A kind of chimeric recombinant antigen and its use
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 being unsuitable for promotion in grassroots units, discontinuous epitopes in the peptide library, and impact on stimulation effects.
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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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