鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2023.03.036

摘要/Abstract

摘要： 【目的】试验旨在建立以鸡白痢沙门氏菌的毒力相关基因所编码的IPAJ蛋白为抗原的间接ELISA方法。【方法】PCR扩增IPAJ基因并连接到pCzn1表达载体上,转化大肠杆菌BL21(DE3)感受态细胞,IPAJ基因重组蛋白用IPTG进行诱导表达并纯化,通过Western blotting验证蛋白的反应原性。用纯化的IPAJ蛋白免疫60日龄试验兔,共免疫3次,制备其多克隆抗体,通过ELISA方法检测抗体效价。采用方阵滴定法优化以IPAJ蛋白作为诊断抗原的ELISA条件,初步建立以IPAJ蛋白为诊断抗原的间接ELISA方法,并与平板凝集试验结果进行比较。【结果】试验成功构建鸡白痢沙门氏菌IPAJ原核表达载体,并以包涵体形式纯化出重组蛋白,具有良好的反应原性。Western blotting分析结果显示,成功表达出32 ku的IPAJ蛋白,具有良好的特异性。间接ELISA检测结果显示,多克隆抗体效价为1∶25600,表明成功制备出多克隆抗体。建立的以IPAJ蛋白为诊断抗原的间接ELISA方法的最佳优化条件为:抗原包被浓度为5 μg/mL,5%脱脂奶粉封闭1.5 h,一抗最佳稀释浓度为1∶250(孵育1 h),二抗最佳稀释浓度为1∶10000(孵育1 h),避光显色15 min。与平板凝集试验结果的符合率为91.00%。【结论】鸡白痢沙门氏菌IPAJ重组蛋白具备良好的特异性及反应原性,初步建立的鸡白痢沙门氏菌间接ELISA诊断法具有一定的实用性,可应用于临床大批量检测,为后续建立快捷的诊断方法提供依据。

关键词: 鸡白痢沙门氏菌; IPAJ蛋白; 多克隆抗体; 间接ELISA

Abstract: 【Objective】 This study was aimed to establish an indirect ELISA method using IPAJ protein encoded by virulence related genes of Salmonella Pullorum as antigen.【Method】 The IPAJ gene was amplified by PCR and connected to pCzn1 expression vector to transform into Escherichia coli BL21 (DE3) competent cells.The recombinant protein of IPAJ gene was induced to express by IPTG and purified,and the reactivity of the protein was verified by Western blotting.The purified IPAJ protein was used to immunize rabbits at 60 days of age for three times to prepare polyclonal antibodies.The antibody titer was detected by ELISA method.ELISA conditions of IPAJ protein as diagnostic antigen were optimized by square titration.An indirect ELISA method using IPAJ protein as diagnostic antigen was established and compared with the results of plate agglutination assay.【Result】 The IPAJ prokaryotic expression vector of Salmonella Pullorum was successfully constructed,and the recombinant protein was purified as inclusion body with good reactivity.Western blotting analysis results showed that IPAJ protein was successfully expressed at 32 ku,indicating good specificity.Indirect ELISA results showed that the titer of polyclonal antibody against IPAJ was 1∶25 600,indicating that polyclonal antibody was successfully prepared.The optimal condition for indirect ELISA using IPAJ protein as diagnostic antigen was preliminarily established as follows:The antigen coating concentration was 5 μg/mL,and 5% skim milk powder was used for 1.5 h,the optimum dilution of the primary antibody was 1∶250 (incubated for 1 h),the optimum dilution of the secondary antibody was 1∶10 000 (incubated for 1 h),and the color was dark for 15 min.Compared with plate agglutination assay results,the coincidence rate was 91.00%.【Conclusion】 The recombinant protein of Salmonella Pullorum IPAJ had good specificity and reactivity.The indirect ELISA method preliminarily established for the diagnosis of Salmonella Pullorum had certain practicability,which could be applied to clinical mass detection,and provide a basis for the establishment of a rapid diagnostic method.

Key words: Salmonella Pullorum; IPAJ protein; polyclonal antibody; indirect ELISA

中图分类号:

S816.48

牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.

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.

