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

A technology of Mycoplasma suis pneumonia and diluent, which is applied in the field of diluent, can solve the problems of massive expression, lack of antigen immunity, inability of pigs to produce P97R1 antibody, etc., and achieves simple operation, good acupuncture, and enhancement of cellular and humoral immune stimulation capabilities. Effect

Active Publication Date: 2013-05-01
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to make sure we don't overdose an organism by giving them specific substances called certain types of bacteria (mycobactenemic cells) which help protect against other diseases like tuberculosis. These agents are very small particles made from living microorganisms found naturally on animal skin. They have been shown to kill off many different forms of harmful germs such as Methanobrever spores. By adding these tiny pieces into this solution they could increase both their defense mechanisms and how well they fight disease-causing viruses effectively.

Problems solved by technology

This patented technical problem addressed in this patents relating to improving Portebrumpyne monocerpasty virus vaccology involves developing improved methods for producing lyophillosporium spp., including introducing specific elements called PrEP717 onto certain strains during their cultivations. These techniques aimed towards reducing harmfulness associated with traditional chemical agents while maintaining its benefits over conventional ones.

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

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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

[0046] Example 2 Preparation of Immunostimulatory Complex ISCOM-P97R1 Containing Recombinant P97R1 Protein

[0047] (1) Preparation of 20% Mega-10 stock solution

[0048] 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.

[0049] (2) Preparation of lipid mixture stock solution

[0050]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.

[0051] (3) Preparation of Quil A solution

[0052] Dissolve 25 mg of Quil A in 4.5 ml of phosphate buffered saline, pH 7.2-7.4.

[0053] (4) Mycoplasma hyopneumoniae P97R1 protein solution

[0054] 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°...

Embodiment 3

[0057] Embodiment 3 Preparation of special diluent for swine mycoplasma pneumonia vaccine

[0058] 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.

[0059] 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 invention discloses a special diluent for swine mycoplasmal pneumonia vaccines. The special diluent mainly contains a recombinant P97R1 protein immunostimulating complex (ISCOM-P97R1); and in addition, the special diluent also possibly contains other water-based adjuvants comprising levomisole, astragalus polysaccharide or/and carbomer. 1 ml of diluent solution contains 0.1-1 mg of QuilA, 0.02-0.2 mg of cholesterol, 0.02-0.2 mg of phosphatidylcholine and 0.4-4 mg of P97R1 antigen; and in addition, 1 ml of diluent solution also possibly contains 2-20 mg of levomisole, 20-100 mg of astragalus polysaccharide or/and 3-10 mg of carbomer and a phosphate buffer solution for the rest. The diluent is specially used for dissolving or diluting the swine mycoplasmal pneumonia vaccines comprising a live vaccine and an inactivated vaccine, has the double effects of complementing vaccine antigen protection spectrums and improving the vaccine cell and humoral immunostimulation capability, can remarkably improve the toxic attack protection efficacy of the vaccines, and also has the advantages of being simple and convenient to operate, good in syringeability and free from remarkable toxic side reaction due to the adoption of a hydrosolvent system at the same time.

Description

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Claims

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Application Information

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Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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