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Recombinant main outer membrane protein of chlamydi trachomatis and preparation method and application thereof

A major outer membrane protein, Chlamydia trachomatis technology, applied in the direction of Chlamydia antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as poor sensitivity and subjective influence

Inactive Publication Date: 2008-01-30
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it has poor sensitivity in the population with low infection rate and is susceptible to the subjective influence of the experimenter. The advantages are that it is fast, cheap, easy to operate, convenient to store and transport the specimen, and the Chlamydia trachomatis in the specimen does not have to be live or is infectious

Method used

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  • Recombinant main outer membrane protein of chlamydi trachomatis and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment one , Preparation of Ct MOMP

[0030] 1. Utilize the PCR method to amplify the whole DNA of MOMP, and its primers are:

[0031] 1: 5'-CATGCCATGGTGCCTGTGGGGAATCCTGCT-3';

[0032] 2: 5'-ACGCGTCGACTTAGAAGCGGAATTGTGCAT-3'.

[0033] 2. Cut out the target fragment from the T vector, connect it with the pET32a(+) vector cut with the same enzyme, and transfer it into the expression-type bacterium BL21(DE3).

[0034] 3. Pick positive colonies, the ratio of bacteria solution to culture solution is 1:50, add Amp (1:1000) at the same time, and culture on a shaker at 37°C and 250rpm for 4 hours until the bacteria solution is OD 600 =0.7-0.9, add IPTG to a final concentration of 1 mM, continue culturing for 5 hours, and collect bacteria by centrifugation.

[0035] 4. Resuspend in a solution containing 30% sucrose, 50mM Tris-HCl, 1mM EDTA pH8.0, bathe in ice for 30min, and centrifuge to collect bacteria.

[0036] 5. Use ultrasonic buffer (50mM NaH 2 PO 4 pH 7.8, 300...

Embodiment 2

[0049] Embodiment two , the immune evaluation of rabbits with the vaccine of Ct recombinant MOMP of the present invention as active ingredient

[0050] The Ct recombinant MOMP obtained in Example 1 was diluted to 0.25 mg / ml and used for double amplification to determine the serum titer. Six big-eared white rabbits whose serum test was negative were randomly divided into experimental group and control group. Big-eared white rabbits, 30 days old, male, weighing about 2kg / piece, were provided by the Experimental Animal Center of our hospital. The experimental group was immunized with the Ct recombinant MOMP of the present invention, and the prepared Ct recombinant MOMP was fully stirred and mixed with Freund's adjuvant to make a latex solution, and the abdomen was subcutaneously injected into 3 rabbits, each injected with 2 mg, with the same dose at intervals of 20 days An additional injection was given to the site and 30 days later, for a total of 2 additional injections. In ...

Embodiment 3

[0051] Embodiment three , Immunoblotting to detect the antigenicity of Ct recombinant MOMP

[0052] (1) Protein sample: purified Ct recombinant MOMP.

[0053] (2) Electrophoresis: prepare a gel and perform SDS-PAGE electrophoresis.

[0054] (3) Transfer:

[0055] 1. After electrophoresis, cut the gel strip to a suitable size, equilibrate with transfer buffer (39mM glycine, 48mM Tris base, 0.037% SDS, 20% methanol), 5min×3 times.

[0056] 2. Membrane treatment: pre-cut the filter paper and NC membrane with the same size as the strip, and immersed in the transfer buffer for 10 minutes.

[0057] 3. Membrane transfer: The membrane transfer device is placed in the order of anode carbon plate, 4-layer filter paper, NC membrane, gel, 4-layer filter paper, and cathode carbon plate from bottom to top. The filter paper, gel, and NC membrane are precisely aligned. Remove air bubbles in one step. Turn on the power supply, 230mA, and transfer for 1.5h. After the transfer, disconnect...

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Abstract

The invention discloses a recombinant major outer membrane protein of Chlamydia trachomatis and a coding gene and a preparation method thereof. The recombinant major outer membrane protein of Chlamydia trachomatis provided by the invention is a protein with an amino acid residue sequence of sequence 2 in the sequence list, or a protein derives form the sequence 2 that one or more amino acid residues of the amino acid residues sequence are substituted or lost, or that are added with the same activity as the amino acid residues sequence of the sequence 2. The coding gene of the recombinant major outer membrane protein of Chlamydia trachomatis is one of the nucleotide sequences: (1) the DNA sequences of the sequence 1 of the sequence list; (2) the DNA sequence of the functional protein that has above 90 percent of homology with the DNA sequence confined in the sequence 1 of the sequence list and has the same code. The antigens of the invention have excellent immunogenicity and wide market application prospect.

Description

technical field [0001] The present invention relates to a recombinant main outer membrane protein of Chlamydia trachomatis and its encoding gene, its preparation method and application, in particular to a recombinant main outer membrane protein of Chlamydia trachomatis and its encoding gene, its preparation method and its application in vaccine preparation and Chlamydia detection in the application. Background technique [0002] Chlamydia trachomatis (Chlamydiat rachomatis, Ct) has 15 serotypes, which can cause various human diseases, among which types A~C are the main pathogens causing trachoma, and types D~K cause human genitourinary tract infection, and in men it is manifested as abnormal Gonococcal urethritis, prostatitis, proctitis, etc.; in women, it manifests as cervicitis, salpingitis, ectopic pregnancy and infertility, and L1-L3 types mainly cause lymphogranuloma, a venereal disease. Studies have shown that sexually transmitted diseases (STDs) caused by Ct infectio...

Claims

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Application Information

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IPC IPC(8): C07K14/295A61K39/118A61P31/04C12N15/31C12N15/63
Inventor 端青刘光桥朱虹宋立华檀华何君左庭婷
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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