绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究

doi:10.12160/j.issn.1672-5190.2022.01.001

畜牧与饲料科学 ›› 2022, Vol. 43 ›› Issue (1): 1-7.doi: 10.12160/j.issn.1672-5190.2022.01.001

• 基础研究 • 下一篇

绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究

戴伶俐, 王娜, 刘威, 白帆, 张帆, 张月梅, 宋越, 杨斌, 陈伟, 赵世华, 达来宝力格

内蒙古自治区农牧业科学院,内蒙古呼和浩特 010031

收稿日期:2021-11-01 出版日期:2022-01-30 发布日期:2022-02-10
通讯作者: 赵世华（1962—）,男,研究员,主要从事动物传染病防控和营养代谢病研究工作。达来宝力格（1979—）,男,助理研究员,博士,主要从事动物传染病防控研究工作。
作者简介:戴伶俐（1983—）,女,助理研究员,硕士,主要从事动物传染病防控研究工作。
基金资助:
内蒙古自治区重大专项（2021ZD0023,2021ZD0024）; 内蒙古自治区自然科学基金面上项目联合基金（2019LH03004）; 内蒙古自治区科技计划项目（2020GG0041）; 内蒙古自治区农牧业创新基金项目（2014CXJJM03）

Cloning,Expression of DnaJ Gene and Immunogenicity Analysis of DnaJ Protein in Mycoplasma ovipneumoniae Isolated from Inner Mongolia

DAI Ling-li, WANG Na, LIU Wei, BAI Fan, ZHANG Fan, ZHANG Yue-mei, SONG Yue, YANG Bin, CHEN Wei, ZHAO Shi-hua, Dalaibaolige

Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China

Received:2021-11-01 Online:2022-01-30 Published:2022-02-10

摘要/Abstract

摘要： [目的] 克隆表达绵羊肺炎支原体DnaJ基因,研究其表达产物的免疫原性。[方法] 以绵羊肺炎支原体内蒙古分离株NM03-MO株基因组DNA为模板,克隆其DnaJ基因序列,对蛋白质序列进行分析比对及三维结构预测;将该基因片段连接至pColdⅠ表达载体,构建pColdⅠ-DnaJ原核表达载体表达重组DnaJ蛋白并纯化,通过Western blot技术检测其与支原体抗体阳性血清的结合活性。[结果] NM03-MO株DnaJ蛋白序列与绵羊肺炎支原体序列同源性最高,但三维结构存在一定差异,重组DnaJ蛋白与绵羊肺炎支原体阳性血清有较好的结合活性,初步证明该蛋白具有免疫原性。[结论] 绵羊肺炎支原体DnaJ蛋白被首次成功克隆表达,且具有较好的免疫原性,为后续分子致病机制研究及新型疫苗研制奠定基础。

关键词: 绵羊肺炎支原体, DnaJ蛋白, 序列分析, 分子克隆, 免疫原性

Abstract: [Objective] To clone and express the DnaJ gene in Mycoplasma ovipneumoniae, and to assess the immunogenicity of its expression product. [Method] Using genomic DNA extracted from Mycoplasma ovipneumoniae NM03-MO strain isolated from Inner Mongolia as template, DnaJ gene was cloned. The DnaJ protein sequence was analyzed and its three-dimensional structure was predicted. A pColdⅠ-DnaJ prokaryotic expression vector was constructed with the cloned DnaJ gene fragment being connected to pColdⅠ vector to express the recombinant DnaJ protein. The obtained DnaJ protein was purified, and its binding activity to Mycoplasma antibody positive serum was evaluated by Western blot. [Result] The NM03-MO strain′s DnaJ protein had the highest homology to Mycoplasma ovipneumoniae, while there were minor differences in the three-dimensional structure. The recombinant DnaJ protein displayed high binding activity to Mycoplasma ovipneumoniae positive serum, indicating that it was a immunogenic protein. [Conclusion] For the first time, the DnaJ protein of Mycoplasma ovipneumoniae was successfully cloned and expressed and had good immunogenicity. This research lays the groundwork for the further molecular pathogenesis research and the development of novel vaccines.

Key words: Mycoplasma ovipneumoniae, DnaJ protein, sequence analysis, molecular cloning, immunogenicity

中图分类号:

S858.262.4

戴伶俐, 王娜, 刘威, 白帆, 张帆, 张月梅, 宋越, 杨斌, 陈伟, 赵世华, 达来宝力格. 绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究[J]. 畜牧与饲料科学, 2022, 43(1): 1-7.

