Preparation method of recombinant grass carp interleukin-6 active protein
A technology of active protein and interleukin, applied in the biological field, can solve the problems of low yield and low activity of recombinant grass carp interleukin-6 active protein, and achieve the effects of high yield, reduced salt precipitation and high activity
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[0043] A preferred embodiment of the present invention provides a method for preparing recombinant grass carp interleukin-6 active protein, the specific steps are as follows:
[0044] 1. Inoculate BL21(DE3) Escherichia coli containing expression plasmid pET-30 / gcIL-6 in 6 mL of LB medium containing kanamycin (final concentration: 30 mg / mL). Activate overnight at 37°C in a 50mL BD tube at 180rpm.
[0045] 2. The next day, inoculate 4 mL of activated E. coli into 180 mL of LB medium containing kanamycin (final concentration: 30 mg / mL). Cultivate in a 500 mL Erlenmeyer flask at 180 rpm at 37° C. until the light absorption value at 600 nm is 0.6-0.8. Add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, and induce protein expression at 16° C. for 20 hours at 180 rpm.
[0046] 3. Collect the fermentation broth and divide it into two 50mL BD tubes, centrifuge at 4°C and 10,000×g centrifugal force for 3 minutes, discard the supernatant, collect the bacteria, and rep...
experiment example 1
[0060] Denaturing polyacrylamide gel electrophoresis analysis of the purification process of grass carp and recombinant grass carp interleukin-6, the results are as follows figure 1 and figure 2 As shown, it shows that the recombinant grass carp interleukin-6 protein with high purity and large amount can be obtained by the method of the present invention.
experiment example 2
[0062] The interleukin-6 protein obtained by the method of the present invention, the interleukin-6 protein obtained by the Pichia eukaryotic expression system and the activity of the interleukin-6 protein in the prokaryotic expression supernatant of Escherichia coli were detected respectively, and the results showed that ( image 3 ) The recombinant protein obtained in the present invention can significantly up-regulate the gene expression of grass carp SOCS3 in grass carp head kidney lymphocytes, so the grass carp recombinant interleukin-6 protein obtained by the method of the present invention has high biological activity. Grass carp interleukin-6 protein obtained from Pichia pastoris eukaryotic expression system, only 1000ng / mL concentration of interleukin-6 recombinant protein can up-regulate the expression of SOCS3 gene in grass carp head kidney leukocytes by 1.4 times; Escherichia coli prokaryotic expression supernatant Grass carp interleukin-6 protein in the method, whe...
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