参考文献

[1] 陈飞,成大荣,田志高,等.鸡蛋中沙门氏菌的快速检测[J].江苏农业科学,2009,5:284-285. CHEN F,CHENG D R,TIAN Z G,et al.Rapid detection of Salmonella in eggs[J]. Jiangsu Agricultural Sciences,2009,5:284-285.(in Chinese)
[2] AHIER C.Genetic and environmental control of Salmonella invasion[J]. Journal of Microbiology,2005,43:85-92.
[3] 刘德.鸡白痢的临床症状与中西医治疗[J].养殖技术顾问,2014,6:200-201. LIU D.Clinical symptoms and treatment of pullorosis in chicken with traditional Chinese and Western medicine[J]. Aquaculture Technology Consultant,2014,6:200-201.(in Chinese)
[4] KOSA K M,CATES S C,BRADLEY S,et al.Consumer-reported handling of raw poultry products at home:Results from a national survey[J].Journal of Food Protection,2015,78(1):180-186.
[5] 陈伟,赵学峰,徐鸿,等.山东省部分规模化鸡场鸡白痢血清学调查[J].家禽科学,2021,10:38-41. CHEN W,ZHAO X F,XU H,et al.Serological investigation on pullorosis in some large-scale chicken farms in Shandong province[J].Poultry Science,2021,10:38-41.(in Chinese)
[6] GAO W,HUANG H,ZHU P,et al.Recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of Salmonella,in shellfish[J].Bioprocess & Biosystems Engineering,2018,41(5):603-611.
[7] ARORA D,KUMAR S,JINDAL N,et al.Prevalence and epidemiology of Salmonella enterica serovar Gallinarum from poultry in some parts of Haryana,India[J]. Veterinary World,2015,8(11):1300-1304.
[8] BARROW P A,JONES M A,SMITH A L,et al.The long view:Salmonella-the last forty years[J].Avian Pathology,2012,41(5):413-420.
[9] 任行星.广东省动物源沙门氏菌流行特点及HRM检测鸡白痢和鸡伤寒沙门氏菌方法的建立[D].广州:华南农业大学,2016. REN X X.Epidemic characteristics of Salmonella from animals in Guangdong province and establishment of HRM method for detection of Salmonella pullorum and Salmonella typhimurium[D].Guangzhou:South China Agricultural University,2016.(in Chinese)
[10] 徐耀辉,焦新安,李求春,等.鸡白痢沙门氏菌基因组差异片段的筛选与分析[J].中国兽医科学,2007,37(7):563-567. XU Y H,JIAO X A,LI Q C,et al.Screening and analysis of different genomic fragments of Salmonella pullorum[J].Veterinary Science in China,2007,37(7):563-567.(in Chinese)
[11] LI Q,HU Y,XU Y,et al.A gene knock-in method used to purify plasmid pSPI12 from Salmonella enterica serovar Pullorum and characterization of IpaJ[J].Journal of Microbiological Methods,2014,98:128-133.
[12] 彭永刚,于倩,刘建华.乌鲁木齐某农场沙门氏菌的分离鉴定[J].中国动物检疫,2014,31(3):55-57. PENG Y G,YU Q,LIU J H.Isolation and identification of Salmonella from a farm in Urumqi[J]. China Animal Quarantine,2014,31(3):55-57.(in Chinese)
[13] 马晨,陈雪华,李建国.国标法与快速法检测沙门氏菌的结果比较[J].食品科技,2015,9:283-289. MA C,CHEN X H,LI J G.Comparison of results between national standard method and rapid method for Salmonella detection[J]. Food Science and Technology,2015,9:283-289.(in Chinese)
[14] YIN C,XU L,LI Y,et al.Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as animmune response modulator[J]. Avian Pathology,2018,47(4):410-417.
[15] LI Q C,XU Y H,JIAO X A.Identification of Salmonella pullorum genomic sequences using suppression subtractive hybridization[J].Journal of Industrial Microbiology & Biotechnology,2009,19(9):898-903.
[16] LI Q,XU Y,HUANG J,et al.Cloning and identification of IPAJ gene in Salmonella pullorum[J].Acta Microbiologica Sinica,2010,50(11):1545-1549.
[17] XU L J,LIU Z J,LI Y,et al.A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorun using a specific target gene IPAJ[J].Avian Pathology:Journal of the WVPA,2018,47(3):238-244.
[18] 朱春红,吴娟,张伟娟,等.肠炎沙门氏菌SEFA基因表达和间接ELISA检测方法的初步建立[J].中国预防兽医学报,2010,32(1):44-48. ZHU C H,WU J,ZHANG W J,et al.Preliminary establishment of SEFA gene expression and indirect ELISA method for detection of Salmonella enteritidis[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(1):44-48.(in Chinese)
[19] MA Z,YANG X,FANG Y,et al.Detection of Salmonella infection in chickens by an indirect enzyme-linked immunosorbent assay based on presence of PagC antibodies in sera[J].Foodborne Pathogens and Disease,2018,15(2):109-113.
[20] GONG J S,ZHANG J Q,XU M,et al.Prevalence and fimbrial genotype distribution of poultry Salmonella isolates in China(2006 to 2012)[J].Applied and Environmental Microbiology,2014,80(2):687-693.