猪多杀性巴氏杆菌皮肤坏死毒素亚单位蛋白的制备及免疫原性研究

doi:10.16431/j.cnki.1671-7236.2019.11.030

Abstract

Abstract: In order to obtain the high immunogenicity antigen of the main pathogen of porcine atrophic rhinitis,Pasteurella multocida toxin,the subunit protein antigen of the Pasteurella multocida toxin was constructed,expressed and verified.The PMT gene of porcine Pasteurella multocida toxin was linked and transformed to pMD19-T vector,and identified by sequence analysis.BamHⅠ,HindⅢ and BlpⅠ restriction endonucleases were used to digest three gene fragments.Fragment 1 (Tox1),fragment 2 (Tox2) and fragment 3 (Tox3) were subcloned into prokaryotic expression vectors pET32-b,pET32-a and pET32-b,respectively,and three recombinant expression vectors were constructed.The recombinant expression vector was transformed into E.coli BL21 (DE3) competent cells.After induction by IPTG,SDS-PAGE and Western blotting were performed respectively.The immunogenicity of the recombinant expression vector was preliminarily studied in mice and guinea pigs.The results showed that the length of three gene fragments were 776,409 and 410 bp,which were highly homologous with the related sequences in GenBank.Three protein fragments were expressed normally,with the expression levels reaching 379.95,447.62 and 459.82 μg/mL,respectively.The SDS-PAGE validation bands were 75,77 and 53 ku,respectively.Because of the different positions of the three fragments,only Tox3 had Western blotting detection bands,which was consistent with the theoretical prediction.After immunizing mice and guinea pigs with three expressed proteins,the positive rate of serum was 100% on the 14th day after immunization,and the protected rate was 93% to mice on the 14th day after immunization.In this study,three subunit active fragments of PMT were successfully constructed,and they had good immunogenicity.

Key words: Pasteurella multocida toxin (PMT); subunit active fragment; immunogenicity

CLC Number:

S858.28

HE Hai, HAO Chengwu, LING Chen, ZHANG Fei, HOU Feng, HE Sun. Study on Preparation and Immunogenicity of Subunit Proteins of Pasteurella multocida Toxin from Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3396-3403.

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Study on Preparation and Immunogenicity of Subunit Proteins of Pasteurella multocida Toxin from Pigs

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