丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2022.01.034

Abstract

Abstract: [Objective] The purpose of this study was to establish an indirect ELISA method based on Mycoplasma mycoides subsp. capri (Mmc) membrane lipoprotein LPPA. [Method] In this study, the Mmc LPPA gene was amplified and cloned into pCold-Ⅰ vector. The recombinant plasmid pCold-Ⅰ-LPPA was constructed. After sequencing identification, it was transformed into E. coli DH5α competent cells. The recombinant protein of Mmc LPPA was purified after induced expression by IPTG. The expression of the recombinant protein was verified by SDS-PAGE. Its reactivity with Mmc positive serum was analyzed by Western blotting test and traditional classical agar diffusion serological test. Using the recombinant protein as the coating antigen, an indirect ELISA antibody detection method for Mmc recombinant LPPA protein was established. After the reaction conditions were optimized, the specificity and repeatability tests were carried out, and it was preliminarily applied to 184 goat serum samples. [Result] The Mmc LPPA gene was amplified by PCR, and the recombinant plasmid pCold-Ⅰ-LPPA was successfully constructed. After induced expression, the recombinant protein Mmc LPPA was obtained. SDS-PAGE result showed that the recombinant fusion protein of LPPA with the size of about 23 ku was obtained. Agar diffusion and Western blotting showed that the protein had good reactivity. The optimized reaction conditions of ELISA showed that the coating concentration of LPPA antigen protein of 2.0 μg/100 μL, serum dilution of 1:300 and blocking with 3% BSA for 1 h were the optimum reaction conditions, and the critical value was 2.738. Intra assay and inter assay coefficients of variation of the indirect ELISA method established in this test were both less than 10%, which proved that the method had good repeatability. The standard positive sera of Mo, BTV, PPRV, FMDV, Toxoplasma gondii and GPV were negative, indicating that the method had strong specificity. The clinical application results of 184 clinical sera showed that the positive coincidence rate between the method and the positive indirect hemagglutination diagnostic kit was 93.33%, the negative coincidence rate was 75.23%, and the relative coincidence rate was 82.61%. [Conclusion] This study successfully established an indirect ELISA method for the detection of antibodies for Mmc LPPA protein, which laid a foundation for the monitoring of serum antibody level of clinical Mmc and the investigation of serum epidemiology of Mmc.

Key words: Mycoplasma mycoides subsp. capri (Mmc); LPPA protein; indirect ELISA; prokaryotic expression

CLC Number:

S852.62

CHEN Jing, WU Yan, LI Mei, YUE Jun, ZHU Erpeng, WEN Ming, ZHANG Shuangxiang, CHENG Zhentao. Prokaryotic Expression of LPPA Protein of Mycoplasma mycoides subsp. capri and Establishment of Indirect ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 318-327.

