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Mycoplasma hyopneumoniae P97R1 gene recombined Pichia pastoris and expression protein

A technology of Mycoplasma hyopneumoniae and Pichia hyopneumoniae is applied in the construction and expression of Mycoplasma hyopneumoniae P97R1 Pichia saccharomyces expression vector, Mycoplasma hyopneumoniae P97R1 gene recombination Pichia saccharomyces and the field of expression protein, which can solve the problems of application interference, cumbersome purification and the like , to achieve the effect of easy construction, good antigenicity, and favorable purification

Inactive Publication Date: 2009-10-28
JIANGSU ACAD OF AGRI SCI
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Problems solved by technology

[0004] The current research is mainly to express it in Escherichia coli, and most of the expressed proteins exist in the form of fusion proteins, which may interfere with subsequent applications, and due to the presence of Escherichia coli components, the expressed protein needs cumbersome purification to be used in later research

Method used

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  • Mycoplasma hyopneumoniae P97R1 gene recombined Pichia pastoris and expression protein
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  • Mycoplasma hyopneumoniae P97R1 gene recombined Pichia pastoris and expression protein

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Embodiment Construction

[0045] The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0046] 1. Primer Design

[0047] According to the Mycoplasma hyopneumoniae P97R1 gene sequence (AE017243) reported in GenBank, two primers were designed with reference to the partial codons of Pichia pastoris and synthesized by TaKaRa Company. The primer sequences are:

[0048] P1: 5′ATA GAATTC TTACCTCAGCCGCCAGCAG 3′ EcoR I

[0049] P2: 5′CGC TCTAGA AAGCCATTGGGAAATAG 3′ Xba I

[0050] 2. Obtain the P97R1 target gene by PCR

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Abstract

The invention discloses Mycoplasma hyopneumoniae P97R1 gene recombined Pichia pastoris and expression protein, and relates to the field of bioprotein preparation. The invention is mainly obtained by gene acquisition, establishment of an expression vector and expression of target protein. A method for preparing the protein comprises the following steps that: a PCR method and uses a designed primer to obtain the target protein from Mycoplasma hyopneumoniae genome DNA by amplification; the target gene is cloned into a yeast expression vector pPICZalpha-A through enzyme restriction and connection; a secretion type recombined yeast expression vector pPICZalpha-A / P97R1 is established; and the pPICZalpha-A / P97R1 is electrically converted into a Pichia pastoris strain GS115 through Sac I enzyme restriction and linearization. The recombined Pichia pastoris expresses the P97R1 protein through methanol induction and secretion. The Mycoplasma hyopneumoniae P97R1 protein prepared by the method is pure, has good immunoreaction, and can be used for researching Mycoplasma hyopneumoniae P97 protein and developing Mycoplasma hyopneumoniae immunodetection kits and genetic engineering vaccines.

Description

1. Technical field [0001] The invention relates to a recombinant Pichia pastoris and expressed protein of mycoplasma hyopneumoniae P97R1 gene, belonging to the technical field of biological protein preparation. In particular, it relates to the construction and expression method of the expression vector of mycoplasma hyopneumoniae P97R1 Pichia pastoris. The prepared protein can be used for P97R1 research, kit and development of genetic engineering vaccine. 2. Background technology [0002] Mycoplasma hyopneumoniae (Mhp) is the pathogen that causes swine asthma (MPS). The latter is one of the most prevalent, fastest-spreading, and most difficult diseases in pig herds. Mycoplasma hyopneumoniae spreads through the respiratory tract. After infecting the respiratory epithelium, the cilia of the respiratory tract will shrink, fall off, and be damaged. According to reports, the disease can reduce the feed conversion rate of pigs by 10%, reduce the growth rate by 12% to 15%, and p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P21/02C12R1/84
Inventor 刘茂军邵国青祝永琴冯志新王海燕吴叙苏甘源
Owner JIANGSU ACAD OF AGRI SCI
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