马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2018.06.032

Abstract

Abstract:

In order to express Bc48 gene of Babesia caballi from Xinjiang Yili,and prepare its polyclonal antibody,a pair of specific primers was designed according to Bc48 gene sequence of Babesia caballi in GenBank.The genomic DNA was extracted from Babesia caballi,and Bc48 gene was cloned into pET-28a(+) expression vector.The positive plasmid was transformed to Escherichia coli BL21(DE3).After IPTG induction,the product was analyzed by SDS-PAGE,six weeks old female BALB/c mice were immunized with purified Bc48 protein to prepare polyclonal antibodies.Meanwhile Bc48 gene was cloned into eukaryotic expression vector pCMV-N-flag.The positive plasmid was transformed to Escherichia coli DH5α,eukaryotic expression of pCMV-N-flag-Bc48 plasmid was transfected into 293T cells.The results showed that the recombinant protein with 15 ku was obtained,which was consistent with the expected protein size;Western blotting analysis showed that polyclonal antibodies could specifically identify the corresponded antigens;ELISA analysis showed that the antibodies had high titer (1:128 000);And eukaryotic expression plasmids expressed Bc48 protein in 293T cells.This test could lay the foundation for further study on the functional genes and establish quick detection method of Babesia caballi.It had important significance in the study of strain taxonomy and the development of candidate vaccine.

Key words: Babesia caballi; Bc48 gene; prokaryotic expression; eukaryotic expression; polyclonal antibody

CLC Number:

S852.72

WANG Panju, AIRIDENG Caicike, XU Zhengmao, WEN Xiuxiu, DANG Nana, SONG Jingjing, ZHANG Mengyuan, BA Yinchahan. Cloning and Expression of Bc48 Gene of Babesia caballi Xinjiang Yili Strain and Preparation of Its Polyclonal Antibody[J]. , 2018, 45(6): 1677-1682.

