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丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析

张靖鹏 江锦秀 林裕胜 游伟 胡奇林

张靖鹏,江锦秀,林裕胜,等. 丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析 [J]. 福建农业学报,2019,34(10):1124−1128. doi: 10.19303/j.issn.1008-0384.2019.10.002
引用本文: 张靖鹏,江锦秀,林裕胜,等. 丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析 [J]. 福建农业学报,2019,34(10):1124−1128. doi: 10.19303/j.issn.1008-0384.2019.10.002
ZHANG J P, JIANG J X, LIN Y S, et al. Cloning and Expression of Specific Protein Gene, Mmc -3740 , of Mycoplasma mycoides [J]. Fujian Journal of Agricultural Sciences,2019,34(10):1124−1128. doi: 10.19303/j.issn.1008-0384.2019.10.002
Citation: ZHANG J P, JIANG J X, LIN Y S, et al. Cloning and Expression of Specific Protein Gene, Mmc - 3740 , of Mycoplasma mycoides [J]. Fujian Journal of Agricultural Sciences,2019,34(10):1124−1128. doi: 10.19303/j.issn.1008-0384.2019.10.002

丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析

doi: 10.19303/j.issn.1008-0384.2019.10.002
基金项目: 国家重点研发计划项目(2016YFD0500906);福建省农业科学院科技创新团队建设项目(2017-09);福建省科技计划公益类专项(2019R1026-17);福建省农业科学院青年人才创新基金(YC-20180-3)福建省农业科学院科技创新团队建设项目(PC2018-8)
详细信息
    作者简介:

    张靖鹏(1988−),男,硕士,主要从事动物传染病研究(E-mail: 870063543@qq.com

    通讯作者:

    胡奇林(1963−),男,硕士,研究员,主要从事动物传染病学和免疫学研究(E-mail: hql562713@163.com

  • 中图分类号: S 852.62

Cloning and Expression of Specific Protein Gene, Mmc-3740, of Mycoplasma mycoides

  • 摘要:   目的  构建表达丝状支原体山羊亚种特异性蛋白基因Mmc-3740的重组表达菌,为Mmc-3740蛋白的进一步研究与应用奠定基础。  方法  采用PCR技术从Mmc-47中扩增出该基因,进行克隆、测序,将克隆质粒与pET-28a分别双酶切,回收正确的片段进行连接构建重组表达质粒,转化至表达菌BL21(DE3)中,IPTG诱导表达,产物用SDS-PAGE 检测,用镍柱对其纯化后免疫小鼠获得高免血清,并进行Western-blot验证。  结果  成功构建了克隆和表达载体,重组菌表达目的蛋白大小约21 ku。表达产物通过镍柱纯化后可得到单一的目的蛋白条带,Western-blot检测结果显示所表达的目的蛋白有较好的免疫原性。  结论  成功在BL21(DE3)宿主菌内表达了Mmc-3740重组蛋白。
  • 图  1  目的片段的PCR扩增

    注:1. DNA 标准 DL2000;2. 目的基因片段;3. 阴性对照。

    Figure  1.  PCR amplification of target fragment

    Note: 1. DNA maker DL2000; 2. product of target gene fragments; 3. negative control.

    图  2  重组质粒的双酶切

    注:1. DNA 标准 DL5000;2. 重组质粒双酶切产物;3. 阴性对照。

    Figure  2.  Double-enzyme digestion of recombinant plasmids

    Note: 1. DNA maker DL2000; 2. products of recombinant plasmid digestion; 3. negative control.

    图  3  诱导表达的结果

    注:1. 蛋白分子量标准;2. 空载体;3. 诱导的pET-28a-3740。

    Figure  3.  Results of induced expression

    Note: 1. protein molecular weight marker; 2. blank vector; 3. expression vector pET-28a-3740.