DAI Ling-li, WANG Na, LIU Wei, BAI Fan, ZHANG Fan, ZHANG Yue-mei, SONG Yue, YANG Bin, CHEN Wei, ZHAO Shi-hua, Dalaibaolige. Cloning,Expression of DnaJ Gene and Immunogenicity Analysis of DnaJ Protein in Mycoplasma ovipneumoniae Isolated from Inner Mongolia[J]. Animal Husbandry and Feed Science, 2022, 43(1): 1-7.

参考文献 24

[1]	韩笑. 绵羊肺炎支原体HSP70基因遗传多样性及其在分子检测中的应用[D].成都:西南民族大学,2012.
[2]	ZHAO J Y, DU Y Z, SONG Y P, et al.Investigation of the prevalence of Mycoplasma ovipneumoniae in southern Xinjiang, China[J]. Journal of Veterinary Research,2021,65(2):155-160.
[3]	凤英,高娃,赵世华,等.绵羊肺炎支原体内蒙古毒株的分离与鉴定[J].中国兽医杂志,2014,50(10):22-24,50.
[4]	吴锦艳,尚佑军,陈妍,等.国内部分地区羊支原体肺炎血清学调查及分析[J].中国预防兽医学报,2017,39(2):156-158.
[5]	姜新基,孟柯其其格,付登胜,等.羊支原体肺炎诊断及治疗[J].畜牧兽医科技信息,2021(6):94.
[6]	郭长明,吴植,吴双,等.无乳链球菌dnaJ基因缺失株的构建及其致病性研究[J].中国畜牧兽医,2019,46(9):2507-2515.
[7]	BALISH M F.Mycoplasma pneumoniae, an underutilized model for bacterial cell biology[J]. Journal of Bacteriology,2014,196(21):3675-3682.
[8]	LIU Y, WANG H, ZHANG S, et al.Mucosal immunization with recombinant fusion protein DnaJ-△A146Ply enhances cross-protective immunity against Streptococcus pneumoniae infection in mice via interleukin 17A[J]. Infection and Immunity,2014,82(4):1666-1675.
[9]	JIANG F, HE J, NAVARRO-ALVAREZ N, et al.Elongation factor Tu and heat shock protein 70 are membrane-associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J].PLoS One,2016,11(8):e0161170.
[10]	GHAZAEI C.Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens[J]. Journal of Medical Microbiology,2017,66(3):259-265.
[11]	OHTSUKA K, HATA M.Molecular chaperone function of mammalian Hsp70 and Hsp40[J].International Journal of Hyperthermia,2000,16(3):231-245.
[12]	CUI J, MA C, YE G, et al.DnaJ (hsp40) of Streptococcus pneumoniae is involved in bacterial virulence and elicits a strong natural immune reaction via PI3K/JNK[J].Molecular Immunology,2017,83:137-146.
[13]	BARRIOT R, LATOUR J,CASTANIÉ-CORNET M P, et al. J-Domain proteins in bacteria and their viruses[J].Journal of Molecular Biology,2020,432(13):3771-3789.
[14]	ZHANG B, HAN X, YUE H, et al.Molecular characterization of the heat shock protein 70 gene in Mycoplasma ovipneumoniae[J].The Veterinary Journal,2013,198(1):299-301.
[15]	周怡,王柏林,杨美,等.绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒的构建及对小鼠细胞免疫应答的影响[J].中国畜牧兽医,2019,46(11):3387-3395.
[16]	ZHANG Y, MEI S, ZHOU Y, et al.TIPE2 negatively regulates Mycoplasma pneumonia-triggered immune response via MAPK signaling pathway[J]. Scientific Reports,2017, 7(1):13319.
[17]	EINARSDOTTIR T, GUNNARSSON E, HJARTARDOTTIR S.Icelandic ovine Mycoplasma ovipneumoniae are variable bacteria that induce limited immune responses in vitro and in vivo[J].Journal of Medical Microbiology,2018, 67(10):1480-1490.
[18]	LUO H, WU X, XU Z, et al.NOD2/c-Jun NH2-terminal kinase triggers Mycoplasma ovipneumoniae-induced macrophage autophagy[J]. Journal of Bacteriology,2020,202(20):e00689-19 .
[19]	崔瑾,董杰,姜慧,等.肺炎链球菌dnaJ基因缺陷对小鼠肺炎感染模型天然免疫应答的影响[J].中国生物制品学杂志,2013,26(2):160-165.
[20]	张晓燕. DnaK家族蛋白DnaJ介导猪链球菌2型的热应激及其对上皮细胞的黏附[J]. 杭州:浙江大学, 2014.
[21]	CLOWARD J M, KRAUSE D C.Mycoplasma pneumoniae J-domain protein required for terminal organelle function[J].Molecular Microbiology,2009, 71(5):1296-1307.
[22]	KHAN M N, BANSAL A, SHUKLA D, et al .Immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae against lethal infection in mice[J]. Vaccine,2006,24(37/39):6225-6231.
[23]	CUI Y, ZHANG X, GONG Y, et al.Immunization with DnaJ (hsp40) could elicit protection against nasopharyngeal colonization and invasive infection caused by different strains of Streptococcus pneumoniae[J]. Vaccine,2011,29(9):1736-1744.
[24]	CUI J, SHAO F.Biochemistry and cell signaling taught by bacterial effectors[J]. Trends in Biochemical Sciences,2011,36(10):532-540.

绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究

Cloning,Expression of DnaJ Gene and Immunogenicity Analysis of DnaJ Protein in Mycoplasma ovipneumoniae Isolated from Inner Mongolia

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献 24

相关文章 15

编辑推荐

Metrics

本文评价

[1]	马爱军, 李佳, 金敏, 金依璇, 刘诗语, 刘凯强, 刘燕, 甘露, 巴音查汗·盖力克. 新疆维吾尔自治区阿克苏地区牛羊体表蜱携带斑点热群立克次体调查[J]. 畜牧与饲料科学, 2024, 45(1): 107-113.
[2]	戴伶俐, 王娜, 白帆, 张帆, 宋越, 张月梅, 达来宝力格, 李晓艳, 王根云, 赵世华. 9株绵羊肺炎支原体内蒙古分离株P113基因克隆及蛋白序列分析[J]. 畜牧与饲料科学, 2022, 43(6): 1-5.
[3]	辜彦霏, 殷瑛, 李玉杰, 宰晓东, 徐俊杰. 重组布鲁菌外膜蛋白Omp19的制备方法及免疫原性研究[J]. 畜牧与饲料科学, 2022, 43(3): 1-7.
[4]	戴伶俐, 王娜, 张帆, 宋越, 白帆, 刘威, 杨斌, 赵世华, 张月梅. 绵羊肺炎支原体分子致病机理研究进展[J]. 畜牧与饲料科学, 2022, 43(2): 124-128.
[5]	李勇, 李生虎, 贺林芝. PRRSV分离株GP3蛋白ORF3基因克隆及序列分析[J]. 畜牧与饲料科学, 2020, 41(1): 7-12.
[6]	潘孝成, 沈学怀, 赵瑞宏, 戴银, 胡晓苗, 侯宏艳, 周学利, 张丹俊. 6株猪流行性腹泻病毒N基因的克隆与分析[J]. 畜牧与饲料科学, 2019, 40(9): 23-26.
[7]	黄玉欣, 李鹏昊, 刘鹏, 朱巧燕, 李向东, 田克恭. 利用总蛋白评价猪肺炎支原体HN0613株免疫原性的研究[J]. 畜牧与饲料科学, 2019, 40(6): 1-5.
[8]	侯宏艳,张丹俊,赵瑞宏,周学利. 绵羊肺炎支原体安徽株p113基因PCR检测与序列分析[J]. 畜牧与饲料科学, 2019, 40(1): 101-103.
[9]	侯宏艳,惠文巧,张丹俊,胡晓苗,赵瑞宏. 绵羊肺炎支原体的培养和PCR鉴定[J]. 畜牧与饲料科学, 2016, 37(11): 97-97.
[10]	宫利娜,郝瑞峰,王业华,张岩,高航飞,李琛,海日汗,李平安,吴太平,关平原. 牛支原体内蒙古分离株脂蛋白vspY基因的克隆和序列分析[J]. 畜牧与饲料科学, 2015, 36(3): 9-9.
[11]	李锋,窦洪举,孙素珍,牧其尔,刘秀丽,侯勇跃. 犬皮肤癣菌病病原的分离及其ITS区序列分析[J]. 畜牧与饲料科学, 2014, 35(12): 17-17.
[12]	何建文,党娟,关平原,李平安,高魁. 禽呼肠孤病毒C-98、T-98分离株S2基因的克隆及序列分析[J]. 畜牧与饲料科学, 2013, 34(1): 18-18.
[13]	徐华敏,希尼尼根,申之义. 羊传染性脓疱病毒内蒙古分离株VIR基因的克隆与序列分析[J]. 畜牧与饲料科学, 2012, 33(4): 1-1.
[14]	鲁燕,申之义,关平原,希尼尼根. 羊传染性脓疱病毒内蒙古分离株（OV/nm-05）GIF基因的克隆及序列分析[J]. 畜牧与饲料科学, 2012, 33(4): 11-11.
[15]	孙喜安,韩敏,刘晓松,凤英,高娃,杨鼎. 绵羊肺炎支原体分离株交叉反应性的测定[J]. 畜牧与饲料科学, 2012, 33(1): 112-112.