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Mycoplasma pneumoniae recombinant antigen, and preparation method and application of mycoplasma pneumoniae recombinant antigen

A technology of Mycoplasma pneumoniae and recombinant antigens, applied in the biological field, can solve the problems of poor antigen sensitivity, missed treatment of patients, false negatives, etc.

Active Publication Date: 2013-09-04
WUHAN CHANGLI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the antigen sensitivity of P1 protein as a detection antibody is poor, especially for the detection of IgM produced by MP in the early stage. Since the content of IgM in human serum is low, detection with P1 protein as antigen is prone to false negative misjudgment , so that patients miss the golden period of treatment

Method used

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  • Mycoplasma pneumoniae recombinant antigen, and preparation method and application of mycoplasma pneumoniae recombinant antigen
  • Mycoplasma pneumoniae recombinant antigen, and preparation method and application of mycoplasma pneumoniae recombinant antigen
  • Mycoplasma pneumoniae recombinant antigen, and preparation method and application of mycoplasma pneumoniae recombinant antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Amplification of the target gene of the present invention and construction of a recombinant vector

[0043] 1. Amplify the target band

[0044] The nucleotide sequence shown in SEQ ID NO: 2 is synthesized by the whole gene as an amplification template.

[0045] Design primers for PCR amplification, the primer sequence is as follows:

[0046] lan-MP-501: 5'-GGATCCGATGACGACGATAAAG-3' (SEQ ID NO: 3)

[0047] lan-MP-304: 5'-GAATTCCTAGCGTTTCGGCGGAAAACCTG-3' (SEQ ID NO: 4)

[0048] PCR amplification system:

[0049]

[0050] PCR amplification conditions:

[0051] Pre-denaturation, 94℃, 5min;

[0052] A total of 30 cycles of denaturation, annealing and extension, 94℃, 60s; 58℃, 60s; 72℃, 60s;

[0053] Keep warm, 72°C, 6min.

[0054] 2. Recombinant vector construction

[0055] The amplified target band was constructed into the pET-30a (purchased from Novagen) prokaryotic expression vector, and the restriction enzymes BamHI (purchased from NEB) and EcoRI (purchased from NEB) were used...

Embodiment 2

[0063] Example 2: Induced expression, purification, dialysis and concentration of the MP recombinant antigen of the present invention

[0064] Transform the recombinant plasmid into E. coli BL-21, and induce 0.4mM-1.0mM IPTG at 37°C for 2-4h;

[0065] Collect E. coli BL-21 cells by centrifugation, suspend the cells with 20mM-50mM, pH8.0 tris, sonicate them, and collect the supernatant by centrifugation;

[0066] Purify the supernatant on a Ni column. Wash the column with a binding buffer solution containing 20mM imidazole, and then use a binding buffer solution to gradient elution of the MP recombinant antigen protein. The binding buffer solution is pH8.0 with 20mM-50mM tris and 200-500mM The solution composed of NaCl; the purified protein was detected by polyacrylamide gel electrophoresis, the result is figure 2 , Plus the label of the carrier itself, about 50KD, consistent with the strip position in the figure;

[0067] Put the purified MP recombinant antigen protein into a dialys...

Embodiment 3

[0068] Example 3: Elisa identification of the MP recombinant antigen of the present invention and the existing MP antigen (RP14 protein)

[0069] Coating: Dilute the antigen with PBS (pH7.4) buffer to a protein content of 10μg / mL. Add 0.1 mL to the reaction well of each polystyrene plate at 4°C overnight. Discard the solution in the well and wash 5 times with washing buffer for 2 minutes each (referred to as washing, the same below);

[0070] Blocking: Use 5% bovine serum albumin as a blocking agent, add 0.1 mL to each well, incubate at 37°C for 2 hours, discard the solution in the well, and wash 5 times with washing buffer;

[0071] Add sample: add 0.1 mL of diluted positive serum and negative serum (1:100-1:400) to the above-mentioned coated reaction wells, and incubate at room temperature for 1 hour. Then wash 5 times with washing buffer;

[0072] Add enzyme-labeled antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody (goat anti-human HRP1:2000) to each reaction well. ...

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Abstract

The invention relates to the technical field of biology and discloses a mycoplasma pneumoniae (MP) recombinant antigen, and a preparation method and an application of the mycoplasma pneumoniae recombinant antigen. An amino acid sequence of the mycoplasma pneumonia recombinant antigen is shown as SEQ ID No: 1. According to the antigen, a section of amino acid is taken as connecting oligopeptide, and an MP specific antigen P1 proteantigen dominant epitope and a P30 antigen dominant epitope form a recombinant protein. Tests prove that the recombinant protein has higher specificity and sensitivity in comparison with the existing MP antigen, and can be used for preparing MP antibody testing products.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Mycoplasma pneumoniae antigen and its preparation method and application. Background technique [0002] Mycoplasma pneumonia is an acute respiratory infectious pneumonia caused by Mycoplasma pneumoniae (MP), which can cause epidemics, accounting for about 10% of all types of pneumonia, and can lead to death in severe cases. Therefore, it is particularly important to study an effective MP diagnostic kit. [0003] At present, the diagnostic methods for MP include immunofluorescence test, indirect hemagglutination ELISA test, complement fixation test, culture of Mycoplasma pneumoniae, polymerase chain reaction, etc. However, these existing detection methods generally require the use of specific instruments on specific occasions. Detection is not only not convenient and fast but also takes a long time, which is not conducive to taking decisive protection and preventive measures f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C12N15/31G01N33/68
Inventor 兰军
Owner WUHAN CHANGLI BIOLOGICAL TECH CO LTD
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