References

[1] 林裕胜,江锦秀,张靖鹏,等.丝状支原体山羊亚种SYBR Green Ⅰ qRT-PCR快速检测方法的建立[J].农业生物技术学报,2017,25(11):1895-1902. LIN Y S,JIANG J X,ZHANG J P,et al.Establishment of a SYBR Green Ⅰ qRT-PCR for rapid detection of Mycoplasma mycoides subsp.capri[J].Journal of Agricultural Biotechnology,2017,25(11):1895-1902.(in Chinese)
[2] 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,2017,9(2):156-158. WU J Y,SHANG Y J,CHEN Y,et al.Serological investigation of Mycoplasmal pneumonia of sheep and goats in China[J].Chinese Journal of Preventive Veterinary Medicine,2017,9(2):156-158.(in Chinese)
[3] 徐春光,郝永清,王明莹,等.羊支原体肺炎防治研究进展[J].动物医学进展,2014,35(1):81-85. XU C G,HAO Y Q,WANG M Y,et al.Advance in treatment and prevention of Mycoplasmal pneumonia of sheep and goat[J].Progress in Veterinary Medicine,2014,35(1):81-85.(in Chinese)
[4] 贾凡,王建银,胡军勇,等.猪胸膜肺炎放线杆菌ApxⅡ-ELISA抗体检测试剂盒的研制与应用[J].中国兽医学报,2009,29(3):283-287. JIA F,WANG J Y,HU J Y,et al.Development and application of ApxⅡ-ELISA diagnostic kit for Actinobacillus pleuropneumoniae[J].Chinese Journal of Veterinary Science,2009,29(3):283-287.(in Chinese)
[5] 吴娅琴,颜新敏,郝华芳,等.山羊传染性胸膜肺炎免疫学诊断方法的研究进展[J].中国兽医学报,2020,40(9):1876-1880. WU Y Q,YAN X M,HAO H F,et al.Advances in immunological diagnosis of contagious caprine pleuropneumonia[J].Chinese Journal of Veterinary Science,2020,40(9):1876-1880.(in Chinese)
[6] 王素果,曾焱华.支原体脂质相关膜蛋白致病机制的研究进展[J].微生物学免疫学进展,2016,44(4):42-47. WANG S G,ZENG Y H.Pathogenic mechanisms on Mycoplasma lipid-associated membrane proteins[J].Progress in Microbiology and Immunology,2016,44(4):42-47.(in Chinese)
[7] 麻昌姣,郑佳琪,黄海碧,等.绵羊肺炎支原体p60蛋白N端与C端的表达及其免疫学特性研究[J].中国畜牧兽医,2017,44(2):371-376. MA C J,ZHENG J Q,HUANG H B,et al.Expression of N-terminal domain and C-terminal domain of Mycoplasma ovipneumoniae p60 protein and initial identification on their immunologic activity[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):371-376.(in Chinese)
[8] 倪博,白方方,刘茂军,等.支原体脂蛋白及其变异与宿主互作研究进展[J].生物技术通报,2013,2:49-54. NI B,BAI F F,LIU M J,et al.Research progress on Mycoplasma lipoprotein and its variation interaction with host[J].Biotechnology Bulletin,2013,2:49-54.(in Chinese)
[9] 张懿,邢小勇,温峰琴,等.牛支原体pdhc基因的克隆、表达及其亚细胞定位[J].中国畜牧兽医,2018,45(9):2377-2385. ZHANG Y,XING X Y,WEN F Q,et al.Cloning,expression of pdhc gene of Mycoplasma bovis and its subcellular localization[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2377-2385.(in Chinese)
[10] 凌晨,郝成武,张飞,等.牛支原体新疆分离株P48基因克隆及分子特征分析[J].中国畜牧兽医,2021,48(2):457-466. LING C,HAO C W,ZHANG F,et al.Cloning and bioinformatics analysis of P48 gene of Mycoplasma bovis isolated strain in Xinjiang[J].China Animal Husbandry & Veterinary Medicine,2021,48(2):457-466.(in Chinese)
[11] 江锦秀,林裕胜,游伟,等.丝状支原体山羊亚种FJ-GT株的分离和鉴定[J].中国农学通报,2016,32(29):11-16. JIANG J X,LIN Y S,YOU W,et al.Isolation and identification of Mycoplasma mycoides subsp.capri FJ-GT[J].Chinese Agricultural Science Bulletin,2016,32(29):11-16.(in Chinese)
[12] MANIMARAN K,SINGH V P.Rapid detection of infection due to Mycoplasma mycoides subsp.capri in experimental goats by PCR by assay[J].Indian Journal of Animal Research,2017,52(5):758-760.
[13] LIN Y,JIANG J,ZHANG J,et al.Development of a TaqMan Real-time PCR for detection of the Mycoplasma mycoides subsp.capri[J].Small Ruminant Research,2019,175(6):31-36.
[14] 周而璇,范慧,延君芳,等.猪塞尼卡病毒VP2 ELISA抗体检测方法的建立及初步应用[J].中国预防兽医学报,2020,42(11):1123-1127. ZHOU E X,FAN H,YAN J F,et al.Development of an indirect ELISA assay for detecting the antibodies against VP2 of Senecavirus A[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(11):1123-1127.(in Chinese)
[15] 易华山,赵瑶,马鲜平,等.基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用[J].畜牧兽医学报,2020,51(12):3133-3140. YI H S,ZHAO Y,MA X P,et al.Establishment and preliminary application of indirect ELISA based on recombinant NS4 protein for detection of Bluetongue virus antibodies[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):3133-3140.(in Chinese)
