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Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody as well as preparation method and application of natural antigen P1

A technology of Mycoplasma pneumoniae and natural antigen is applied in the field of biological detection, which can solve the problems of long time for stable cell expression, low protein expression, instability between batches, etc., and achieves easy expansion of production, low preparation cost, and mass production. Effect

Pending Publication Date: 2021-12-21
山东硕景生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antigenic site of P1 is mainly the spatial site of the spatial structure, so in vitro expression must depend on cell expression, and the transfection efficiency and mass culture of cells expressing forward transfection reagents are a big test; at the same time, the protein expression level is low and the batches are not consistent Stability is also a thorny issue. It takes a long time to stabilize the expression of cells, and the high cost has become the main reason for restricting mass production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:

[0026] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture solution, centrifuge at 16,000 rpm for 0.5 h at 4°C to obtain Mycoplasma pneumoniae bacteria, wash twice with 10 mM PBS washing solution, and centrifuge at 4°C, 16,000 rpm Centrifuge for 0.5h, discard the supernatant, repeat washing twice, and the precipitate obtained is Mycoplasma pneumoniae;

[0027] (2) Freeze-thaw crushing treatment: first prepare a lysate containing 10 mM PBS, 5 mM Tcep, 2.5 mM EDTA, a final concentration of 2% arginine, and a final concentration of 2% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 0.5 h, freeze at -20°C for 12 h, rethaw, centrifuge at 16,000 rpm for 0.5 h at 4°C, and discard the supernatant ,...

Embodiment 2

[0031] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:

[0032] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture solution, centrifuge at 10,000 rpm for 2 h at 4°C to obtain Mycoplasma pneumoniae cells, wash with 20 mM PBS washing solution for 3 times, and centrifuge at 4°C, 10,000 rpm Centrifuge for 2 h, discard the supernatant, repeat washing once, and the precipitate obtained is Mycoplasma pneumoniae;

[0033] (2) Freeze-thaw crushing treatment: first prepare a lysate containing 20 mM PBS, 0.5 mM Tcep, 0.5 mM EDTA, a final concentration of 1% arginine, and a final concentration of 1% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 2 hours, freeze at -20°C for 2 hours and rethaw, centrifuge at 10,000 rpm for 2 hours at 4°C, discard the superna...

Embodiment 3

[0037] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:

[0038] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture medium, centrifuge at 12000 rpm for 1 h at 4°C to obtain Mycoplasma pneumoniae cells, wash once with 15 mM PBS washing solution, centrifuge at 4°C, 12000 rpm Centrifuge for 1 h, discard the supernatant, repeat washing twice, and the precipitate obtained is Mycoplasma pneumoniae;

[0039] (2) Freeze-thaw crushing treatment: first prepare a lysate whose components are 15 mM PBS, 1 mM Tcep, 1 mM EDTA, a final concentration of 5% arginine, and a final concentration of 5% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 1 hour, freeze at -20°C for 4 hours and rethaw, centrifuge at 12,000 rpm for 1 hour at 4°C, discard the supernatant, The...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a natural antigen P1 for detecting an anti-mycoplasma pneumoniae antibody as well as a preparation method and application of the natural antigen P1. The preparation method comprises the following steps of taking an inactivated human mycoplasma pneumoniae antigen culture solution, centrifuging to obtain mycoplasma pneumoniae thalli, washing with a mycoplasma washing solution, centrifuging, and discarding supernatant and precipitate to obtain mycoplasma pneumoniae; putting into a lysis solution, carrying out water bath incubation at 37 DEG C, carrying out cryopreservation at -20 DEG C, then carrying out re-melting, carrying out resuspension on the centrifuged precipitate by using a mycoplasma washing solution, and carrying out centrifugation to leave the precipitate; resuspending the precipitate in the lysis solution, carrying out ultrasonic treatment, and centrifuging to obtain a supernatant which is an antigen crude product; and carrying out centrifugal ultrafiltration on the supernatant obtained by filtering the crude product, and collecting the protein on the membrane, namely the natural antigen P1. The method is simple in step and suitable for popularization and mass production, and the prepared natural antigen P1 has good specificity and sensitivity to the mycoplasma pneumoniae antibody, so that the clinical detection sensitivity and specificity of the mycoplasma pneumoniae antibody in disease diagnosis are further improved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody and its preparation method and application. Background technique [0002] Mycoplasma pneumoniae (M.pneumoniae, MP) is the pathogen that causes human mycoplasma pneumonia, and mycoplasma pneumonia accounts for more than one-third of acellular pneumonia. MP is mainly transmitted through the air by nasal and oral secretions, and causes respiratory tract infection through droplets. The incubation period is 2-3 weeks, and it mainly occurs in autumn and winter. Most of the onset groups are children and young people. The clinical manifestations are cough, fever, headache, and sore throat And muscle pain, without typical clinical symptoms, it is not easy to distinguish from bacterial pneumonia. However, the treatment of mycoplasma pneumonia and bacterial pneumonia infection is very different....

Claims

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Application Information

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IPC IPC(8): C07K14/30C07K1/34G01N33/569
CPCC07K14/30G01N33/56933
Inventor 刘万建刘向祎杨帆杜金芳王婷李林
Owner 山东硕景生物科技有限公司
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