汉赛巴尔通体17-kDa蛋白的表达及间接ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2013.10.015

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (10): 1616-1621.doi: 10.11843/j.issn.0366-6964.2013.10.015

汉赛巴尔通体17-kDa蛋白的表达及间接ELISA方法的建立

程王琨，常晨，马培培，陈攀，郑芳园，李玉峰^*

(南京农业大学农业部动物细菌学重点开放实验室，南京 210095)

收稿日期:2013-04-08 出版日期:2013-10-23 发布日期:2013-10-23
通讯作者: 李玉峰，副教授，E-mail: yufengli@njau.edu.cn, Tel: 025-84395504
作者简介:程王琨（1986- ），男，安徽潜山人，硕士研究生，主要从事畜禽传染病防治研究，E-mail: 20070590cwk@163.com
基金资助:
公益性行业（农业）科研专项（200903036-10）

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

CHENG Wang-kun, CHANG Chen, MA Pei-pei, CHEN Pan, ZHENG Fang-yuan, LI Yu-feng^*

(Key Laboratory of Animal Bacteriology of Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China)

Received:2013-04-08 Online:2013-10-23 Published:2013-10-23

摘要/Abstract

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0.4 μg·mL^－1,37 ℃包被2 h后4 ℃过夜，1%BSA 37 ℃封闭3 h，待检血清1∶100稀释后，37 ℃孵育1 h，二抗1∶15 000稀释，37 ℃作用30 min，抗体临界值OD_{450 nm}≥0.392判为阳性，OD_{450 nm}≤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。

Abstract:

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 constructed 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 0.4 μg·mL^－1 concentration and then stored overnight at 4 ℃; The plates were blocked with 1% 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 OD_{450 nm} ≥ 0.392 and OD_{450 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.

中图分类号:

S852.64

程王琨，常晨，马培培，陈攀，郑芳园，李玉峰. 汉赛巴尔通体17-kDa蛋白的表达及间接ELISA方法的建立[J]. 畜牧兽医学报, 2013, 44(10): 1616-1621.

CHENG Wang-kun, CHANG Chen, MA Pei-pei, CHEN Pan, ZHENG Fang-yuan, LI Yu-feng. Expression of 17-kDa Protein of Bartonella henselae and Its Usage in the Development of an Indirect ELISA[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1616-1621.

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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

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