Protein rv2818c for specific detection of Mycobacterium tuberculosis infection
A technology of Mycobacterium tuberculosis and rv2818c, which is applied in the field of biomedicine, can solve the problems that active pulmonary tuberculosis cannot be diagnosed and the positive detection rate is low.
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Embodiment 1
[0038] Example 1, prokaryotic expression of target protein Rv2818c
[0039] Preparation of the target protein Rv2818c: Introduce the recombinant vector pET28a-Rv2818c expressing the Rv2818c protein coding gene into Escherichia coli BL21 to obtain the recombinant strain BL21 / pET28a-Rv2818c; then induce the recombinant strain BL21 / pET28a-Rv2818c with IPTG, and collect the supernatant, namely For the target protein Rv2818c.
[0040] details as follows:
[0041] 1. Construction of recombinant bacteria expressing the target protein
[0042] The amino acid sequence of the Rv2818c protein is sequence 2, and the nucleotide sequence of its coding gene is sequence 1.
[0043] Insert the Rv2818c protein coding gene shown in Sequence 1 into the NdeI and EcoRI sites of the pET28a vector to obtain the recombinant vector pET28a-Rv2818c;
[0044] The recombinant vector was introduced into Escherichia coli BL21 to obtain the recombinant bacterium BL21 / pET28a-Rv2818c.
[0045] 2. Protein ex...
Embodiment 2
[0060] Example 2, the application of Mycobacterium tuberculosis marker antigen Rv2818c in the detection of Mycobacterium tuberculosis
[0061] 1. T-SPOT kit detection
[0062] The specific method is as follows, and some solutions inside are all from the T-SPOT kit of Oxford immunotec (UK):
[0063] 1) Use heparin vacuum blood collection tubes to directly collect peripheral veins from different patients to be tested (61 patients with tuberculosis clinically detected as shown in Table 1 and 10 cases of clinically detected non-tuberculosis patients shown in Table 2, and informed). blood, to obtain anticoagulated blood samples;
[0064] 2) Mix the anticoagulated blood sample with RPMI1640 culture medium at a volume of 1:1; carefully add the blood sample to the upper layer of Ficoll lymphocyte separation medium (GE Healthcare 17-1440-02) at a ratio of 3:1 (using Ficoll-PaqueTM plus Separation of PBMCs according to the operating instructions); room temperature 25 ° C, 1000g centri...
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