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Mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity

A technology of HSP70-P113 and Mycoplasma pneumoniae, applied in the field of recombinant protein

Pending Publication Date: 2022-08-05
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no commercial subunit vaccine for Mycoplasma ovis pneumoniae (Movi).

Method used

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  • Mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity
  • Mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity
  • Mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 HSP70-P113 Fusion gene design and synthesis

[0021] Analysis of Heat Shock Protein Gene of Mycoplasma Sheepneumoniae FJ-01CL Isolate by Protean Software in DNAStar 7.0 Software HSP70 and the adhesin gene of Mycoplasma ovine pneumonia ATCC 29419 isolate P113 The predominant antigen region in , the gene sequences of the two predominant antigen regions obtained are optimized by rare codons and then spliced, and the GGGGSGGGGS sequence linker is added in the middle to obtain HSP70-P113 fusion gene. HSP70-P113 The full-length fusion gene is 1392 bp, and has the nucleotide sequence shown in SQE ID NO.1 and the amino acid sequence shown in SEQ ID NO.2. HSP70-P113 The fusion gene synthesis was completed by Nanjing GenScript Co., Ltd.

Embodiment 2

[0022] Example 2 HSP70-P113 - Construction of recombinant plasmid pET-30a(+)

[0023] by gene cloning HSP70-P113 The fusion gene was inserted into the E. coli plasmid pET-30a Nde I and BamH I Between the sites, the recombinant plasmid was constructed HSP70-P113 -pET-30a(+). HSP70-P113 -pET-30a(+) plasmid map such as figure 1 shown.

Embodiment 3

[0024] Example 3 HSP70-P113 -Prokaryotic expression of pET-30a(+)

[0025] Put the stored BL21(DE3) on ice for 30 min, add 100 ng of recombinant plasmid HSP70-P113 -pET-30a(+), mix well by gently pipetting, react on ice for 30 min; heat shock at 42°C for 90 s; place on ice for 3 min after heat shock; then add 100 mL of LB liquid medium; Incubate at 37°C in a bed for 60 min with shaking at 200 rpm; mix the bacterial solution and spread it onto a 50 μg / mL kanamycin-resistant plate, invert the plate and incubate at 37°C overnight. The next day, pick 3 single clones, inoculate them into test tubes containing 50 μg / mL kanamycin in LB liquid medium, and culture in a shaker at 37°C; 600 When it reached 0.6~0.8, 0.5 mM IPTG was added to two of the test tubes, respectively, and incubated at 15 °C for 16 h and 37 °C for 4 h, and the last tube was the negative reference; the samples were prepared for SDS-PAGE analysis to detect proteins expression.

[0026] Sample preparation for S...

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Abstract

The invention discloses a mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity, which is encoded by a fusion gene formed by splicing a mycoplasma ovipneumoniae heat shock protein gene HSP70 and a mycoplasma ovipneumoniae adhesin gene P113, and the amino acid sequence of the mycoplasma ovipneumoniae HSP70-P113 fusion protein is a sequence shown as SQE ID NO.2. A Western Blot test proves that the mycoplasma ovipneumoniae HSP70-P113 fusion protein has immunogenicity and can be used for preparing a mycoplasma ovipneumoniae subunit vaccine.

Description

technical field [0001] The invention belongs to the technical field of recombinant proteins, in particular to an immunogenic Mycoplasma ovine pneumonia HSP70-P113 fusion protein. Background technique [0002] Mycoplasma ovine pneumonia ( Mycoplasma ovipneumoniae , Movi) is one of the main pathogens causing mycoplasma pneumonia in sheep and goats, which is distributed worldwide and has brought huge economic losses to the world sheep breeding industry. The treatment of Mycoplasma ovine pneumonia is mainly based on antibiotics. However, the drug resistance caused by antibiotics makes the treatment effect of Mycoplasma ovine pneumoniae unsatisfactory, so the prevention of Mycoplasma ovine pneumonia should be the main focus. However, the current domestic Movi vaccines are mainly inactivated vaccines, which are mainly inactivated by whole bacterial antigens. However, Mycoplasma that is prevalent in different places has genetic diversity, and the cross-protection between strains ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/30C12N15/62C12N15/70C12N1/21G01N33/569A61K39/02A61P31/04C12R1/19
CPCC07K14/30C12N15/70G01N33/56933A61K39/0241A61P31/04C07K2319/00G01N2333/30A61K2039/552Y02A50/30
Inventor 江锦秀胡奇林林裕胜张靖鹏
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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