Mycoplasma hyopneumoniae genetic engineering subunit vaccine as well as preparation method and application thereof

A technology of gene and coding gene, which is applied in the field of animal immune drugs, can solve the problems of high difficulty in operation, difficult cultivation, strong virulence, etc., and achieve the effects of reducing production costs, good application prospects, and high expression levels

Active Publication Date: 2020-11-13
苏州世诺生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inactivated vaccine can reduce the pathological damage of the lungs and stimulate the body's humoral immunity, but it hardly produces cellular immunity and cannot prevent the infection and spread of Mhp in pigs; the attenuated vaccine has a high degree of difficulty in operation, low yield, and strong virulence And other issues
Mhp has very strict requirements on the conditions of the medium. It is a kind of mycoplasma animal that is difficult to cultivate. It is easily contaminated by other bacteria and mycoplasma during the cultivation process, which also makes the production cost of the above two vaccines remain high.
Therefore, in order to prevent and treat MPS more effectively, it is extremely important to develop a new genetically engineered subunit vaccine of Mycoplasma hyopneumoniae with strong immunogenicity and high safety, but there are still few related reports

Method used

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  • Mycoplasma hyopneumoniae genetic engineering subunit vaccine as well as preparation method and application thereof
  • Mycoplasma hyopneumoniae genetic engineering subunit vaccine as well as preparation method and application thereof
  • Mycoplasma hyopneumoniae genetic engineering subunit vaccine as well as preparation method and application thereof

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preparation example Construction

[0061] For example, in a specific embodiment of the embodiments of the present invention, a method for preparing a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae may specifically include:

[0062] (1) Prepare nucleic acid molecules for encoding P36-DnaK-ffh and P97-P46-DnaJ fusion proteins respectively;

[0063] (2) Clone each nucleic acid molecule prepared in step (1) into a shuttle vector to obtain a corresponding recombinant shuttle vector containing the target gene;

[0064] (3) Transform the recombinant shuttle vectors obtained in step (2) into DH10Bac bacteria, select the recombinant bacteria, extract the genome and transfect Sf9 cells (or other insect cells mentioned above) to obtain recombinant baculoviruses;

[0065] (4) Cultivate the Sf9 cells (or other aforementioned insect cells) and then recombinantly express and produce P36-DnaK-ffh, P97-P46-DnaJ fusion proteins;

[0066] (5) Mixing the P36-DnaK-ffh and P97-P46-DnaJ fusion proteins into an ad...

Embodiment 1

[0071] Example 1 Transfer vector pF-P36-DnaK-ffh construction and identification

[0072] 1. Amplification and purification of P36-DnaK-ffh gene

[0073] The codon-optimized P36-DnaK-ffh gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC17 vector to obtain the pUC-P36-DnaK-ffh plasmid vector. Using pUC-P36-DnaK-ffh plasmid as template, P36-DnaK-ffh-F, P36-DnaK-ffh-R as upstream and downstream primers for PCR amplification (P36-DnaK-ffh-F, P36-DnaK-ffh- The gene sequence of R is shown in SEQ ID NO: 3, 4), and the amplification system is shown in Table 1.

[0074] Table 1 P36-DnaK-ffh gene amplification system

[0075]

[0076] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0077] Perform gel electrophoresis on the PCR product to verify the size of the target gene...

Embodiment 2

[0093] Example 2 Construction and Identification of Transfer Vector pF-P97-P46-DnaJ

[0094] 1. Amplification and purification of P97-P46-DnaJ gene

[0095] The codon-optimized P97-P46-DnaJ gene (SEQ IDNO:5) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC17 vector to obtain the pUC-P97-P46-DnaJ plasmid vector. Using pUC-P97-P46-DnaJ plasmid as template, P97-P46-DnaJ-F, P97-P46-DnaJ-R as upstream and downstream primers for PCR amplification (P97-P46-DnaJ-F, P97-P46-DnaJ- The gene sequence of R is shown in SEQ ID NO: 7, 8), and the amplification system is shown in Table 5.

[0096] Table 5 P97-P46-DnaJ gene amplification system

[0097]

[0098] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0099] Perform gel electrophoresis on the PCR product to verify the size of the target gen...

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Abstract

The invention discloses a mycoplasma hyopneumoniae genetic engineering subunit vaccine as well as a preparation method and an application thereof. The vaccine comprises a protein composition and a pharmaceutically acceptable carrier, wherein the protein composition comprises two fusion proteins with sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6 respectively. The vaccine provided by the invention has no toxicity; the vaccine is high in safety, good in immunogenicity and capable of generating strong humoral immunity in pig bodies, immunized animals can resist poison attacking of strong poisonand are comprehensively protected, and the vaccine can be prepared through large-scale serum-free suspension culture of a bioreactor and has the advantages of being easy to control in quality, stablebetween batches, low in production cost and the like.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a genetic engineering subunit vaccine of mycoplasma hyopneumoniae, its preparation method and application, and belongs to the technical field of animal immune medicines. Background technique [0002] Mycoplasmal pneumonia of swine (MPS), also known as swine panting disease and swine enzootic pneumonia (SEP), is a contact and chronic respiratory infection caused by Mycoplasma hyopneumoniae (Mhp) Its clinical symptoms are cough, wheezing, stunted growth and low feed-to-meat ratio. The disease is mainly transmitted through carrier pigs and diseased pigs. Pigs of different breeds, ages, and sexes can be infected. The transmission route is direct contact and airborne transmission. When diseased pigs and healthy pigs are mixed, large-scale outbreaks often occur. The infection spreads. MPS is an immunosuppressive disease. After infection, it often causes damage to porcine bronchial cil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N5/10A61K39/02A61P11/00A61P31/04
CPCA61K39/0241A61K2039/552A61P11/00A61P31/04C07K14/30C07K2319/00C12N15/86C12N2710/14043
Inventor 曹文龙孔迪滕小锘张大鹤
Owner 苏州世诺生物技术有限公司
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