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Mycoplasma pneumoniae recombinant antigen and its application

A technology of Mycoplasma pneumoniae and recombinant antigen, applied in the biological field, can solve the problems of high price of natural antigen, cumbersome steps of natural antigen, indeterminate yield and the like

Active Publication Date: 2020-09-11
GUANGDONG WESAIL BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the incidence of Mp in my country continues to rise. However, most of the current clinical serological tests use Mp natural antigens. However, natural antigens are expensive, and the steps to extract natural antigens are cumbersome, with no definite yield, and high risk to the human body. Therefore, it is necessary to study recombinant antigens to lay the foundation for basic Mp biological research and the development of fast and accurate Mp detection kits
[0006] However, the traditional Mycoplasma pneumoniae recombinant antigen with multiple antigenic epitopes is difficult to stably display different types of antigenic dominant epitopes as required
The paired screening of antigens is cumbersome, and the detection rate and specificity are low when used to detect Mycoplasma pneumoniae

Method used

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  • Mycoplasma pneumoniae recombinant antigen and its application
  • Mycoplasma pneumoniae recombinant antigen and its application
  • Mycoplasma pneumoniae recombinant antigen and its application

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preparation example Construction

[0051] In addition, the present application also provides a method for preparing a Mycoplasma pneumoniae recombinant antigen according to an embodiment, comprising the following steps:

[0052] Construct the gene expression sequence of the recombinant antigen of Mycoplasma pneumoniae, optimize its codons, and select the codons preferred by Escherichia coli. The final gene sequence is obtained by chemical synthesis, and the upstream primers are introduced into the BamHI site, and the downstream primers are introduced into The EcoRI site and the coding sequence of 6 His amino acids and the stop codon TAA before the EcoRI site, the specific amino acid sequence of the Mycoplasma pneumoniae recombinant antigen is shown in SEQ ID NO1. After synthesizing the target fragment, use the upstream and downstream primers for PCR amplification. After the amplified PCR fragment is recovered, it is digested with BamHI and EcoRI, and connected to the expression vector pET-32a digested with BamHI...

Embodiment 1

[0071] 1. Construction of Mycoplasma pneumoniae Recombinant Antigen Expression Plasmid

[0072] First, the sequence of the Mycoplasma pneumoniae gene was collected in Genbank, the gene data of Mycoplasma pneumoniae was established, and it was analyzed by computer software to obtain the Mycoplasma pneumoniae adhesion protein P1 (GenBank: AAB95661.1) and adhesion protein P30 (NCBI Reference Sequence : NP_110141.1) the segment with the most detection activity, the segment with the most detection activity of the P1 protein (the 1219th amino acid segment to the 1422th amino acid segment, referred to as the P1A segment, the 1583rd amino acid segment to the 1627th amino acid segment segment, referred to as P1B), and the segment with the most detectable activity of the P30 protein (the 168th amino acid to the 274th amino acid, referred to as the P30 segment) is connected in series through the connecting peptide GGGGS, and the series sequence from the C-terminal to the N-terminal is P1A...

Embodiment 2

[0094] The preparation of embodiment 2 Mycoplasma pneumoniae antibody colloidal gold detection test paper

[0095] 1. Preparation of coated membrane (nitrocellulose membrane)

[0096] Preparation of coating buffer: 6% methanol, 0.01M pH7.2 PBS buffer as coating buffer, filtered through 0.22 μm membrane, set at 4°C for later use, valid for one week.

[0097] 1000mL 0.01M pH 7.2 PBS buffer solution with 6% methanol: 8g NaCL, 0.2g KCL, 2.9g Na2HPO4·12H2O, 0.2g KH2PO4, 60mL methanol, distilled deionized water to 1000mL.

[0098]Preparation of the coated membrane (nitrocellulose membrane): Dilute the Mycoplasma pneumoniae recombinant coated antigen MP-2 to 1-2 mg / mL with the coating buffer, adjust the machine, and draw a line on the detection area of ​​the coated membrane, which is Mycoplasma pneumoniae Antibody detection line (T line). Dilute the rabbit anti-mouse IgG antibody (manufactured by Feipeng Biological Co., Ltd., product number BA-PAB-MU0002) to 1-5 mg / mL with coating ...

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Abstract

The invention relates to a mycoplasma pneumoniae recombinant antigen and application thereof. The mycoplasma pneumoniae recombinant antigen comprises a PIA fragment, a first binding peptide, a PIB fragment, a second binding peptide and a P30 fragment. Experiment results show that the recombinant antigen can obtain different types of antigen dominant epitopes after being purified in different ways,and the various types of antigen dominant epitopes are stable in expression, namely the different types of antigen dominant epitopes can be stably expressed as required. The matching and screening works of the antigen can be greatly reduced during detection; the detection rate is high, and the specificity is high during the detection of mycoplasma pneumoniae.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mycoplasma pneumoniae recombinant antigen and its application. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma Pneumoniac, Mp) is a pathogenic mycoplasma, and the primary atypical pneumonia caused by it accounts for about 1 / 2 of non-bacterial pneumonia, and the incidence of Mp infection is on the rise in recent years. In addition to primary atypical pneumonia, Mp can still cause other respiratory infectious diseases such as bronchitis, pharyngitis, etc., as well as complications in the nervous system, blood system, cardiovascular system, skin, muscle, and joints. About 30% of pneumonia in the general population is caused by Mycoplasma pneumoniae. The respiratory damage and various extrapulmonary complications caused by Mp infection have attracted widespread attention. [0003] The diagnosis of Mycoplasma pneumoniae mainly relies on isolation and culture and serolog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/30C07K1/22C12N15/31C12N15/70C12N1/21G01N33/569
CPCC07K14/30C12N15/70G01N33/56933
Inventor 朱碧银刘春艳罗沛黄荣生杨耿周魏钟杰覃素妮吴仁贞宗雪陈洁
Owner GUANGDONG WESAIL BIOTECH CO LTD
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