References

[1] RVEGG S R,TORGERSON P,DEPLAZES P,et al.Age-dependent dynamics of Theileria equi and Babesia caballi infections in Southwest Mongolia based on IFAT and/or PCR prevalence data from domestic horses and ticks[J].Parasitology,2007,134(7):939-947.
[2] MACHADO R Z,TOLEDO C Z,TEIXEIRA M C,et al.Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil[J].Veterinary Parasitology,2012,186(3-4):461-465.
[3] BRVNING A,PHIPPS P,POSNETT E,et al.Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis[J].Veterinary Parasitology,1997,68(1-2):11-26.
[4] IKADAI H,TAMAKI Y,XUAN X,et al.Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi[J]. International Journal for Parasitology,1999,29(11):1785-1791.
[5] FRIEDHOFF K T,TENTER A M.Haemoparasites of equines:Impact on international trade of horses[J].Revue Scientifique Et Technique,1990,9(4):1187-1194.
[6] BASHIRUDDIN J B,CAMMÀ C,REBÊLO E.Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene[J].Veterinary Parasitology,1999,84(1-2):75-83.
[7] 罗金,刘光远,谢俊仁,等.驽巴贝斯虫病PCR检测方法的建立和应用[J].中国畜牧兽医,2011,38(7):193-195. LUO J,LIU G Y,XIE J R,et al.Establishment and application of Babesia caballi PCR detection method[J].China Animal Husbandry & Veterinary Medicine,2011,38(7):193-195.(in Chinese)
[8] LI Y,TENG L,SHANG L,et al.Detection of Theileria and Babesia sp in Ixodid ticks from Qinghai province,Northwestern China[J].Journal of Animal & Veterinary Advances,2013,12(6):775-778.
[9] 薛书江,于龙政,曹世诺,等.驽巴贝斯虫PCR检测方法的建立及应用[J].河南农业科学,2007,4:106-109. XUE S J,YU L Z,CAO S N,et al.Establishment and application of Babesia cballi PCR detection method[J].Journal of Henan Agricultural Sciences,2007,4:106-109.(in Chinese)
[10] 赵祥平,王玉玲,侯艳梅.驽巴贝斯虫病巢式PCR检测方法的研究[J].中国动物检疫,2009,26(10):37-38. ZHAO X P,WANG Y L,HOU Y M.A study on the detection method of Babesia caballi in the nested PCR[J].Chinese Animal Quarantine,2009,26(10):37-38.(in Chinese)
[11] 王群,郑小龙,朱来华,等.马媾疫锥虫SYBR GreenⅠ荧光定量PCR检测方法的建立[J].中国预防兽医学报,2012,34(9):724-727. WANG Q,ZHENG X L,ZHU L H,et al.Establishment of Trypanosoma equiperdum SYBR Green Ⅰ Real-time fluorescence quantitative PCR method[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(9):724-727.(in Chinese)
[12] FRIEDHOFF K T.Piroplasmas of horses——Impact on the international horse trade[J].Berl Munch Tierarztl Wochenschr,1982,95(19):368-374.
[13] IKADAI H,XUAN X,IGARASHI I,et al.Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay[J].Journal of Clinical Microbiology,1999,37(11):3475-3480.
[14] BÖSE R,PEYMANN B.Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay (ELISA) and Western blot[J].International Journal for Parasitology,1994,24(3):347-356.
[15] SAM YELLOWE T Y.Rhoptry organelles of the apicomplexa:Their role in host cell invasion and intracellular survival[J].Parasitol Today,1996,12(8):308-316.
[16] 高慎阳,查恩辉,王珅,等.一种"高性价比"切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26. GAO S Y,CHA E H,WANG K,et al.A method of purifying prokaryotic expression protein with high cost performance SDS-PAGE cutting[J].Chinese Agricultural Science Bulletin,2010,26(22):24-26.(in Chinese)
[17] 吕绘倩,蒋洁兰,姜志强,等.太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备[J].大连海洋大学学报,2017,32(2):127-133. LV H Q,JIANG J L,JIANG Z Q,et al.Prokaryotic expression of the antifreeze gene and preparation of polyclonal antibody[J].Journal of Dalian Fisheries University,2017,32(2):127-133.(in Chinese)
[18] MEHLHORN H,SCHEIN E.Redescription of Babesia equi Laveran,1901 as Theileria equi Mehlhorn,Schein 1998[J].Parasitology Research,1998,84(6):467-475.
[19] UILENBERG G.Babesia——A historical overview[J].Veterinary Parasitology,2006,138(1-2):3-10.
[20] 张杨.马驽巴贝斯虫二温式、荧光PCR检测方法的建立及其初步应用[D].乌鲁木齐:新疆农业大学,2015. ZHANG Y.Establishment of a two temperature PCR assay and Real-time fluorescence quantitative PCR method for detection of Babesia caballi[D].Urumqi:Xinjiang Agricultural University,2015.(in Chinese)
[21] SCHWINT O N,UETI M W,PALMER G H,et al.Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential[J].Antimicrobial Agents & Chemotherapy,2009,53(10):4327-4332.
[22] 薛书江,于龙政,曹世诺,等.驽巴贝虫BC-48基因的克隆及其在大肠杆菌中的表达[J].中国兽医科学,2007,37(3):214-217. XUE S J,YU L Z,CAO S N,et al.Cloning and expression of BC-48 gene of Babesia caballi Jilin strain and its expression in Escherichia coli[J].Chinese Veterinary Science,2007,37(3):214-217.(in Chinese)

Cloning and Expression of Bc48 Gene of Babesia caballi Xinjiang Yili Strain and Preparation of Its Polyclonal Antibody

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]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[5]	PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.
[6]	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.
[7]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[8]	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.
[9]	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.
[10]	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.
[11]	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.
[12]	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.
[13]	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.
[14]	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.
[15]	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.