ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2012, Vol. 43 ›› Issue (3): 431-437.doi:

• 预防兽医 • Previous Articles     Next Articles

Expression of P46 Protein of Mycoplasma hyopneumoniae in Escherichia coli and Used for Indirect ELISA

SHEN Qingchun1,2, WANG Fang2, HAN Mingyuan2, QIN Qingsong2,
FAN Xuezheng2, YANG Hanchun1*, NING Yibao2*   

  1. 1. Key Laboratory of Zoonosis of Ministry of Agriculture, China Agricultural University, Beijing 100193 , China; 2.Institute of Veterinary Drug Control of China, Beijing 100081, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-03-28 Published:2012-03-28
  • Contact: YANG Hanchun, NING Yibao

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Abstract: Mycoplasma hyopneumoniae is the agent of Enzootic pneumonia of swine. Part of the hydrophilic region of the surface protein P46 of M. hyopneumoniae was cloned, and three TGA coding Trp in the ORF of P46 gene were point mutated to TGG. The modified DNA fragement was coloned to vector pET28a(+), and highefficiency expressed in E. coli BL21 (DE3). SDSPAGE showed that the target DNA was expressed in inclusion body, the molecular weight of the recombinant protein was 31 kDa, owed 35% of the total proteins. The recombinant protein was proved to be well reacted with the high immune serum against M. hyopneumoniae from rabbit specially by western blotting. After wash and purification with Ni2+ column, the recombinant protein was used as coating antigen of indirect ELISA for detection the antibody against M. hyopneumoniae in swine serum. The ELISA detection kid was developed by optimizing the relational parameters and reagents. The further compare detection test indicated the recombinant P46 protein based ELISA was satisfactory and high complicated with the marketable products.