    图  4  重组蛋白Western bolt结果

    注:1.蛋白分子量标准;2. 重组蛋白与其高免血清的免疫印迹;3.阴性对照。

    Figure  4.  Immunoblotting of recombinant protein

    Note:1. protein molecular weight marker; 2. immunoblotting of recombinant protein with its antiserum; 3. negative control.

  • [1] KUMAR H, PARIHAR N S, CHARAN K, et al. Clinical pathology of intratracheal Mycoplasma mycoides subsp. capri infection in goats [J]. Indian Journal of Animal Sciences, 2013, 65(2): 133−136.
    [2] COTTEW G S, BREARD A, DAMASSA A J, et al. Taxonomy of the Mycoplasma mycoides cluster. [J]. Israel Journal of Medical Sciences, 1987, 23(6): 632−635.
    [3] FISCHER A, SHAPIRO B, MURIUKI C, et al. The origin of the ‘Mycoplasma mycoides cluster’ coincides with domestication of ruminants [J]. PLoS One, 2012, 7(4): e36150. doi: 10.1371/journal.pone.0036150
    [4] KINDE H, DAMASSA A J, WAKENELL P S, et al. Mycoplasma infection in a commercial goat dairy caused by Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (caprine biotype) [J]. Journal of Veterinary Diagnostic Investigation, 1994, 6(4): 423−427. doi: 10.1177/104063879400600404
    [5] HERNANDEZ L, LOPEZ J, ST-JACQUES M, et al. Mycoplasma mycoides subsp. capri associated with goat respiratory disease and high flock mortality [J]. Can Vet J, 2006, 47(4): 366−369.
    [6] CHU Y F, ZHAO P, GAO P C, et al. Mycoplasmas and mycoplasmosis in sheep and goats [J]. Animal Husbandry and Veterinary, 2010, 2(3): 128−131.
    [7] THIAUCOURT F, BOLSKE G, LENEGUERSH B, et al. Diagnosis and control of contagious caprine pleuropneumonia [J]. Revue Scientifique et Technique (International Office of Epizootics), 1996, 15(4): 1415−1429.
    [8] 朱建勋, 郝华芳, 陈胜利, 等. 山羊传染性胸膜肺炎分子诊断方法的研究进展 [J]. 畜牧与兽医, 2018, 50(6):132−136.

    ZHU J X, HAO H F, CHEN S L, et al. Progress in research on molecular diagnosis of contagious caprine pleuropneumonia in goats [J]. Animal Husbandry & Veterinary Medicine, 2018, 50(6): 132−136.(in Chinese)
    [9] THIAUCOURT F, MANSO-SILVAN L, SALAH W, et al. Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective [J]. BMC Genomics, 2011, 12(1): 114. doi: 10.1186/1471-2164-12-114
    [10] 朱淑萍. 丝状支原体山羊亚种乳酸脱氢酶的克隆表达及其免疫学特性的研究[D]. 重庆: 西南大学, 2014.

    ZHU S P. Cloning, expression and immunological characterization of lactate dehydrogenae of Mycoplasma Mycoplasma mycoides subsp. capricolum [D]. Chongqing: Southwest University, 2014. (in Chinese)
    [11] 张靖鹏, 江锦秀, 林裕胜, 等. 丝状支原体山羊亚种特异性蛋白基因Mmc-3740生物信息学分析 [J]. 福建农业学报, 2018, 33(9):883−887.

    ZHANG J P, JIANG J X, LIN Y S, et al. Bioinformatics of Specific Gene Mmc-3740 of Mycoplasma mycoides subsp. capri [J]. Fujian Journal of Agricultural Sciences, 2018, 33(9): 883−887.(in Chinese)
    [12] 党斌, 潘玉坤, 吴禹熹. 山羊主要支原体感染的研究 [J]. 黑龙江畜牧兽医, 2016(1):155−158.

    DANG B, PAN Y K, WU Y X. Study on Infection of Mycoplasma in Goats [J]. Heilongjiang Animal Science and Veterinary Medicine, 2016(1): 155−158.(in Chinese)
    [13] 韩赟, 郭晗, 逯忠新. 青海地区丝状支原体山羊亚种的流行病学调查 [J]. 现代农业科技, 2010(2):325, 327.

