Protein of tuberculosis in use for diagnosing tuberculosis

A technology of mycobacterium tuberculosis and tuberculosis, applied in the application, use of vectors to introduce foreign genetic material, biochemical equipment and methods, etc., can solve problems that affect the actual application of proteins, cannot meet the requirements, and the 38KDa protein is less

Active Publication Date: 2005-11-16
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this 38KDa protein has good allergen properties, there is a problem that Mycobacterium tuberculosis synthesizes less 38KDa protein, accounting for only one thousandth of the total protein
At present, although the production of 38KDa protein through genetic engineering has greatly increased its yield, for ex

Method used

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  • Protein of tuberculosis in use for diagnosing tuberculosis
  • Protein of tuberculosis in use for diagnosing tuberculosis
  • Protein of tuberculosis in use for diagnosing tuberculosis

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1: Preparation of Mycobacterium tuberculosis protein of the present invention

[0057] According to the Pab gene sequence, the Pab gene was mutated by PCR technology, and the author designed and synthesized a pair of PCR primers. The upstream primer a1 is 37 bases long, as shown in sequence 4, and the 5' end is designed with a protective base and an NCOI restriction site (including the start codon ATG), and the downstream primer a2 is 34 bases long, as shown in sequence 5 Shown, a BamHI restriction site and a stop codon TAA were designed at the 5' end. Using the Mycobacterium tuberculosis H37Rv genomic DNA as a template, the polynucleotide encoding the protein of the present invention (wherein the 5' end base sequence is ATGGGCTCT- (sequence 1) or ATGGGCTCA- (sequence 2) was obtained by high-fidelity DNA polymerase PCR technology , where the last base is mutated from the original base G. The PCR amplification product of this primer was subjected to 1.2% agar...

Embodiment 2

[0059] Embodiment 2: Purification, renaturation and identification of Mycobacterium tuberculosis protein of the present invention

[0060] Add lysis buffer (1g bacteria plus 3ml lysis buffer) to suspend the bacteria. Sonicate the bacteria with a power of 200W, ultrasonic for 20 seconds with an interval of 20 seconds, a total of 80 times; centrifuge at 4°C and 12,000rpm for 15 minutes, discard the supernatant, and wash the precipitate 2-3 times with 1% Triton-X100. 20mmol / LTris.Cl / 8M urea (pH8.0) was used to dissolve the inclusion body, centrifuged at 12000rpm at 4°C for 15min, and the supernatant was collected.

[0061] The inclusion body solubilized supernatant was loaded on the processed DEAE-52 anion exchange column. After loading the sample, fully wash away the unbound protein with 20mmol / L Tris.Cl / 8 M urea (pH8.0), then 0-0.3M NaCl gradient elution, and collect the protein SDS-PAGE electrophoresis to detect the corresponding collection of the elution peak The solution i...

Embodiment 3

[0069] Embodiment 3: Mycobacterium tuberculosis protein of the present invention is used for guinea pig experiment of skin allergen

[0070] Guinea pig DTH test: 100 guinea pigs of 300-350 g were randomly divided into two groups, 80 guinea pigs were subcutaneously injected with 0.2 ml of inactivated Mycobacterium tuberculosis H37Rv, and DTH test was carried out 4 weeks after the injection. Guinea pigs in the control group were injected with 0.2ml of normal saline. All guinea pigs were depilated on their backs, and 0.1ml of the protein of the present invention and 0.1ml of PPD were injected intradermally at the corresponding parts of the back on both sides of the spine (requiring that the injection site appeared as the criterion), and 24 hours and 48 hours after the injection, the blush or the injection site was detected. The horizontal and vertical diameters of induration (mm), the injection local blush (or induration) with an average horizontal and vertical diameter ≥ 5 mm is...

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Abstract

A tuberculosis mycobacterium protein used for diagnosing tuberculosis and epidemiologic survey and monitor of tuberculosis, the polynucleotide for coding it, the hybrid plasmid and procaryotic host cell containing said polynucleotide, and the process for preparing said protein are disclosed.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis protein prepared by genetic engineering, and the application of the protein in tuberculosis diagnosis, tuberculosis epidemiological investigation and monitoring, and its application as an antigen component in vaccines. Background technique [0002] Mycobacterium tuberculosis is the causative bacterium of tuberculosis, and about three million people in the world suffer from tuberculosis caused by tuberculosis every year. my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of patients ranks second in the world, second only to India. In recent years, the incidence of tuberculosis in my country has been on the rise. [0003] Tuberculosis is a respiratory infectious disease, therefore, early and accurate diagnosis of the disease is the key to effectively control the spread of the disease. [0004] In the 1920s, foreign researchers made the allergen (old...

Claims

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Application Information

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IPC IPC(8): C12N15/11
Inventor 庄玉辉何秀云张小刚
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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