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Recombinant adenovirus expressing mycoplasma hyopneumoniae P102 protein and applications thereof

A recombinant adenovirus, cgmccno.2987 technology, applied in application, virus/phage, recombinant DNA technology, etc., can solve the problems of inactivated vaccine and attenuated vaccine promotion and application, achieve low human pathogenicity and broad application prospects , good stability

Inactive Publication Date: 2011-10-12
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the prevention and control of Mhp mainly adopts drug treatment, immunization and population purification. Vaccine immunization mainly uses inactivated vaccines and attenuated vaccines. However, due to various factors, the promotion and application of inactivated vaccines and attenuated vaccines are affected.

Method used

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  • Recombinant adenovirus expressing mycoplasma hyopneumoniae P102 protein and applications thereof
  • Recombinant adenovirus expressing mycoplasma hyopneumoniae P102 protein and applications thereof
  • Recombinant adenovirus expressing mycoplasma hyopneumoniae P102 protein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P102

[0039] The mutation of embodiment 1P102 gene

[0040] 1.1 Primer design and PCR amplification

[0041] The 5' end primer of primer A1 is provided with CCACC sequence, ATG promoter, Hind III site, and the 3' end primer is provided with TAA stop codon and Xho I site.

[0042] A1:

A2:

ATGGATCCAAACCTTTTTGGTTAATAA

CAGGTCGACATAATCAGAGAGGCTAAT

Primers for amplifying the A-segment gene

a1:

a2:

AAAAAATGGTCGGCAAAAACAGT

CCGACCATTTTTTAAAATTATCTTTAATT

Mutation at position 241-243

Primer

a3:

a4:

GCTCTTTCAAGTCGTGAAAACCAATTAC

TTCACGACTTGAAAGAGCTTTGGTTG

Mutation at position 610-612

Primer

a5:

a6:

TCTAAAATTCTAAAATCCTGGCTTGAAA

ATTTTAGAATTTTAGAAATTTCCTGATTTG

Mutation at position 877-879

Primer

B1:

B2:

CAGGAATTCTGGTATGGATCTCCGAATTCA

CAGGTCGACAAAAGTGCTTACCTCTGTT

Amplify B segment gene primers

C1:

C2:

CAGGGATCCGCAGAAGAAGCTAAG

CCGGGTCGACTTAC...

Embodiment 2

[0051] Example 2 Construction and Expression of Mycoplasma Hyopneumoniae P102 Gene Recombinant Adenovirus

[0052] 1 Materials and methods

[0053] 1.1 Strains, plasmids, cells, gene fragments

[0054] Mycoplasma hyopneumoniae Yin-1 strain was isolated and identified in 2005 by our laboratory; P102 gene (mutated sequence SEQ ID NO: 1 obtained in Example 1); E.coliDH5α is a product of Novagen; adenovirus Expression vector system (AdEasy TM XLAdenoviral Vector System) including the shuttle vector pShuttle-CMV, electroporation competent cells BJ5183-AD-1, packaging cells AD-293 and super competent cells XL-10 were purchased from Stratagene.

[0055] 1.2 Main reagents

[0056] Tissue / cell DNA extraction kits, small plasmid extraction kits and gel recovery kits are all products of Shanghai Huashun Company; protein molecular quality standards, restriction endonucleases NotI, HindIII and Pyrobest TM DNA polymerase was produced by Fermentas MBI Company; restriction enzymes Pme I...

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Abstract

The invention discloses a recombinant adenovirus expressing mycoplasma hyopneumoniae P102 protein and applications thereof. The recombinant adenovirus is obtained by firstly carrying out mutation on gene of the mycoplasma hyopneumoniae P102 protein, cloning the whole genome (SEQ ID No:1) after the mutation onto a shuttle vector pShuttle-CMV, obtaining a recombinant shuttle vector, carrying out electro-transformation into competent cells containing an adenovirus backbone vector after linearization and carrying out homologous recombination in bacteria. The indirect immunofluorescence and the Western Blot identification prove that the recombinant adenovirus successfully expresses the P102 protein. The recombinant adenovirus is used for immunizing female Balb / c mice with 6-8 weeks by two waysof muscle injection and nasal dropping, and the recombinant virus can induce the mice to produce the specific humoral immune and the cellular immune response, thereby indicating that the recombinant adenovirus can be applied in the diagnosis, the prevention or the treatment of mycoplasma pneumoniae of swine.

Description

technical field [0001] The present invention relates to a rearranged cDNA sequence and a recombinant adenovirus containing the cDNA sequence, in particular to a cDNA sequence obtained by rearranging and optimizing the gene encoding the Mycoplasma hyopneumoniae P102 protein, and the present invention also relates to a recombinant adenovirus containing the cDNA The sequenced recombinant adenovirus and their application in the diagnosis, prevention or treatment of swine mycoplasma pneumonia belong to the field of prevention and treatment of swine mycoplasma pneumonia. Background technique [0002] Mycoplasma hyopneumoniae (Mycoplasma, hyopneumoniae, Mhp) is the main pathogen of swine mycoplasma pneumonia (MPS), which is also called porcine endemic pneumonia (EPP). . MPS is characterized by contact, highly contagious, chronic, high morbidity, and low mortality. The main manifestations are cough, dyspnea, and fleshy or marbled lesions in the lung tissue can be seen on dissection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/63C12N7/01C07K14/30C12N15/70C12Q1/68G01N33/53A61K48/00A61K39/02A61P11/00A61P31/04C12Q1/70C12R1/93C12R1/19C12R1/35
Inventor 辛九庆李媛郭丹郭军陈超曹培丽张美晶董惠姜海芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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