Secretory Expression of Mycoplasma hyopneumoniae P97R1 Gene in Pichia pastoris and Primary Application of the Expression Product

[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae(Mhp) in Pichia pastoris expression system and the primary application of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 g...

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Bibliographic Details
Published inHunan agricultural science & technology newsletter : HASTN Vol. 14; no. 5; p. 710
Main Authors Liu, Maojun, Zhu, Yongqin, Feng, Zhixin, Wu, Xusu, Shao, Guoqing
Format Journal Article
LanguageEnglish
Published Changsha Wu Chu (USA-China) Science and Culture Media Corporation 01.05.2013
Subjects
Antibodies
Antigens
Gene amplification
Hogs
Infections
Plasmids
Proteins
Vaccines
Yeast
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Summary:[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae(Mhp) in Pichia pastoris expression system and the primary application of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZα-A yeast expression vector to construct the secretory recombinant expression vector pPICZα-A-P97R1. The plasmid pPICZα-A-p97R1 linearized by Sac I was transformed into P. pastoris GS115 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting analysis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secretory amount of 499µg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for antibody detection and genetically engineered vaccine to Mhp. [PUBLICATION ABSTRACT]
ISSN:1009-4229