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A Strain of Mycoplasma Hyopneumoniae and Its Application

A technology of Mycoplasma hyopneumoniae and Mycoplasma is applied in the directions of bacteria, antibacterial drugs, bacterial antigen components, etc., and can solve the problems of unsatisfactory culture effect and the like

Active Publication Date: 2018-02-09
北京中海生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still more or less deficiencies in these cultivations, and the cultivation effect is still unsatisfactory.

Method used

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  • A Strain of Mycoplasma Hyopneumoniae and Its Application
  • A Strain of Mycoplasma Hyopneumoniae and Its Application
  • A Strain of Mycoplasma Hyopneumoniae and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] ——Cultivation of Mycoplasma hyopneumoniae S strain

[0050] Inoculate 10% of the S strain into the Mycoplasma hyopneumoniae LPS medium, culture at 37°C for 72-96 hours, when the pH of the solution reaches 6.6-6.8, it turns yellow or orange, uniformly turbid without precipitation, harvest the bacterial liquid, and passage twice Afterwards, dilute the bacterial solution for CCU counting (colour change unit, CCU), and the number of viable bacteria can reach 10 9 ~10 10 CCU / mL.

Embodiment 2

[0052] ——PCR determination of Mycoplasma hyopneumoniae 16s RNA-specific primers in bacterial liquid of Mycoplasma hyopneumoniae S strain

[0053] 1. Primer Design

[0054] According to the 16s RNA gene sequence of Mycoplasma hyopneumoniae published on genebank, refer to Shen Qingchun et al. Acta Veterinary Medicine, 55-57.) Synthetic primers.

[0055] The sequence of the upstream primer P1 is: 5'-GAGCCTTCAAGCTTCACCAAGA-3' (sequence 1)

[0056] The downstream primer P2 sequence is: 5'-TGTGTTAGTGACTTTTGCCACC-3' (sequence 2)

[0057] The full length of the amplified fragment is expected to be 649bp.

[0058] 2. Template extraction

[0059] Take 1 mL of the cultured bacterial solution and centrifuge at 12,000 r / min for 15 min, discard the supernatant. The precipitate was suspended in 100 μl 1×TEN solution, placed in a boiling water bath for 10 minutes, and allowed to stand at room temperature for 3 minutes. Centrifuge at 12000r / min for 2min, and the supernatant is template D...

Embodiment 3

[0066] ——PCR assay with specific primers for P37 protein of Mycoplasma hyorhinosus

[0067] 1. Primer Design

[0068] The primers were designed and synthesized according to the P37 gene sequence of Mycoplasma hyorhina published on genebank.

[0069] The upstream primer P1 sequence is: 5'-GTAGTCAAGCAAGAGGATGT-3' (sequence 3)

[0070] The sequence of the downstream primer P2 is: 5'-GCTGGAGTTATTATACCAGGA-3' (sequence 4), and the full length of the amplified fragment is 346bp.

[0071] 2. Template extraction

[0072] Take 1 mL of the cultured bacterial solution and centrifuge at 12,000 r / min for 15 min, discard the supernatant. The precipitate was suspended in 100 μl 1×TEN solution, placed in a boiling water bath for 10 minutes, and allowed to stand at room temperature for 3 minutes. Centrifuge at 12000r / min for 2min, and the supernatant is template DNA.

[0073] 3.PCR amplification

[0074] PCR reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd. PCR reac...

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Abstract

The invention relates to a strain of Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), strain S, which is a new isolate of Mycoplasma hyopneumoniae, can grow stably on a cell-free medium, and the growth titer can reach 109-1010CCU / mL. Immunization tests on rabbits and piglets have confirmed that its immunogenicity is good; the amino acids in the R1 region of the P97 gene of the strain contain 11 consecutive amino acid sequences of AA(V / T)PKE / V, which can be used as its unique molecular biological marker. The mycoplasma hyopneumoniae bacterial strain can be used for preparing the inactivated vaccine of the mycoplasma hyopneumoniae.

Description

[0001] Technical Field The present invention relates to a strain of Mycoplasma hyopneumoniae and its application, belonging to the field of veterinary microbiology. Background technique [0002] Mycoplasma hyopneumoniae, also known as swine panting disease, is a contact chronic respiratory disease of pigs caused by Mycoplasma hyopneumoniae (Mhp), which is prevalent in the world. According to data reports, Mycoplasma hyopneumoniae isolated from all over the world belong to the same serotype, but the genomes of different isolates are quite different. In 1992, Frey et al. (Frey, J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains. Int J Syst Bacteriol 42, 275-280) first confirmed the genome of different isolates of Mycoplasma hyopneumoniae The difference between Artiushin and Minion (1996) (Artiushin, S. & Minion, F.C. (1996). Arbitrarily primed PCR analysis of Mycoplasmahyopneumoniae field isolates demonstrates gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K39/02A61P31/04
CPCA61K39/0241C12N1/205C12R2001/35
Inventor 沈青春冯忠泽吴金孙晔李秋菊李聪研魏晶晶宋潇婷孙亚波史文艳
Owner 北京中海生物科技有限公司