Preparation and application of escherichia coli preferred soluble porcine PD-1 recombinant protein

A PD-1, recombinant protein technology, applied in the field of biomedicine, can solve problems such as unsatisfactory effects, and achieve the effect of improving immunity and enhancing immune response

Pending Publication Date: 2021-04-16
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a preparation of Escherichia coli partial tropism soluble porcine PD-1 recombinant protein and its application in PCV2 vaccine, aiming at improving the level of PCV2 antibody and the body Immunity, to solve the problem of unsatisfactory effect after immunization of PCV2 vaccine in the prior art

Method used

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  • Preparation and application of escherichia coli preferred soluble porcine PD-1 recombinant protein
  • Preparation and application of escherichia coli preferred soluble porcine PD-1 recombinant protein
  • Preparation and application of escherichia coli preferred soluble porcine PD-1 recombinant protein

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Experimental program
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Effect test

Embodiment 1

[0039] The preparation method of embodiment 1 soluble porcine PD-1 recombinant protein

[0040] (1) Escherichia coli codon transformation of porcine PD-1 protein extracellular region gene

[0041] Follow the principle of E. coli codon partial tropism table, the G+C content of the gene sequence should not be higher than 60%, and respect the original codon of the gene to transform the gene of the extracellular region of the porcine PD-1 protein, and also carry out the gene sequence primer design The modification of E. coli codon bias tropism, plus the specific primer sequence of restriction site is shown in Table 1. The major amino acid sites were modified as follows: ACA→ACC, GAG→GAA, CCC→CCG, CTC→CTG, CGC→CGT, AGG→CGT. The nucleotide sequence of the DNA molecule of the obtained Escherichia coli partial tropism soluble porcine PD-1 protein is shown in SEQ ID NO:2. Send the designed and modified gene sequence to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.

[004...

Embodiment 2

[0050] Example 2 In vitro activity identification method of soluble porcine PD-1 recombinant protein as a vaccine enhancer

[0051] (1) Western blotting (Western blotting) to identify PD-1 recombinant protein activity

[0052] Western blotting to identify the induced expression of soluble porcine PD-1 recombinant protein, the specific steps are as follows:

[0053] 1) Gel preparation: First, add each component in the beaker according to the system in Table 2 to prepare a 12% separating gel. After the gel is solidified, use filter paper to absorb excess water in the glass plate, and then prepare the concentrated gel according to Table 3.

[0054] Table 2 Preparation of 12% separating gel

[0055] components volume sterile water 4.9mL 30% acrylamide 6.0mL 1.5mM Tris-HCl buffer 3.8mL 10% Sodium Lauryl Sulfate 150.0μL 10% ammonium persulfate 150.0μL Tetramethylethylenediamine 6.0 μL

[0056] Table 3 Preparation of 5% stac...

Embodiment 3

[0087] Example 3 In vivo activity identification method of soluble porcine PD-1 recombinant protein as a vaccine enhancer

[0088] (1) Animal grouping

[0089] By consulting the literature and equivalent dose conversion among animals, it was determined that the immunization dose of the soluble porcine PD-1 recombinant protein was 0.2 mg / kg. Select 40 weaned piglets that were not immunized with PCV2 vaccine at the age of 45 days, and randomly divide them into 4 groups, 10 pigs in each group. Group A is the PCV2 vaccine immunization group, groups B and C are the test groups, and group D is the control group for irrelevant proteins , and the specific grouping conditions are shown in Table 7.

[0090] Table 7 Experimental animal groups and inoculation preparations

[0091] group number of animals Molecular preparation Vaccination method protein immunization dose A 10 heads PCV2 vaccine intramuscular injection 0mg / kg B 10 heads PCV2 vaccine +...

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Abstract

The invention relates to preparation and application of escherichia coli escherichia coli preferred soluble porcine PD-1 recombinant protein. The amino acid sequence of the soluble porcine PD-1 recombinant protein is shown as SEQ ID NO: 1; the soluble recombinant protein, having been subjected to codon preferred modification, of a porcine PD-1 gene is obtained by using a gene cloning technology; and the soluble porcine PD-1 recombinant protein can effectively improve the antibody level and body immunity of PCV2 after PCV2 vaccine immunization, enhance the immune response of pigs to PCV2 vaccines, improve the immunity of inoculated animals, and solve the problem of unsatisfactory effect after PCV2 vaccine immunization in the prior art.

Description

technical field [0001] The invention relates to the preparation and application of Escherichia coli partial tropism soluble porcine PD-1 recombinant protein, belonging to the field of biomedicine. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) disease is one of the important diseases that endanger the pig industry in the world. The pathogenicity of PCV2 is directly related to the strain type. PCV2 typing is based on sequence differences in ORF2. PCV2 mainly includes five subtypes including PCV2a, PCV2b, PCV2c, PCV2d and PCV2e, among which PCV2b is currently the main prevalent genotype. At present, there is a new situation that different gene subtypes are prevalent at the same time in China. The mixed infection of PCV2a and PCV2b is increasing, and the infection of PCV2d is also increasing and becoming the main subtype of infection. In China, the virulence of PCV2b strain among the PCV2 epidemic strains is significantly higher than...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12C12N15/70A61K39/12A61K39/39A61P31/20
Inventor 王选年朱艳平刘佳何勇岳锋孙国鹏郭东光李鹏李文明刘金宵
Owner XINXIANG UNIV
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