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

Abstract

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.

CLC Number:

S852.64

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

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