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Mycoplasma pneumonia mosaic antigen, antigen detection reagent, and preparation method of both

A technology for Mycoplasma pneumoniae and chimeric antigens, which is applied in the field of preparation, kits containing the antigen or antigen composition, and the expression of chimeric recombinant antigens, can solve the problems of high false positives and complicated preparation processes, and achieve increased sensitivity, Easy to culture, purify, and quickly detect the effect

Inactive Publication Date: 2018-01-12
HANGZHOU CLONGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Foreign studies have confirmed that the P1 protein and P30 protein of MP have strong antigenicity, and the antibody detection rate in the serum of MP-infected patients is high, but there are also two shortcomings, one is the high false positive, and the other is the preparation process complex

Method used

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  • Mycoplasma pneumonia mosaic antigen, antigen detection reagent, and preparation method of both
  • Mycoplasma pneumonia mosaic antigen, antigen detection reagent, and preparation method of both
  • Mycoplasma pneumonia mosaic antigen, antigen detection reagent, and preparation method of both

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Construction of recombinant expression vector containing Mycoplasma pneumoniae gene and preparation of recombinant MP chimeric antigen:

[0067] 1. Screening of antigenic epitopes of MP P1 protein, p30 protein and MPN456 protein

[0068] Using software such as ANTHEWIN, the entire amino acid sequence of the MP protein (M. pneumonia FHstrain) was analyzed by computer, and the antigenic protein sequence P1 (residues 1177–1535) of the MP protein was screened out; the antigenic epitope of the P30 protein (residues 170 –254) and the antigenic epitope of MPN456 protein ((residues 765–846), and the flexible polypeptide (GLy 4 Ser) 3 interconnected. Their amino acid sequence is SEQ ID NO:1.

[0069] 2. Acquisition of Mycoplasma pneumoniae gene

[0070] Different from other pathogens, MP uses a unique biased codon system. The universal stop codon UGA encodes tryptophan in MP. If the wild gene of MP is used directly, protein translation can be interrupted, which li...

Embodiment 2

[0077] Example 2. Induced expression and expanded culture of Mycoplasma pneumoniae

[0078] Get the recombinant expression vector pET28a-MP that embodiment one obtains and transfer Escherichia coli BL21 (DE3) competent cell, cultivate on the LB culture plate that contains kanamycin, select the positive that single bacterium colony is inoculated in 5mlLB medium and screens The recombinant bacteria were induced to express, and the product was analyzed by SDS-PAGE, and compared with the control bacteria containing the expression vector pET28a(+), the results were as follows: figure 2 shown in the instruction manual figure 2 Shown is the induced expression SDS-PAGE protein electrophoresis pattern of recombinant Mycoplasma pneumoniae, in which there are many foreign proteins.

[0079] The bacterial supernatant and total protein of the control bacteria without the target gene ( figure 2 Compared with lanes 3 and 4 in ), the recombinant protein band with a molecular weight of ab...

Embodiment 3

[0081] Embodiment three, the separation and purification of Mycoplasma pneumoniae

[0082] Since the recombinant expression vector pET28a-MP constructed in the above-mentioned Example 1 has a fusion tag (His·Tag) gene sequence, the expressed target protein also has a His·Tag tag. Therefore, the target protein can be purified by affinity chromatography using agarose complexed with Ni, and the affinity chromatography column is purchased from GE Healthcare.

[0083] After inducing expression with IPTG according to the method of the above-mentioned Example 2, harvest the bacterial cell pellet by centrifugation at 10000 rpm / min for 5 min; use Buffer A (20 mM Tris, 0.5 M NaCl pH 8.0) (60ml BufferA / L Cells) were resuspended, lysed by ultrasonication at 250 W for 90 times, each time for 5 s, with an interval of 10 s, and centrifuged at 12,000 rpm / min for 20 min at 4°C to collect the supernatant and prepare for loading.

[0084] The specific purification steps are as follows:

[0085...

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Abstract

The invention provides a mycoplasma pneumonia mosaic antigen amino acid sequence containing an amino acid sequence as shown in SEQ ID NO:1 as well as a full-gene synthesized mycoplasma pneumonia mosaic antigen full-gene sequence containing an amino acid sequence as shown in SEQ ID NO:2. The invention also provides a method for constructing the two gene sequences. The invention also provides a preparation method of the mycoplasma pneumonia mosaic antigen containing full-gene synthesis, a mycoplasma pneumonia detection kit and a preparation method thereof. Mp recombinant mosaic antigen is selected as a mark material and is applied to a gold immunochromatography system, and the detection system is directly marked and captured, so the sensitivity is greatly improved, the specificity of the antigen is high, the antigen is easy in cultivation and purification, and cost is reduced; and a new method for detecting mycoplasma pneumoniae IgG rapidly and accurately is provided for clinical use, and a good market prospect is achieved.

Description

technical field [0001] The present invention relates to the field of in vitro diagnosis and immunoassay, in particular to mycoplasma pneumoniae, the construction of a chimeric antigen expression vector of mycoplasma pneumoniae, the expression and preparation of chimeric recombinant antigens, as well as kits and applications containing the antigen or antigen composition. Background technique [0002] Mycoplasma pneumoniae (MP) is an ultra-filtering pathogenic microorganism between bacteria and viruses, and is mainly transmitted in the form of aerosol particles through respiratory droplets. It was first isolated and cultured from the sputum of human primary atypical pneumonia (PAP) patients in 1962. [0003] Since the 1990s, with the changes in the etiology of pneumonia, MP has become an important pathogen of pneumonia in children. MP infection not only causes lung lesions, but also invades other organs such as the heart, brain, liver and kidney, causing various extrapulmonary...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/68G01N33/569
Inventor 殷秀飞郑曙剑刘静
Owner HANGZHOU CLONGENE BIOTECH
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