Amino acid sequence and nucleotide sequence specifically combined with mycobacterium tuberculosis and application thereof
A technology of mycobacterium tuberculosis and nucleotide sequences, which is applied in the direction of medical preparations with non-active ingredients, non-active ingredients of polymer compounds, antibacterial drugs, etc., and can solve the problems of time-consuming, technically difficult, and false positives in culture methods To achieve the effects of improving diagnostic sensitivity and specificity, easy storage and transportation, and simplifying the preparation process
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Embodiment 1
[0036] Example 1: Amplification and purification of phage library
[0037]Inoculate a single colony of E.coli ER2738 in 5-10ml LB liquid medium, incubate at 37°C, 200rpm shaker until mid-logarithmic phase (OD600≈0.5); add 10ul phage (from NEB company), 37°C, 200rpm shaker Shake for 4-5 hours, centrifuge at 10,000 g for 10 min, and take the supernatant; centrifuge again at 10,000 g for 10 min, take 80% of the supernatant, add 1 / 6 volume of PEG8000 / NaCl, and stand overnight at 4°C. Take it out the next day, and the white precipitate is the phage. Centrifuge at 10,000g for 15 minutes, discard the supernatant, and gently suck out the residual solution after brief centrifugation; add 1ml TBS solution to dissolve the white precipitate.
Embodiment 2
[0038] Example 2: Panning and Identification of Heptapeptides Binding to M.tb
[0039] (1) Phage titer determination
[0040] Inoculate a single colony of E.coli ER2738 in 5-10ml LB liquid medium, incubate at 37°C, 250rpm shaker until mid-logarithmic phase (OD600≈0.5); heat and melt the top layer of agarose medium in a microwave oven, divide into 3mL / portion In the sterilized test tube, use one tube for each phage dilution, and store it at 45°C for later use; pre-warm the LB / IPTG / Xgal agar plate at 37°C, and take one plate for each phage dilution gradient; Doubling serial dilution (dilution range: amplified phage culture supernatant: 10 8 -10 11 ; Unamplified panning eluates: 10 1 -10 4 ); change a new tip for each dilution, and use a filter tip to avoid cross-contamination; when the E. Use one tube; add 10 μL of phages with different dilutions to each tube of E. coli liquid, shake and mix quickly, and incubate at room temperature for 1-5 minutes; add the phage-infected E...
Embodiment 3
[0047] Example 3: Identification of M.tb-specific phage
[0048] Randomly pick 20 well-separated single colonies on the titer determination plate after the third round of screening, culture and purify them separately, and use phage ELISA to detect the binding activity of each strain of phage to M.tb. The specific steps are as follows: ELISA plates were coated with M.tb and Blocking respectively, and each group was replicated three times, and blocked overnight at 4°C, washed 6 times with TBST, added 100 μL of purified phage, incubated at 37°C for 1 hour with shaking, TBST Wash 10 times to remove unbound phage, add 100 μL of HRP-labeled mouse anti-M13 monoclonal antibody, incubate with shaking at 37°C for 1 hour, wash 10 times with TBST, add TMB substrate for 5-10 minutes at room temperature in the dark, and stop the reaction with 2 mol / L sulfuric acid , Measure the OD value at a wavelength of 450nm, and take P / N ≥ 2.1 as positive.
[0049] The results of the P-ELISA detection ...
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