汉赛巴尔通体17-kDa蛋白的表达及间接ELISA方法的建立  

Expression of 17-kDa Protein of Bartonella henselae and Its Usage in the Development of an Indirect ELISA

作　　者：程王琨[1] 常晨[1] 马培培[1] 陈攀[1] 郑芳园[1] 李玉峰[1] 

机构地区：[1]南京农业大学农业部动物细菌学重点开放实验室,南京210095

出　　处：《畜牧兽医学报》2013年第10期1616-1621,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：公益性行业(农业)科研专项(200903036-10)

摘　　要：为探讨中国猪群中是否存在巴尔通体感染情况,作者将汉赛巴尔通体17-kDa蛋白基因按大肠杆菌密码子偏嗜性改造后进行全基因人工合成,然后将目的基因克隆到原核表达载体pET-28a中,构建重组质粒pET-28a-17kDa,导入大肠杆菌BL21感受态细胞中,进行诱导表达,并用Ni-NTA纯化试剂进行纯化;纯化后的蛋白应用His单抗进行鉴定。以17-kDa蛋白作为包被抗原,优化ELISA反应条件,建立检测抗汉赛巴尔通体17-kDa蛋白抗体的间接ELISA方法。结果如下:抗原包被量0.4μg·mL-1,37℃包被2h后4℃过夜,1%BSA 37℃封闭3h,待检血清1∶100稀释后,37℃孵育1h,二抗1∶15 000稀释,37℃作用30min,抗体临界值OD450nm≥0.392判为阳性,OD450nm≤0.332 6判为阴性,介于二者之间为可疑。建立的ELISA与猪嗜血支原体、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、猪伪狂犬病病毒、猪口蹄疫病毒等感染血清无交叉反应,批间和批内重复性较好,对379份血清样品进行检测,抗体阳性检出率达51.72%。该方法可用于汉赛巴尔通体抗体检测和流行病学调查。In order to investigate the epidemiology of Bartonella henselae infection, the 17-kDa protein gene of B. henselae was cloned into a prokaryotic expression vector （pET-28a） and con- structed a recombinant plasmid pET-28a-17kDa. Then the pET-28a 17kDa was transformed into competent E. coli strain （BL21）. The 17-kDa protein was expressed with IPTG induction and purified with nickel-agarose by affinity chromatography. Moreover, an indirect ELISA assay was developed to detect anti-B, henselae antibody using the purified protein as coating antigen. The optimized reaction conditions were as follows： antigen protein was coated at 37 ℃ for 2 h with 0.4μg. mL 1 concentration and then stored overnight at 4 ℃ ; The plates were blocked with 10/oo BSA at 37 ℃ for 3 h; The serum sample was incubated at 37 ℃ for 1 h with 1：100 dilution and secondary antibody was incubated at 37 ℃ for 0.5 h with 1 ： 15 000 dilution. The cutoff values of positive and negative results were OD450nm≥ 0. 392 and OD450 nm ≤ 0. 332 6, respectively. The specificity results showed that the protein could not react with positive sera of other six diseases（PRRSV, PCV2, CSFV, PRV, FMDV, M. suis） and the repeatability of intra-and inter-assay were excellent. Furthermore, total of 379 clinical sera samples obtained from swine farms were detected and the positive rate was 51.72%. In conclusion, the results showed that the indirect ELISA could be used for epidemiological surveys and diagnosis on Bartonella henselae.

关 键 词：巴尔通体 17-kDa蛋白 间接ELISA 

分 类 号：S852.64[农业科学—基础兽医学]