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Mycoplasma pneumoniae antigen as well as preparation method and application thereof

A technology of Mycoplasma pneumoniae and antigen, applied in the directions of botanical equipment and method, biochemical equipment and method, application, etc., can solve the problems of uneven product quality between batches, complicated steps of MP whole bacteria antigen, high price, etc. Cost saving, beneficial to industrial production, high sensitivity

Active Publication Date: 2020-06-09
ZHUHAI LIVZON DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of MP whole bacterial antigen is strict, complicated steps, often mixed with other proteins, expensive, and the product quality is not uniform between batches, and there is a risk of infecting humans during production and use.

Method used

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  • Mycoplasma pneumoniae antigen as well as preparation method and application thereof
  • Mycoplasma pneumoniae antigen as well as preparation method and application thereof
  • Mycoplasma pneumoniae antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: The purpose gene synthesis of the present invention and the construction of recombinant vector

[0064] 1. Use software such as ProtScale to analyze the entire amino acid sequence of Mycoplasma pneumoniae antigen P1 (MP FH strain), and screen out the antigenic protein sequence P1M: residues 1340-1518, the amino acid sequence of which is shown in SEQ ID NO:1.

[0065] 2. In the present invention, the above-mentioned amino acid sequence is reverse-translated by using Escherichia coli preferred codons to obtain a recombinant antigen gene composed of Escherichia coli preferred codons. Using the method of gene synthesis, it is connected to the pET28a vector, and the gene sequence encoding the antigen is shown in SEQ ID NO:2.

Embodiment 2

[0066] Example 2: Induced expression, purification and application of Mycoplasma pneumoniae antigen

[0067] 1. Transform the recombinant vector P1M-pET28a in Example 1 into Escherichia coli E.coli Rosetta (DE3) competent cells, on the LB culture plate containing the kanamycin of 50 μg / ml and the chloramphenicol of 50 μg / ml Culture at 37°C for 14-16 hours; screen for positive recombinant bacteria, pick a single colony and inoculate it into 5ml LB medium containing 50μg / ml kanamycin and 50μg / ml chloramphenicol, and culture overnight.

[0068] 2. Inoculate the above-mentioned overnight cultured bacterial solution into LB medium containing 50 μg / ml kanamycin and 50 μg / ml chloramphenicol according to the inoculum amount of 1%, and cultivate to the OD of the bacterial solution at 37°C and 250 rpm 600 1.0-1.3, and then induced with IPTG at a final concentration of 0.5mM-1.0mM at 25°C and 250rpm for 3-4h.

[0069] 3. Collect the Escherichia coli E. coli Rosetta cells obtained in 2 b...

Embodiment 3

[0079] Example 3: Specificity and Sensitivity Detection

[0080] Based on the principle of antigen-antibody specific binding, enzyme-labeled IgM antibody and sulfonated magnetic beads coated with Mycoplasma pneumoniae antigen were used to detect the binding reactivity of Mycoplasma pneumoniae antigen and IgM antibody by chemiluminescence. Adopt immunoserological detection technique, detect the recombinant P1M antigen provided by the present invention and the application effect of P1 antigen (as comparative example), specifically, comprise the following steps:

[0081] Coated sulfonated magnetic beads: mix mycoplasma pneumoniae antigen with sulfonated magnetic beads in a certain ratio, wash, coat and wash with the Buffer independently developed by Zhuhai Livzon Reagent Co., Ltd., and then make the magnetic bead working solution;

[0082] According to the technological process independently developed by Zhuhai Livzon Reagent Co., Ltd., the enzyme-labeled antibody IgM working sol...

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Abstract

The invention relates to the field of in-vitro diagnostic immunoassay, and in particular provides a mycoplasma pneumoniae antigen as well as a preparation method and application thereof. The mycoplasma pneumoniae antigen provided by the invention is protein composed of an antigenic protein sequence P1M:residues 1340-1518, specifically an amino acid sequence shown in SEQ ID NO.1, or protein which is obtained by performing substitution and / or deletion and / or addition on the amino acid sequence shown in the SEQ ID NO.1 by one or more amino acid residues and has same function. The antigen is verified by an immunoserological detection technology to have strong specificity and high sensitivity, and the antigen is easy to culture and purify, is more conducive to industrial production and saves costs; and in addition, the antigen is suitable for preparation of MP antibody detection products, can be used for any forms of products in the field of in-vitro immunodiagnosis, and has broad market prospects.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis and immunoassay, in particular to a mycoplasma pneumoniae antigen and its preparation method and application. Background technique [0002] Mycoplasma pneumoniae (MP) is a kind of pathogenic mycoplasma, which is an important pathogen of community-acquired pneumonia. Its infection can cause primary atypical pneumonia; it can also cause human respiratory infectious diseases such as bronchitis, pharyngitis, etc. Cause complications involving the nervous system, blood system, cardiovascular system and skin, muscles, joints and so on. In conclusion, respiratory damage and various extrapulmonary complications caused by MP infection have attracted widespread attention. [0003] Since MP infection often has no specific clinical manifestations, timely diagnosis of the etiology, prevention of disease progression, and early detection and early treatment have become the top priority for the treatment of MP ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C12N15/31C12N15/70G01N33/569
CPCC07K14/30C12N15/70C12N2800/22G01N33/56933
Inventor 聂金梅何丽娟朱越谭胡志强陈巧红杨海林
Owner ZHUHAI LIVZON DIAGNOSTICS
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