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Chimeric recombinant antigen and application thereof

A technology of recombinant antigens and antigenic regions, applied in the field of diagnosing whether patients are infected with Mycobacterium tuberculosis, and chimeric recombinant antigens, can solve problems such as affecting the accuracy and reliability of diagnostic results, stimulating effects, and being unsuitable for promotion in grassroots units.

Active Publication Date: 2013-05-29
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of using a library of overlapping peptides is that the content of antigenic endotoxin is easy to control, but the disadvantage is that the epitopes in the peptide library are discontinuous and there are significant differences in the solubility of different peptides
In addition, Quanti-FERON Gold uses blood collection tubes to directly stimulate and culture whole blood. Its advantage is that it is easy to operate, but its disadvantage is that it cannot precisely control the amount of blood collected during blood collection, which will have a great impact on the stimulation effect, thereby affecting the diagnostic results. Accuracy and reliability; T-Spot.TBELISpot needs to separate and cultivate PBMC, which has relatively high requirements for operators, equipment and environment, and is not suitable for promotion in grassroots units

Method used

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  • Chimeric recombinant antigen and application thereof
  • Chimeric recombinant antigen and application thereof
  • Chimeric recombinant antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1. Cloning, expression and endotoxin-free purification of recombinant protein EC5

[0123] 1. Construction of an expression vector comprising the coding sequence of the recombinant protein EC5

[0124] a. Amplification of the target gene fragment

[0125] Using the genomic DNA of the standard strain of Mycobacterium tuberculosis H37Rv (gifted by Professor Wu Wenhan of the University of Hong Kong) as a template, the following primers were used to amplify the target gene fragment by PCR:

[0126] For fragment 1 of the gene of interest encoding Rv3875 aa 1-80, use the following primers:

[0127] F: 5'-ttttCATATgCACCACCACCACCACCACATGACAGAGCAGCAGAG-3' (SEQ ID NO: 3),

[0128] R: 5'-ttttGGATCCACCGGCTTCGCTGAT-3' (SEQ ID NO: 4);

[0129] For target gene segment 2 encoding Rv3874 aa 1-100, use the following primers:

[0130] F: 5'-ttttCTGGGATCCATGGCAGAGATGAAGACC-3' (SEQ ID NO: 5),

[0131] R: 5'-ttttGAATTCGAAGCCCATTTGCGAG-3' (SEQ ID NO: 6);

[0132] For fragment 3 ...

Embodiment 2

[0149] Example 2. Selection of non-specific T cell stimulator and determination of its concentration

[0150] During the in vitro stimulation and culture of anticoagulated whole blood, there are certain factors (such as operating errors, low level of cellular immunity of the subject, etc.), which can lead to no change in the level of cytokines after antigen stimulation, and there are certain factors ( For example, too high stimulant concentration), can lead to immune tolerance of the lymphocytes also react. Therefore, in order to ensure the accuracy and reliability of the results of the method of the present invention, it is preferable to set a positive control during the experiment. Such controls may be whole blood stimulated with a non-specific stimulant.

[0151] Both phytohemagglutinin (PHA) and concanavalin A have a strong ability to promote lymphocyte transformation, and can effectively stimulate T cells in peripheral blood and make them respond. Therefore, both phytoh...

Embodiment 3

[0154] Embodiment 3. the selection of blood collection tube

[0155] To ensure the accuracy and reliability of experimental results, blood collection tubes used to obtain anticoagulated whole blood should contain anticoagulants and be low in endotoxin. A variety of commercially available blood collection tubes can be used to collect anticoagulated whole blood, such as heparin lithium vacuum blood collection tubes produced by BD Company.

[0156] In this example, the whole blood of healthy subjects (N1) and tuberculosis patients (T1) was collected using lithium heparin vacuum blood collection tubes produced by BD Company. Then, the above two kinds of whole blood samples were stimulated with recombinant protein EC5 (1 ml each, the final concentration of recombinant protein EC5 was 2 μg / ml), and cultured at 37° C. for 22 hours. Unstimulated whole blood was used as a negative control. The whole blood plasma of each sample (including the negative control) was then collected by ce...

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Abstract

The invention relates to the field of molecular biology, immunology, disease diagnosis and the like and in particular relates to a chimeric recombinant antigen. The chimeric recombinant antigen comprises three antigen areas which are respectively derived from mycobacterium tuberculosis specific proteins Rv3875, Rv3874 and TB7.7. The invention further relates to a composition of the chimeric recombinant antigen and an application of the chimeric recombinant antigen. The invention further provides a method for detecting the immune response of a specific cell T by using the chimeric recombinant antigen. The invention further provides a method for diagnosing whether a patient is affected by mycobacterium tuberculosis, wherein the method comprises the step of using the chimeric recombinant antigen disclosed by the invention.

Description

technical field [0001] The invention relates to the fields of molecular biology, immunology and disease diagnosis. In particular, the present invention relates to a chimeric recombinant antigen comprising three antigenic regions derived from Mycobacterium tuberculosis specific proteins Rv3875, Rv3874 and TB7.7, respectively, and also to a combination comprising said chimeric recombinant antigen and the use of the chimeric recombinant antigen. The invention also provides a method for using the chimeric recombinant antigen to detect specific T cell immune response in vitro. The present invention also provides a method for diagnosing whether a patient is infected with Mycobacterium tuberculosis, which comprises using the chimeric recombinant antigen of the present invention. Background technique [0002] Using the cellular immune response of antigen-specific T cells for in vitro diagnosis of pathogenic microorganism infection is a new detection method developed in recent year...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/569
CPCC12Q1/68G01N33/53G01N2333/35G01N33/6854C07K14/35A61K39/04G01N33/5695C12N15/11
Inventor 葛胜祥熊君辉符美娟许荣均乔杉李芳罗文新张军夏宁邵
Owner XIAMEN UNIV
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