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Special diluent for swine mycoplasmal pneumonia vaccines and preparation method of special diluent

A technology of Mycoplasma porcine pneumonia and diluents, applied in the field of diluents, can solve the problems of pigs’ inability to produce P97R1 antibodies, large-scale expression, lack of antigen immunity, etc., to enhance the ability of cell and humoral immune stimulation, easy to operate, and good needle penetration Effect

Active Publication Date: 2014-07-23
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, a number of studies have found that pigs cannot produce P97R1 antibodies after being immunized with several existing commercialized inactivated vaccines against Mycoplasma porcine pneumonia, indicating that P97 (including P97R1 fragments) cannot be expressed in large quantities under in vitro culture conditions. Lack of immunity is a major defect of existing vaccines against Mycoplasma pneumoniae

Method used

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  • Special diluent for swine mycoplasmal pneumonia vaccines and preparation method of special diluent
  • Special diluent for swine mycoplasmal pneumonia vaccines and preparation method of special diluent
  • Special diluent for swine mycoplasmal pneumonia vaccines and preparation method of special diluent

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Preparation of recombinant Mycoplasma hyopneumoniae P97R1 protein

[0039] According to the genome sequence of Mycoplasma hyopneumoniae published by GenBank, primers were designed to amplify the P97R1 fragment. Upstream primer: 5'-CCGAATTCTTTACCTCAGCCGCCAGCAG-3' (SEQ ID No.3); Downstream primer: 5'-GCGAAGCTTAAGCCATTGGGAAATAGTCT-3' (SEQ ID No.4). The upstream and downstream primers contain EcoR I site and Hind III site. Using 168 F344 strains of Mycoplasma hyopneumoniae genomes as templates, the P97R1 gene fragment was amplified by PCR. EcoR I and Hind After Ⅲ double enzyme digestion, it was inserted into the pET-32a(+) vector to obtain the prokaryotic plasmid vector pET-32a(+)-P97R1 expressing the recombinant P97R1 protein. Escherichia coli BL-21 (DE3) was transformed, positive clones were screened and verified by DNA sequencing, and recombinant genetically engineered bacteria expressing recombinant P97R1 protein were obtained.

[0040] The gene se...

Embodiment 2

[0045] Example 2 Preparation of Immunostimulatory Complex ISCOM-P97R1 Containing Recombinant P97R1 Protein

[0046] (1) Preparation of 20% Mega-10 stock solution

[0047] Dissolve 0.4 g of Mega-10 (also known as "N-quinoyl-N-methylglucamine") dry powder in 1.6 ml of distilled water, dissolve at 37°C and dilute to 2 ml.

[0048] (2) Preparation of lipid mixture stock solution

[0049] Add 100 mg phosphatidylcholine and 100 mg cholesterol to 10 ml 20% Mega-10, heat slightly (30-60°C) and stir slowly to dissolve completely, filter and aliquot, store at -20°C for later use.

[0050] (3) Preparation of Quil A solution

[0051] Dissolve 25 mg of Quil A in 4.5 ml of phosphate buffered saline, pH 7.2-7.4.

[0052] (4) Mycoplasma hyopneumoniae P97R1 protein solution

[0053] The recombinant P97R1 protein was prepared and purified by the method described in Example 1, dissolved in phosphate buffer, the protein concentration was measured and diluted to 10 mg / ml, and stored at -20°C...

Embodiment 3

[0056] Embodiment 3 Preparation of special diluent for swine mycoplasma pneumonia vaccine

[0057] When preparing the special diluent for swine mycoplasma pneumonia vaccine containing only ISCOM-P97R1 components, the preparation method is as follows: take an appropriate amount of ISCOM-P97R1 stock solution with a Quil A concentration of 1.67 mg / ml, and dilute it with an appropriate amount of phosphate buffer to obtain Each ml solution contains: Quil A 0.1-1 mg, cholesterol 0.02-0.2 mg, phosphatidylcholine 0.02-0.2 mg, P97R1 antigen 0.4-4 mg, ISCOM-P97R1 vaccine special diluent.

[0058] When preparing the special diluent for mycoplasma pneumonia vaccine containing ISCOM-P97R1 and other water adjuvant components, the preparation method is as follows: first prepare ISCOM-P97R1 mother solution, levamisole mother solution, astragalus polysaccharide mother solution, and carbomer mother solution respectively, Then dilute to the required concentration with phosphate buffer solution t...

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Abstract

The special diluent for swine mycoplasma pneumonia vaccine of the present invention mainly contains recombinant P97R1 protein immunostimulatory complex (ISCOM-P97R1), and may also contain other aqueous adjuvant components, including levamisole, or / and astragalus polysaccharide, or / and carboxy bohm. Each ml solution of the diluent contains 0.1-1 mg of QuilA, 0.02-0.2 mg of cholesterol, 0.02-0.2 mg of phosphatidylcholine, 0.4-4 mg of P97R1 antigen, and may further contain 2-20 mg of levamisole , or / and astragalus polysaccharide 20-100 mg, or / and carbomer 3-10 mg, and the rest is phosphate buffer. This diluent is specially used for dissolving or diluting Mycoplasma pneumoniae vaccine, including live vaccine and inactivated vaccine. It has the dual effects of supplementing the protective spectrum of vaccine antigens and enhancing the immune stimulating ability of vaccine cells and humoral, which can significantly improve the protective efficacy of vaccines against viruses. At the same time, due to the use of the water solvent system, it also has the advantages of simple operation, good needle penetration, and no obvious toxic and side effects.

Description

technical field [0001] The invention relates to a diluent specially used for a swine mycoplasma pneumonia vaccine, in particular to a diluent for a swine mycoplasma pneumonia vaccine suitable for live vaccines and inactivated vaccines and capable of specifically enhancing the protective ability of the vaccine. Background technique [0002] Mycoplasma swine pneumonia, commonly known as swine asthma, is caused by Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae , Mhp) is a chronic respiratory infectious disease of pigs, the main symptoms of which are cough and wheezing. The disease mainly causes the feed conversion rate and growth retardation of pigs, with high morbidity and low mortality. Mycoplasma suis pneumonia is widely prevalent in the world, causing significant economic losses to the modern pig industry. [0003] Mycoplasma pneumoniae in swine is a stubborn chronic infectious disease. The effect of drug treatment is not satisfactory, and it is easy to relapse after...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/02A61K39/39A61P31/04
Inventor 熊祺琰邵国青韦艳娜刘茂军冯志新白方方
Owner JIANGSU ACAD OF AGRI SCI
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