Fusion protein of streptavidin and Gaussian luciferase, and application of fusion protein

[0103] Example 1: Construction of Fusion Expression Vector

[0104] Such as figure 1 As shown, the SA gene and the Gaussian luciferase gene are used as templates, and synthetic primers are designed, and PCR amplification is performed to obtain separate gene amplification products of SA and Gaussian luciferase, respectively. Based on this PCR product, a second round of bypass PCR was performed to obtain the SA and Gauss luciferase fusion gene amplification product (sequence such as SEQ ID NO: 13). The PCR amplification product was detected by electrophoresis on a 1% agarose gel, and the product was recovered with a gel recovery kit. The recovered product and the vector plasmid pcDNA3.1(+) were subjected to double enzyme digestion reaction with EcoR I and Hind III respectively. After the reaction, the product was detected by electrophoresis on 1% agarose gel, and the product was recovered with a gel recovery kit . The product recovered by PCR digestion and the product recovered by p

[0106] Example 2: Transfection, expression and purification of fusion expression vector

[0107] Mix 25μg of the constructed pcDNA3.1-SA-Gluc fusion expression plasmid with 1ml cell culture medium. Take another 920μL cell culture medium and 80μL lipo2000 transfection reagent (invitrogen) and mix well. Mix the diluted expression vector and the diluted transfection reagent, and place them at room temperature for 20 minutes. Prepare 25ml concentration as 2×10 6 / ml cells, add the mixed pcDNA3.1-SA-Gluc fusion expression plasmid and transfection reagent mixture to CHO cells, and shake gently. 37°C, 8% CO 2 Cultivate for 3 days under conditions.

[0108] Collect the cell culture fluid, centrifuge at 6000 rpm for 10 min, and collect the supernatant. The supernatant was filtered and transferred to a nickel column, and 10 ml of PBS was equilibrated. Then wash 10ml with buffer (20mM Tris, pH 8.0, 150mM NaCl, 20mM imidazole) to remove impurities, and finally elution with buffer (20mM Tris

[0110] Example 3: Fusion protein luciferase bioluminescence detection

[0111] The SA-Gluc fusion protein and SA-G2L fusion protein were diluted to 3 nM with a substrate diluent (50 mM Tris, pH 8.0, 100 mM NaCl) respectively, and 10 μL was added to a 96-well microplate. Then add 90 μL of the substrate coelenterazine diluted to 10 μM with the same solution, and read the luminous intensity with the self-luminous module of the microplate reader, the result is as follows image 3 As shown, the luminescence intensity of the wild-type SA-Gluc fusion protein is equivalent to that of the Gluc protein at the same concentration, but slightly weaker. The luminescence intensity of the mutant fusion protein SA-G2L is equivalent to that of Gluc, but it clearly shows the characteristic of continuous luminescence, and the luminescence is not significantly weakened after 30s.