[16] KILIC A,EROKSUZ H,MUZ A,et al.Identification by culture,PCR,and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey[J].Tropical Animal Health Production,2013,45(7):1525-1531.
[17] 邹联斌,郭建刚,刘棋,等.应用聚合酶链反应检测绵羊肺炎霉形体[J].畜牧与兽医,2007,34(4):48-49. ZOU L B,GUO J G,LIU Q,et al.Detection of Mycoplasma ovipneumoniae by polymerase chain reaction[J].Animal Husbandry and Veterinary Medicine,2007,34(4):48-49.(in Chinese)
[18] 储岳峰,高鹏程,赵萍,等.应用双重PCR方法检测羊支原体肺炎病原[J].畜牧与兽医,2009,41(12):23-26. CHU Y F,GAO P C,ZHAO P,et al.Development of a duplex polymerase chain reaction assay for the detection of pathogens for Mycoplasmal pneumonia of sheep and goats[J].Animal Husbandry and Veterinary Medicine,2009,41(12):23-26.(in Chinese)
[19] 凌蒙蒙,孙艺学,邓效禹,等.猪伪狂犬病病毒gE37~243蛋白的原核表达及gE蛋白抗体间接ELISA的建立[J].中国兽医学报,2021,41(5):853-857. LING M M,SUN Y X,DENG X Y,et al.Establishment of an indirect ELISA for detection antibodies against Pseudorabies virus gE protein[J].Chinese Journal of Veterinary Science,2021,41(5):853-857.(in Chinese)
[20] MONNERAT M P,FRANCOIS T,POVEDA J B,et al.Genetic and serological analysis of lipoprotein LPPA in Mycoplasma mycoides subsp.mycoides LC and Mycoplasma mycoides subsp.capri[J].Clinical and Diagnostic Laboratory Immunology,2011,6(2):224-230.
[21] DEDIEU L,TOTTE P,RODRIGUES V,et al.Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides small colony identifies LPPA as a major T-cell antigen[J].Comparative Immunology,Microbiology and Infectious Diseases,2010,33(4):279-290.
[22] 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 mutation and prokaryotic expression of LPPA gene of Mycoplasma mycooides subsp.capriclonum[J].Animal Husbandry and Veterinary Medicine,2013,45(10):74-76.(in Chinese)
[23] 朱翔宇,蔡熙姮,鲁荣光,等.水貂细小病毒IgG抗体间接ELISA半定量检测方法的初步建立[J].中国兽医学报,2021,41(4):661-669. ZHU X Y,CAI X H,LU R G,et al.Development of an indrect ELISA-assay for semi-quantitative detection of specific IgG antibodies against Mink enteritis virus[J].Chinese Journal of Veterinary Science, 2021,41(4):661-669.(in Chinese)
[24] 钱炳旭,白雪雁,张成成,等.猪德尔塔冠状病毒抗体间接ELISA检测方法的建立及初步应用[J].中国兽医学报,2021,41(7):1234-1241. QIAN B X,BAI X Y,ZHANG C C,et al.Establishment and preliminary application of indirect ELISA for detection of Porcine deltacoronavirus antibody[J].Chinese Journal of Veterinary Science, 2021,41(7):1234-1241.(in Chinese)
[25] 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,2007,267(10):64-66. CHU Y F,LU Z X,ZHAO P,et al.Establishment and application of indirect hemagglutination detection method for Mycoplasma mycooides subsp.capriclonum antibody[J].Animal Husbandry and Veterinary Medicine,2007,34(10):64-66.(in Chinese)
[26] 李敏杰,许智强,刘彦玲,等.A型口蹄疫抗体固相竞争ELISA检测方法的建立[J].中国预防兽医学报,2020,42(9):899-904. LI M J,XU Z Q,LIU Y L,et al.A study of solid phase competition ELISA for detection of A-type Foot-and-mouth disease virus antibody[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(9):899-904.(in Chinese)
[27] 张焕容,张贤宇,杨发龙.基于重组P113蛋白的山羊抗绵羊肺炎支原体抗体间接ELISA检测方法的建立及应用[J].中国预防兽医学报,2020,42(12):1244-1249. ZHANG H R,ZHANG X Y,YANG F L,et al.Development of an indirect ELISA for detection of Mycoplasma ovipneumoniae antibody in goats based on recombinant protein P113[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(12):1244-1249.(in Chinese)

Prokaryotic Expression of LPPA Protein of Mycoplasma mycoides subsp. capri and Establishment of Indirect ELISA Method

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[5]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[6]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[7]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[8]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[9]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[10]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[11]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[12]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[13]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[14]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[15]	HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3508-3519.