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Construction method of multi-pathogen mycoplasma ovine pneumonia nucleic acid vaccine

A technology of mycoplasma pneumonia and nucleic acid vaccine is applied in the field of construction of multi-pathogen Mycoplasma sheep pneumonia nucleic acid vaccine, which can solve the problems of high cost, no cross immune protection, low immune protection efficacy and the like

Inactive Publication Date: 2021-09-24
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the invention is to overcome the problems of low immune protection efficacy, long production cycle, high cost, no cross immune protection between Mo and Mmc in the existing Mycoplasma pneumonia inactivated vaccine

Method used

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  • Construction method of multi-pathogen mycoplasma ovine pneumonia nucleic acid vaccine
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  • Construction method of multi-pathogen mycoplasma ovine pneumonia nucleic acid vaccine

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Effect test

Embodiment 1

[0032] The invention provides a method for constructing a multi-pathogenic mycoplasma pneumoniae nucleic acid vaccine, comprising the following steps:

[0033] Step 1: Extraction of Mo / Mmc genome:

[0034] S1: Recovery and cultivation of Mo Y98 / Mmc PG3 standard strain:

[0035] Use an alcohol cotton ball to wipe the surface of the single-tube freezing tube for storing the Mo Y98 strain and the Mmc PG3 standard strain in the ultra-clean workbench, open the tube opening with scissors, add the improved Hayflick's medium to dissolve the freeze-dried bacteria, and inoculate the solution in 5 mL of Hayflick's culture culture medium at 37°C for 3 to 5 days in a constant temperature shaking incubator, when the color of the medium turns yellow, take 200 μL for subculture, pass for 3 consecutive generations, and store each subculture at 4°C for later use;

[0036] S2: Extraction of Mo Y98 / Mmc PG3 standard strain DNA:

[0037] The third-generation culture product was extracted with ref...

Embodiment 2

[0048] The applicant's research team successfully isolated several strains of Mycoplasma ovis pneumoniae (Mo) and one strain of Mycoplasma mycoplasma goat subsp.

[0049] 1 Acquisition of the target gene

[0050] Primer design and synthesis

[0051] The upstream and downstream primers of Mo P113 and Mmc LppA were designed based on the nucleotide sequence of the genome of the isolate. The nucleotide sequences of the primers and the size of the amplified fragments are shown in Table 1. They were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0052] Table 1 Primer Sequence

[0053] Tab.1 Primersequence

[0054]

[0055] 1.2 Amplification of the target gene

[0056] The C-terminus of the Mo P113 gene and the N-terminus of the Mmc LppA gene were obtained by the PCR method, and a 50 μL reaction system was established:

[0057]

[0058] After mixing evenly, carry out PCR amplification of the target gene according to the following reaction conditions: pre-denatura...

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Abstract

The invention discloses a construction method of a multi-pathogen mycoplasma ovine pneumonia nucleic acid vaccine. The construction method comprises the following steps of: step 1, extracting a Mo / Mmc genome; step 2, obtaining a MoP113 / MmcLppA gene; and step 3, constructing a pVAX1-P113-LppA co-expression vector and expressing in mammalian cells. According to the construction method disclosed by the invention, the pVAX1-P113-LppA co-expression vector is constructed through a gene engineering method on the basis of mycoplasma ovine pneumonia multi-epitope antigen genes MoP113 and MmcLppA which are screened out at an early stage; an indirect immunofluorescence technology is used for verifying an expression condition of recombinant plasmids in MDBK (MDBK); the nucleic acid vaccine is developed by selecting two different antigen genes and an immune protection effect can be effectively provided aiming at mycoplasma ovine pneumonia caused by Mo and Mmc infection; and a plurality of problems of current vaccine research and development are solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a multi-pathogenic mycoplasma pneumoniae nucleic acid vaccine. Background technique [0002] Mycoplasma pneumonia is caused by Mycoplasma mycoides subsp. Capri (Mmc), Mycoplasma ovipneumoniae (Mo) and Mycoplasma capricolum subsp. Capricolum (Mcc). It is a contagious infectious disease prevalent in sheep flocks. Fibrinous pneumonia and pleurisy are its main features, and it is listed as a B-type infectious disease by the World Organization for Animal Health (OIE). In recent years, there have been reports of the occurrence and prevalence of Mycoplasma ovis pneumonia in many provinces and regions with developed sheep raising industry in China. Loss, and the main pathogens of this disease in my country are Mycoplasma ovis pneumoniae and Mycoplasma mycoides goat subspecies. [0003] Immunization is still one of the important measures to prevent and treat such in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/85C12N5/10A61K39/02A61P31/04
CPCC07K14/30C12N15/85A61K39/0241A61P31/04A61K2039/53
Inventor 张双翔杨鹏程振涛岳筠吴燕袁林
Owner GUIZHOU UNIV
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