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Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

A technology of mycoplasma haemophilus and protein, which is applied in the field of recombinant Mh-PGK protein and its application in the detection of mycoplasma haemophilus from swine, can solve the problems of unreported research and achieve good specificity and repeatability

Active Publication Date: 2022-04-15
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the prokaryotic expression of the PGK protein of the pig-derived Mycoplasma haemophilus

Method used

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  • Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma
  • Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma
  • Recombinant Mh-PGK protein and application thereof in detection of swine haemophilus mycoplasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Preparation of swine-derived mycoplasma antibody indirect ELISA antigen and detection of antigenicity

[0075] 1. Antigen preparation

[0076] 1) Optimization of mh-pgk gene

[0077] According to the codon preference of Escherichia coli, the mh-pgk gene (its nucleotide sequence is shown in SEQ ID NO.3, obtained by Composed of 1233 nucleotides, the coding amino acid sequence is as shown in the 22-431st position of SEQ ID NO.1 The new species of Mycoplasma suis PGK protein (abbreviated as Mh-PGK protein) carries out codon optimization, and three TGA (due to The specificity of the gene coding system of Mycoplasma, TGA encoding tryptophan non-stop codon) was optimized as the codon TGG that can encode tryptophan in E. coli and part of the codons were optimized as E. The mh-pgk gene (its nucleotide sequence is shown in the 7th-1239th position of SEQ ID NO.2, consisting of 1233 nucleotides, and the amino acid sequence of the encoded Mh-PGK protein remains unchanged...

Embodiment 2

[0096] Example 2 Establishment and Condition Optimization of Indirect ELISA Detection Method for Antibody of Mycoplasma Haemophilus of Porcine Origin

[0097] 1. Determination of the optimum coating concentration and serum dilution

[0098] 1) Coating antigen: using the square matrix titration method, the recombinant Mh-PGK protein was coated with a coating solution (carbonate buffer solution at pH 9.60.01M, 1.59g Na 2 CO 3 and 2.93g NaHCO 3 Add deionized water to 1000mL) for doubling dilution (antigen concentrations are 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078μg / mL), 100μL / well, each concentration is coated with 1 line, coated with 7 Concentration, the last line of blank control, after incubation at 37°C for 1h, coat the microplate overnight at 4°C, and remove it with washing solution (PBST, 8.0g NaCl, 0.2g KCl, 2.86g NaCl 2 HPO 4 12H 2 O, 0.27g KH 2 PO 4 and 0.5mL Tween-20 plus deionized water to 1000mL) washed 3 times, 3min each time;

[0099] 2) Blocking: add 25...

Embodiment 3

[0151] Example 3 Specificity and Sensitivity Test of Indirect ELISA Detection Method for Antibody of Mycoplasma Haemophilus of Porcine Origin

[0152] 1. Specificity test

[0153] The indirect ELISA detection method of the swine-derived Mycoplasma antibody established in Example 2 detects M.suis, M.parvum, M.haemosuis, porcine circovirus (PCV), swine fever (CSFV), porcine reproductive and respiratory syndrome respectively Positive serum for 8 common swine diseases (PRRS), porcine toxoplasma gondii (PT) and foot-and-mouth disease (FMD), determine OD 450 Value, in order to determine the specificity of serum detection by indirect ELISA detection method for the Mycoplasma porcine antibody.

[0154] The results of the specificity test are shown in Table 7, and the results show that there is no cross-reaction with common swine disease (porcine circovirus disease, swine fever, porcine reproductive and respiratory syndrome, porcine toxoplasma gondii and foot-and-mouth disease) serum,...

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Abstract

The invention discloses a recombinant Mh-PGK protein and an application of the recombinant Mh-PGK protein in detection of swine haemophilus mycoplasma. The invention firstly discloses a recombinant Mh-PGK protein with an amino acid sequence as shown in SEQ ID NO.1. The recombinant Mh-PGK protein disclosed by the invention has the The invention further discloses an indirect ELISA (enzyme-linked immunosorbent assay) detection kit and a detection method for the swine haemophilus mycoplasma antibody, which take the recombinant Mh-PGK protein as an elisa plate of a coating antigen. The swine haemophilus mycoplasma antibody indirect ELISA detection method established based on the recombinant Mh-PGK protein is used for detecting the swine haemophilus mycoplasma antibody level, has no cross reaction with part of other swine disease serum, has good specificity and repeatability, and provides an effective means for clinical diagnosis, epidemiological research and immunodetection of Mycoplasma suis, Mycoplasma parvum and Mycoplasma haemosuis.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, it relates to a recombinant Mh-PGK protein and its application in detecting mycoplasma haemophilus of pig origin. Background technique [0002] Hemotropic mycoplasma (Hemotropic mycoplasma, also known as Eperythrozoon) is a kind of mycoplasma that infects a variety of mammals and parasitizes on the surface of red blood cells and in bone marrow (Lu Chengping, editor-in-chief. Veterinary Microbiology [M]. Fourth Edition. Beijing : China Agricultural Press 2010: 240-241). HM destroys red blood cells and causes hemolysis and infectious anemia in the host, which can cause abortion and stillbirth in sows, anemia in piglets, jaundice and emaciation (Hoelzle L E, Felder K M, Hoelzle K.Porcineeperythrozoonosis: from Eperythrozoon suis to Mycoplasma suis[J]. Grosstiere Nutztiere.2011,39(4):215-220.; Messick, J.B., Hemotrophic mycoplasmas (hemoplasmas): a review and new insights int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/70C12N15/54G01N33/569G01N33/543G01N33/58
CPCC12N9/1217C12N15/70G01N33/56933G01N33/54306G01N33/581C12Y207/02003G01N2333/30Y02A50/30
Inventor 付媛石团员袁秀芳孙洪超王德前徐丽华李军星苏菲郝力力薛红叶刘娱霏李天恩余斌
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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