    HAN Y, GUO H, LU Z X. Serological investigation of Mycoplasma. mycoides subsp.Capri. mmc in Qinghai region [J]. Modern Agricultural Sciences and Technology, 2010(2): 325, 327.(in Chinese)
    [14] 凤英, 高娃, 陈伟, 等. 一株致病性丝状支原体山羊亚种支原体的分离鉴定 [J]. 畜牧与饲料科学, 2015, 36(12):4−8. doi: 10.12160/j.issn.1672-5190.2015.12.002

    FENG Y, GAO W, CHEN W, et al. Characterization of a Pathogenic Mycoplasma mycoides subsp.capri Isolate [J]. Animal Husbandry and Feed Science, 2015, 36(12): 4−8.(in Chinese) doi: 10.12160/j.issn.1672-5190.2015.12.002
    [15] 江锦秀, 林裕胜, 游伟, 等. 福建省羊支原体性肺炎分子流行病学研究 [J]. 福建农业学报, 2017, 32(1):12−16.

    JIANG J X, LIN Y S, YOU W, et al. A molecular epidemiological study on Mycoplasma pneumonia of sheep and goats in Fujian [J]. Fujian Journal of Agricultural Sciences, 2017, 32(1): 12−16.(in Chinese)
    [16] 张建华. 山羊传染性胸膜肺炎病原的分离及其中国分离株的分子特征分析[D]. 北京: 中国农业科学院, 2007.

    ZHANG J H. Isolation of Contagious Caprine Pleuropneumonia Pathogen and Molecular Characteristic Analysis of China Strains [D]. Beijing: Chinese Academy of Agricultural Sciences, 2007. (in Chinese)
    [17] 田青. 丝状支原体山羊亚种PG3分离培养及LppA基因克隆[D]. 重庆: 西南大学, 2006.

    TIAN Q. Isolation and Culture of Mycoplasma Mycoides Subsp. capri and Cloning of LppA Gene[D]. Chongqing: Southwest University, 2006. (in Chinese)
    [18] 徐春光, 郝永清, 郝瑞霞, 等. 丝状支原体山羊亚种PG3株的全基因组序列测定与分析 [J]. 微生物学通报, 2017, 44(11):2669−2678.

    XU C G, HAO Y Q, HAO R X, et al. Complete genome sequence and analysis of Mycoplasma mycoides subsp. capri str. PG3 [J]. Microbiology China, 2017, 44(11): 2669−2678.(in Chinese)
    [19] 张玲, 郝永清, 徐春光, 等. 丝状支原体山羊亚种脂蛋白LPPA基因的定点突变及原核表达 [J]. 畜牧与兽医, 2013, 45(10):74−76.

    ZHANG L, HAO Y Q, XU C G, et al. Site-directed mutagenesis and prokaryotic expression of LppA Gene of Mycoplasma Mycoides Subsp. capri [J]. Animal Husbandry & Veterinary Medicine, 2013, 45(10): 74−76.(in Chinese)
    [20] 储岳峰, 逯忠新, 赵萍, 等. 丝状支原体山羊亚种抗体间接血凝检测方法的建立及应用 [J]. 畜牧与兽医, 2007, 39(10):64−66. doi: 10.3969/j.issn.0529-5130.2007.10.022

    CHU Y F, LU Z X, ZHAO P, et al. Establishment and Application of Indirect Hem agglutination Detection of Mycoplasma Mycoides Subsp. capri [J]. Animal Husbandry & Veterinary Medicine, 2007, 39(10): 64−66.(in Chinese) doi: 10.3969/j.issn.0529-5130.2007.10.022
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出版历程
  • 收稿日期:  2019-07-15
  • 修回日期:  2019-09-01
  • 刊出日期:  2019-10-01

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