Recombinant protein, expression vector, recombinant engineering bacterium and application of mycobacterium tuberculosis
A technology of Mycobacterium tuberculosis and recombinant engineering bacteria, which can be used in application, genetic engineering, recombinant DNA technology and other directions, and can solve the problems of unclear regulatory relationship and other problems
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Embodiment 1
[0038] Example 1 Primer Design and Synthesis
[0039] Referring to the rv0324 (Gene ID: 886548) gene sequence SEQ ID NO: 1 in the GenBank database, specific primers SN221f and SN221r were designed respectively, and the primer information is shown in Table 1.
[0040] Table 1 Primer Information Table
[0041]
Embodiment 2
[0042] Example 2 Expression of recombinant protein Rv0324
[0043] 1. BCG genome extraction
[0044] Bacterial Genome Extraction Kit (Tiangen) was used to extract.
[0045] (1) Centrifuge the BCG bacterial liquid at 10 000 rpm for 1 min, discard the supernatant, and collect the bacterial liquid precipitate.
[0046] (2) Add about 1 mL lysozyme (20 mg / mL) to 500 mL bacterial solution and divide into two tubes, 500 μL / tube. Breaking up bacteria: Ultrasonic: 35% amplitude, on 3s, off 5s, 2min, then grinding with a grinding rod for 2min. 37°C water bath for 1h.
[0047] (3) Add 4 μL RNase to each tube, and let it react for 5 minutes at room temperature.
[0048] (4) Add 20 μL proteinase K for treatment.
[0049] (5) Add 440μL GB, shake for 15s, place at 70°C for 10min, and centrifuge briefly.
[0050] (6) Add 440 μL of absolute ethanol, shake for 15 s, and centrifuge at 10 000 rpm for 5 min.
[0051] (7) Add the supernatant to the adsorption column CB3, centrifuge at 10 000...
Embodiment 3
[0077] Example 3 In vitro binding experiment between Rv0324 and p / o rv3597c
[0078] Using the Mycobacterium bovis BCG (Tokyo-172) genome as a template and SN395f / r as primers, p / o rv3597c was amplified by PCR, and gel shift assay (EMSA) was performed. Prepare 30 μL of EMSA reaction system: 1 μL 40ng / μL p / orv3597c, 6 μL 5×Binding Buffer, different final concentrations (0.16 μmol / L, 0.33 μmol / L, 0.67 μmol / L, 0.83 μmol / L, 0.99 μmol / L) Rv0324 protein, and with ddH 2 O make up. After incubating at 37°C for 30 min, electrophoresis was performed on 8% polyacrylamide gel at 200V for 3 h to separate the complexes. After staining, the gel imaging system was used for UV transmission imaging to verify that Rv0324 and p / o rv3597c could combine in vitro. Cy5-labeled primer SN395f / r at the 5' end, PCR amplified BCG (Tokyo-172) genome to obtain Cy5-labeled p / o rv3597c, which competed with unlabeled p / o rv3597c for binding to Rv0324, and performed EMSA. EMSA reaction system 30μL: 0.5μL 0.1...
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