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Mycobacterium tuberculosis specific recombinant protein OTC as well as preparation method and application thereof

A technology of Mycobacterium tuberculosis and a recombinant protein is applied to the Mycobacterium tuberculosis-specific recombinant protein OTC and its preparation method and application field, and achieves the effects of low cost, strong specificity and high sensitivity

Inactive Publication Date: 2014-07-09
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no specific kit for detecting anti-tuberculosis antibodies in urine samples at home and abroad.

Method used

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  • Mycobacterium tuberculosis specific recombinant protein OTC as well as preparation method and application thereof
  • Mycobacterium tuberculosis specific recombinant protein OTC as well as preparation method and application thereof
  • Mycobacterium tuberculosis specific recombinant protein OTC as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Cloning of OTC protein coding gene by genetic engineering technology:

[0032] 1. Design and synthesize a pair of primers for amplifying the OTC gene according to the sequence 1 in the sequence list

[0033] Upstream primer (containing restriction endonuclease Nhe I site)

[0034] 5'-CTA GCTAGC ATGATCAGGCATTTCCTGCG-3'

[0035] Downstream primer (contains restriction endonuclease EcoR I site)

[0036] 5'-CCG GAATTC TTATGAGCGCTCCAGCAGCCA-3'

[0037] Amplified fragment: 942bp

[0038] 2. PCR amplification of OTC gene

[0039]Using upstream and downstream primers, under the action of Taq plus I DNA polymerase, using Mycobacterium tuberculosis H37Rv genomic DNA as a template, the OTC gene was amplified. PCR reaction program: 95°C for 5min; 94°C for 1min, 65°C for 1min, 72°C for 2min, cycled 30 times; finally 72°C for 7min. The 942bp amplified DNA fragment was identified by 1% agarose gel electrophoresis.

[0040] 3. Recovery of target gene fragments:

[0041] A...

Embodiment 2

[0089] The OTC recombinant protein constructed and purified in Example 1 of the present invention was applied to the detection of tuberculosis antibodies in urine and blood of tuberculosis patients. It is used as the diagnostic antigen of chemiluminescence immunoassay and applied to the test of clinical specimens, and a good diagnostic effect is obtained. Compared with the three currently commercialized tuberculosis antibody detection kits, it significantly improves the sensitivity of tuberculosis diagnosis. The ELISA detection procedure is as follows:

[0090] 1. Experimental specimens: A total of 103 serum specimens were selected and divided into two groups:

[0091] (1) Tuberculosis group: 56 patients with active tuberculosis diagnosed clinically by imaging, laboratory examination and anti-tuberculosis treatment, including 33 males and 23 females, with an average age of 40.8±19.1 years. Including pulmonary tuberculosis, pulmonary tuberculosis with tuberculous pleurisy, pul...

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Abstract

The invention relates to a mycobacterium tuberculosis specific recombinant protein OTC (Ornithine Carbamoyltransferase) as well as a preparation method and application thereof, and belongs to the field of the medical immunological detection of the tuberculosis. The mycobacterium tuberculosis specific recombinant protein OTC has a nucleotide sequence as shown in the sequence 1 in the sequence table, and is prepared by using the genetic engineering technology, and the protein is utilized as a detection antigen to prepare a blood or urine tuberculosis antibody chemiluminescence enzyme immunodetection kit. Compared with the existing commercial antibody detection kit, the prepared kit has the advantages that sensitivity and specificity are high, two samples are complementary, and has complementation with other antigens on the aspect of the serum or urine antibody detection of the tuberculosis, and thus the prepared kit can be used for detecting specific anti-tuberculosis antibodies in fluid samples such as serum and urine.

Description

technical field [0001] The invention relates to a Mycobacterium tuberculosis-specific recombinant protein OTC and its preparation method and application, in particular to a Mycobacterium tuberculosis-specific recombinant protein OTC prepared by genetic engineering technology and its preparation method, and the application of the protein The invention relates to a blood or urine tuberculosis antibody chemiluminescent enzyme immunoassay kit as a detection antigen composition, which belongs to the technical field of tuberculosis medical immunology detection. Background technique [0002] Tuberculosis is still one of the major infectious diseases that endanger human health in the world. Since 1985, the prevalence of AIDS, tuberculosis-infected immigrants and some people living in poverty have caused the incidence of tuberculosis to rise in the United States and other developed countries in Europe and the United States, especially tuberculosis. Bacterial drug resistance and co-in...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70G01N33/53C12R1/32
CPCC12N9/1018C12N15/70C12Y201/03003G01N33/573G01N2333/91034G01N2800/26
Inventor 吴雪琼阳幼荣张俊仙赵卫国冯金栋梁艳
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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