A kind of adjustable luciferase fusion protein, its preparation method and application
A technology of luciferase and fusion protein, which is applied in the field of molecular biology, can solve the problems of poor real-time performance and differences of dual fluorescence complementary technology, which can only be successfully detected in a few minutes or even a few hours, and achieve high-throughput drug screening and reduce The effect of background interference and high penetration rate
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Embodiment 1
[0047] Example 1 Preparation of Gausssia luciferase fusion fragment
[0048] (1) Select the expression vector of the luciferase fusion gene as pET21a, and then select a suitable insertion site for luciferase, and finally choose to insert an exogenous fragment between Arg93 and Cys94 to form the N fragment NGluc1 (1~ 93aa) and C fragment CGluc1 (94~185aa);
[0049] (2) Using the recombinant vector pET28a-Gluc1 as a template, when designing and amplifying the NGluc1 fragment, introduce BamH Ⅰ and EcoR Ⅰ restriction sites at the 5' end of the primer, and when designing and amplifying the CGluc1 fragment, introduce HindⅢ at the 5' end of the primer and Not I restriction sites, and are designed to introduce flexible peptide chains (GGGGS) 4 To reduce the steric hindrance caused by specific enzyme cleavage;
[0050] The designed primer sequences are as follows:
[0051] Gluc1 upstream primer (BamH Ⅰ):
[0052] 5'-CGCGGATCCATGAAGCCCACCGAGAACAACGAAG-3';
[0053] NGluc1 downstream...
Embodiment 2
[0069] Embodiment 2 Expression and purification of luciferase fusion body and Gaussia luciferase
[0070] (1) Strain activation: inoculate frozen glycerol bacteria pET21a-NGluc1-malE-CGluc1 and pET28a-Gluc1 into LB liquid medium (containing 100 μg / mL ampicillin) at 37°C and 225 rpm to activate overnight strains;
[0071] (2) Strain fermentation: Inoculate the activated bacterial liquid into fresh LB liquid medium (containing 100 μg / mL ampicillin) at a ratio of 1:100 and cultivate to OD 600 When it reaches 0.4-0.6, it is the logarithmic phase of the growth of the strain;
[0072] (3) Induced expression: add inducer isopropyl-β-D-1-thiogalactoside IPTG (final concentration is 1mM) to induce high-speed expression of luciferase fusion at 28°C, centrifuge at 11000rpm at 4°C Collect the thalli within 10min, discard the supernatant LB medium, add a certain amount of binding buffer (20mM Na 3 PO 4 , 0.5M NaCl, 40mM imidazole, pH 7.4) suspended bacteria;
[0073] (4) Ultrasonic dis...
Embodiment 3
[0075] Example 3 Construction of the dynamic interaction model of luciferase fusion protein
[0076] (1) Realize the ON mode of protein dynamic interaction
[0077] Add 1×PBS (pH7.4) buffer solution to a 96-well white microtiter plate, add luciferase fusion protein diluted to a certain concentration with buffer solution, and quickly add intestinal Coelenterazine (Coelenterazine) substrate, to ensure that the total volume of the system is 200 μL, after thorough mixing, immediately use a microplate reader to detect the bioluminescent intensity in the system (the emission spectrum of the fluorescein surface is shown in image 3 As shown, the detection wavelength is 480-500nm), and the ON mode of the protein dynamic interaction model is realized. The model was evaluated with Gaussia luciferase as a control group;
[0078] (2) Realize the OFF mode of protein dynamic interaction
[0079] Add a certain concentration of cutting enzyme Factor Xa Protease to the reaction system, mix ...
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