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

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (1): 271-281.doi: 10.11843/j.issn.0366-6964.2024.01.025

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

谢青云^1,3, 邢蕙萱^1,2, 于岩飞^1,3, 袁厅^1,3, 熊祺琰^1,3, 熊富强^2,4*, 冯志新^1,2,3,4*

1. 江苏省农业科学院兽医研究所, 农业农村部兽用生物制品工程重点实验室, 南京 210014;
2. 西藏农牧学院, 动物科学学院, 林芝 850400;
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300;
4. 南京农学大学动物医学院, 南京 210095

收稿日期:2023-04-13 出版日期:2024-01-23 发布日期:2024-01-24
通讯作者: 熊富强,主要从事动物病原菌致病机制研究,E-mail:xiongfuqiang@njau.edu.cn;冯志新,主要从事动物支原体致病机制研究,E-mail:fzxjaas@163.com
作者简介:谢青云(1990-),女,安徽宣城人,助理研究员,博士,主要从事支原体致病机制研究,E-mail:xqy816626@163.com;Tel:025-84390880;邢蕙萱(1997-),女,安徽马鞍山人,硕士生,主要从事支原体致病机制研究,E-mail:xinghuixuan521@qq.com。谢青云和邢蕙萱为同等贡献作者
基金资助:
国家自然基金（32202812）；江苏省农业科技自主创新项目（CX（22）3195）

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

XIE Qingyun^1,3, XING Huixuan^1,2, YU Yanfei^1,3, YUAN Ting^1,3, XIONG Qiyan^1,3, XIONG Fuqiang^2,4*, FENG Zhixin^1,2,3,4*

1. Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture/Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. College of Animal Science, Tibet Agricultural and Animal Husbandry University, Linzhi 850400, China;
3. GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China;
4. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2023-04-13 Online:2024-01-23 Published:2024-01-24

摘要/Abstract

摘要： 猪肺炎支原体的持续性感染问题频发，同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此，本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白，鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA，经诱导表达和纯化获得RuvA_Mhp重组蛋白；免疫家兔制备抗RuvA_Mhp多克隆抗体，再利用间接ELISA和Western blot试验进行效价测定和特异性验证；利用凝胶迁移试验结合表面等离子共振技术分析RuvA_Mhp的DNA结合活性；利用凝胶迁移试验结合Western blot试验鉴定RuvA_Mhp的寡聚特性。结果显示：原核表达的RuvA_Mhp重组蛋白约为26 ku；制备的抗RuvA_Mhp多克隆抗体效价为1∶256 000，且具有良好的特异性；RuvA_Mhp具有较强的DNA结合活性，对霍利迪连接体的亲和力高达624.4 pmol·L^-1（K_D），并且主要以八聚体形式与其形成稳定复合物。通过RuvA_Mhp的原核表达、多克隆抗体制备及活性鉴定，为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。

关键词: 猪肺炎支原体, 解旋酶RuvA, DNA结合, 多克隆抗体

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

中图分类号:

S852.62

谢青云, 邢蕙萱, 于岩飞, 袁厅, 熊祺琰, 熊富强, 冯志新. 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[J]. 畜牧兽医学报, 2024, 55(1): 271-281.

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.

参考文献 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.

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

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王静瑜, 潘阳阳, 徐庚全, 张瑞, 张文兰, 王筱珊, 乌仁套迪, 照日格图, 崔燕, 余四九. 牦牛Fas相关因子1多克隆抗体制备及初步应用[J]. 畜牧兽医学报, 2023, 54(8): 3369-3382.
[2]	焦正兴, 潘阳阳, 王萌, 王靖雷, 马文斌, 高翔, 张晖, 崔燕, 余四九, 王立斌. 牦牛LC3B蛋白多克隆抗体制备及在生殖器官表达检测中的应用[J]. 畜牧兽医学报, 2023, 54(6): 2436-2447.
[3]	高真贞, 蒙泽菁, 何小兵, 房永祥, 田慧慧, 陈国华, 景志忠. 鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定[J]. 畜牧兽医学报, 2023, 54(6): 2468-2477.
[4]	王国超, 赵亚茹, 张忠辉, 张玉龙, 白鸽, 耿抒贤, 樊洁, 杨吉飞, 关贵全, 殷宏, 罗建勋, 牛庆丽. 非洲猪瘟病毒RNA聚合酶亚基D205R基因生物信息学分析及多克隆抗体制备[J]. 畜牧兽医学报, 2023, 54(5): 2042-2049.
[5]	王冰楠, 孙雪, 贾红菓, 谭纯, 梅四鹏, 周荣琼. 犬弓首蛔虫C型凝集素4的原核表达及组织定位[J]. 畜牧兽医学报, 2023, 54(4): 1641-1651.
[6]	杨洪早, 朱杰, 郭宏元, 汤傲星, 陈少宇, 刘春草, 张达, 袁莉刚, 刘光清. 猫源C11orf96基因表达及其在细胞中的定位分析[J]. 畜牧兽医学报, 2022, 53(7): 2390-2395.
[7]	曹艳桃, 樊江峰, 余四九, 周应聪, 杜培岩, 李一娟, 马进彪, 赵生贤. 牦牛EPF的原核表达及多克隆抗体制备[J]. 畜牧兽医学报, 2022, 53(2): 470-480.
[8]	刘欣杰, 吴晓雪, 刘素平, 袁利明, 陈宁, 赛务加甫. PLC-γ1的多克隆抗体制备及对绵羊早期胚胎发育作用的初步研究[J]. 畜牧兽医学报, 2022, 53(11): 3842-3855.
[9]	潘启东, 曾显成, 杨彬偲, 刘庆华, 徐泉明, 陈吉龙. 山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究[J]. 畜牧兽医学报, 2021, 52(11): 3165-3174.
[10]	孙盼盼, 时建立, 彭喆, 王硕, 王妍, 吴晓燕, 徐绍建, 李俊. 猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用[J]. 畜牧兽医学报, 2020, 51(6): 1419-1428.
[11]	王宁, 谨瑾, 刘璐, 刘欢欢, 姜立波, 辛九庆, 刘恒贵. 猪肺炎支原体P97蛋白单克隆抗体的制备、表位鉴定及其初步应用[J]. 畜牧兽医学报, 2020, 51(5): 1110-1118.
[12]	王艳芳, 周雅坪, 郭婷, 张新竹, 吴倩, 陈昕迪, 郝永清. 牛支原体EF-Tu基因的克隆表达、亚细胞定位及间接ELISA方法建立[J]. 畜牧兽医学报, 2019, 50(1): 134-142.
[13]	朱建波, 杨振兴, 肖雷, 高林, 苗海生, 何于雯, 孟锦昕, 杨恒, 李华春. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7): 1440-1450.
[14]	刘璐, 郭杰, 朱可蒙, 王宁, 辛九庆, 刘玉芬, 刘恒贵. 利用酵母随机展示技术对猪肺炎支原体P97上B细胞表位的筛选和鉴定[J]. 畜牧兽医学报, 2018, 49(7): 1492-1499.
[15]	李成, 张雨迪, 黄小波, 刘浩宇, 刘志鹏, 赵玉佳, 曹三杰, 文心田, 文翼平, 赵勤, 伍锐. 猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备[J]. 畜牧兽医学报, 2018, 49(6): 1256-1264.