologous recombination mediated antigenic variation plays an important role in the frequent persistent infection of Mycoplasma hyopneumoniae of pigs. Allosteric regulation of Holliday junctions by helicase RuvA is a key step involved in homologous recombination. Given that, this study aims at expressing RuvA_Mhp recombinant protein, identifying its DNA binding activity and preparing polyclonal antibody against RuvA_Mhp. Prokaryotic expression plasmid pET21a-RuvA was constructed by molecular cloning. Following expression induction and purification, the RuvA_Mhp recombinant protein was obtained; The polyclonal antibody against RuvA_Mhp was prepared by immunizing rabbit, and the titer and specificity were determined by indirect ELISA and Western blot; The DNA binding activity of RuvA_Mhp was analyzed by electrophoretic mobility shift assay and surface plasmon resonance technique; The oligomerization of RuvA_Mhp was determined by electrophoretic mobility shift assay combined Western blot. RuvA_Mhp recombinant protein expressed by prokaryotic expression was about 26 ku; The prepared polyclonal antibody against RuvA_Mhp had good specificity and high titer of 1:256 000; RuvA_Mhp has strong DNA binding activity with an affinity of 624.4 pmol·L^-1 (K_D) for Holliday junctions, and forms stable complexes with it mainly in octamers. In this study, the prokaryotic expression, polyclonal antibody preparation and activity identification of RuvA_Mhp were achieved, which laid a foundation for further exploration of the molecular mechanism of helicase RuvA mediating antigenic variation of Mycoplasma hyopneumoniae by homologous recombination regulation.

Key words: Mycoplasma hyopneumoniae, helicase RuvA, DNA binding, polyclonal antibody

CLC Number:

S852.62

XIE Qingyun, XING Huixuan, YU Yanfei, YUAN Ting, XIONG Qiyan, XIONG Fuqiang, FENG Zhixin. Prokaryotic Expression, Polyclonal Antibody Preparation and Activity Identification of Helicase RuvA from Mycoplasma hyopneumoniae[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 271-281.

References 26

[1]	宁雅茹, 丁红雷. 猪肺炎支原体与宿主相互作用研究进展[J]. 生物工程学报, 2020, 36(9):1741-1753.NING Y R, DING H L. Interaction between Mycoplasma hyopneumoniae and host-a review[J]. Chinese Journal of Biotechnology, 2020, 36(9):1741-1753. (in Chinese)
[2]	MAES D, SIBILA M, KUHNERT P, et al. Update on Mycoplasma hyopneumoniae infections in pigs:knowledge gaps for improved disease control[J]. Transbound Emerg Dis, 2018, 65(S1):110-124.
[3]	MAES D, BOYEN F, HAESEBROUCK F, et al. Antimicrobial treatment of Mycoplasma hyopneumoniae infections[J]. Vet J, 2020, 259-260:105474.
[4]	HAKIM M S, ANNISA L, JARIAH R O A, et al. The mechanisms underlying antigenic variation and maintenance of genomic integrity in Mycoplasma pneumoniae and Mycoplasma genitalium[J]. Arch Microbiol, 2021, 203(2):413-429.
[5]	MCGOWIN C L, TOTTEN P A. The unique microbiology and molecular pathogenesis of Mycoplasma genitalium[J]. J Infect Dis, 2017, 216(S2):S382-S388.
[6]	QIN L M, CHEN Y W, YOU X X. Subversion of the immune response by human pathogenic mycoplasmas[J]. Front Microbiol, 2019, 10:1934.
[7]	BETLACH A M, MAES D, GARZA-MORENO L, et al. Mycoplasma hyopneumoniae variability:current trends and proposed terminology for genomic classification[J]. Transbound Emerg Dis, 2019, 66(5):1840-1854.
[8]	CAÑAS C, SUZUKI Y, MARCHISONE C, et al. Interaction of branch migration translocases with the Holliday junction-resolving enzyme and their implications in Holliday junction resolution[J]. J Biol Chem, 2014, 289(25):17634-17646.
[9]	FALLON A M. DNA recombination and repair in Wolbachia:RecA and related proteins[J]. Mol Genet Genomics, 2021, 296(2):437-456.
[10]	LOHMAN T M, FAZIO N T. How does a helicase unwind DNA?Insights from RecBCD helicase[J]. BioEssays, 2018, 40(6):1800009.
[11]	BURGOS R, TOTTEN P A. Characterization of the operon encoding the Holliday junction helicase RuvAB from Mycoplasma genitalium and its role in mgpB and mgpC gene variation[J]. J Bacteriol, 2014, 196(8):1608-1618.
[12]	ESTEVÃO S, VAN DER HEUL H U, SLUIJTER M, et al. Functional analysis of the superfamily 1 DNA helicases encoded by Mycoplasma pneumoniae and Mycoplasma genitalium[J]. PLoS One, 2013, 8(7):e70870.
[13]	SECHMAN E V, ROHRER M S, SEIFERT H S. A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae[J]. Mol Microbiol, 2005, 57(2):468-483.
[14]	OTSUJI N, IYEHARA H, HIDESHIMA Y. Isolation and characterization of an Escherichia coli ruv mutant which forms nonseptate filaments after low doses of ultraviolet light irradiation[J]. J Bacteriol, 1974, 117(2):337-344.
[15]	IWASAKI H, TAKAHAGI M, NAKATA A, et al. Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration[J]. Genes Dev, 1992, 6(11):2214-2220.
[16]	SLUIJTER M, ESTEVÃO S, HOOGENBOEZEM T, et al. The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics[J]. PLoS One, 2012, 7(5):e38301.
[17]	ESTEVÃO S, SLUIJTER M, HARTWIG N G, et al. Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium[J]. J Bacteriol, 2011, 193(23):6425-6435.
[18]	SHIBA T, IWASAKI H, NAKATA A, et al. SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli:functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA[J]. Proc Natl Acad Sci U S A, 1991, 88(19):8445-8449.
[19]	CATTANI A M, SIQUEIRA F M, GUEDES R L M, et al. Repetitive elements in Mycoplasma hyopneumoniae transcriptional regulation[J]. PLoS One, 2016, 11(12):e0168626.
[20]	CITTI C, NOUVEL L X, BARANOWSKI E. Phase and antigenic variation in mycoplasmas[J]. Future Microbiol, 2010, 5(7):1073-1085.
[21]	DE CASTRO L A, RODRIGUES PEDROSO T, KUCHIISHI S S, et al. Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains[J]. Vet Microbiol, 2006, 116(4):258-269.
[22]	IWASA T, HAN Y W, HIRAMATSU R, et al. Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation[J]. Sci Rep, 2015, 5:18177.
[23]	BRADLEY A S, BAHAROGLU Z, NIEWIAROWSKI A, et al. Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo[J]. J Biol Chem, 2011, 286(25):22372-22383.
[24]	PRIVEZENTZEV C V, KEELEY A, SIGALA B, et al. The role of RuvA octamerization for RuvAB function in vitro and in vivo[J]. J Biol Chem, 2005, 280(5):3365-3375.
[25]	BAHAROGLU Z, BRADLEY A S, LE MASSON M, et al. ruvA mutants that resolve Holliday junctions but do not reverse replication forks[J]. PLoS Genet, 2008, 4(3):e1000012.
[26]	BROSH R M Jr, MATSON S W. History of DNA helicases[J]. Genes (Basel), 2020, 11(3):255.

Prokaryotic Expression, Polyclonal Antibody Preparation and Activity Identification of Helicase RuvA from Mycoplasma hyopneumoniae

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