Mycoplasma hyopneumoniae P36 gene recombined Pichia pastoris and expression protein

A technology of Mycoplasma hyopneumoniae and Pichia pastoris, applied in recombinant DNA technology, fermentation, microorganism-based methods, etc., can solve problems such as application interference, cumbersome purification, etc., and achieve the effects of easy construction, favorable purification, and good antigenic reactivity

Inactive Publication Date: 2009-10-28
JIANGSU ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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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

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  • Mycoplasma hyopneumoniae P36 gene recombined Pichia pastoris and expression protein
  • Mycoplasma hyopneumoniae P36 gene recombined Pichia pastoris and expression protein
  • Mycoplasma hyopneumoniae P36 gene recombined Pichia pastoris and expression protein

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Embodiment Construction

[0037] The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0038] 1. Primer Design

[0039] According to the complete gene sequence of Mycoplasma hyopneumoniae P36 (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:

[0040] P1: 5′-CCC GAATTC ATGAAACCTATTAAAATAGCTCT-3′ EcoR I

[0041] P2: 5′-GCG TCTAGA TTAAATATTTTTAATTGCATCCTGAT-3′XbaI

[0042] 2. Obtain the P36 target gene by PCR

[0043] Using P1 and P2 as primers, the genomic DNA of Mycoplasma hyopneumoniae JS99 strain (from the National Veterinary Microbiological Culture Collection Management Center) was used as a template for PCR amplification. Reaction system (50 μL): 10×PCR Buffer 5 μL, MgCl 2 (25mmol / L) 2μL, dNTP (10mmol / L) 3μL, primer P1, P2 (20pmol / L) each 0.5μL, Taq TM 0.5 μL, template 4 μ...

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Abstract

The invention discloses Mycoplasma hyopneumoniae P36 gene recombined Pichia pastoris and expression protein, and relates to the field of bioprotein preparation. A PCR method and a primer are designed to obtain a P36 mesh gene, the P36 mesh gene is cloned into a pPICZalpha-A expression vector, recombinant expression plasmid pPICZalpha-A / P36 is converted into competent Pichia pastoris, and recombinant pichia pastoris X-33 / pPICZalpha-A / P36 is screened. A method for preparing the expression protein comprises that: positive yeast is inoculated into BMGY, and the expression protein is in supernatant in a soluble form through methanol induction expression. The Mycoplasma hyopneumoniae P36 protein prepared by the method is pure and has good immunoreaction; and the expression protein can be used for researching P36 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 P36 gene, belonging to the technical field of biological protein preparation. In particular, it relates to the construction of recombinant Pichia pastoris with the P36 gene of mycoplasma hyopneumoniae and the preparation of expressed protein. The prepared protein can be used in the research of mycoplasma hyopneumoniae P36 protein, the development of detection kits and genetic engineering vaccines. 2. Background technology [0002] Mycoplasma hyopneumoniae (Mhp) is the main pathogen causing swine asthma (MPS). Porcine asthma is one of the most widespread, fastest-spreading, and most difficult to purify 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 red...

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 邵国青祝永琴冯志新刘茂军王海燕吴叙苏甘源
Owner JIANGSU ACAD OF AGRI SCI
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