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

CN112159466AInactive Publication Date: 2021-01-01UNIV OF ELECTRONIC SCI & TECH OF CHINA
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2021-01-01
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a preparation method of recombinant grass carp interleukin-6 active protein. The method comprises the following steps of S1, culturing BL21(DE3) escherichia coli containing expression plasmid pET-30 / gcIL-6, and inducing protein expression for 15-25 hours; S2, collecting fermentation liquor, performing centrifugation, discarding supernate, resuspending thalli, and performingfreezing and thawing for 10-20 hours; S3, adding a phenylmethylsulfonyl fluoride solution into bacterial liquid, carrying out ultrasonic treatment, then performing centrifugation, and collecting precipitate, namely an inclusion body; S4, carrying out oscillation washing on the inclusion body by using a washing buffer solution, and then performing centrifugation; S5, carrying out oscillation washing on the inclusion body by using 1% Triton X-114, and performing centrifugation; S6, carrying out oscillation washing on the inclusion body by using 3M urea, and performing centrifugation; S7, dissolving the inclusion body by using a binding buffer solution containing 6M guanidine hydrochloride, performing centrifugal dissolving for 5-15 minutes, and reserving supernate; S8, purifying protein; S9, dropwise adding the purified protein into a folding buffer solution, then performing stirring for 10-20 hours, and then performing filtration and centrifugation to obtain a protein solution; and S10, putting the protein solution into a phosphate buffer solution, and performing centrifugation to obtain the recombinant grass carp interleukin-6 active protein. Through the method, a large amount ofthe recombinant grass carp IL-6 protein with high biological activity can be obtained.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of recombinant grass carp interleukin-6 active protein. Background technique

[0002] In mammals, interleukin-6 plays an important role in both the immune and nervous systems. In addition, interleukin-6 is also involved in the regulation of liver regeneration and metabolism of the body. So interleukin-6 is one of the very important cytokines in the body. In fish, knowledge about the function of interleukin-6 is limited. To study the function of interleukin-6 in fish needs to obtain a large amount of recombinant interleukin-6 protein.

[0003] In order to study the function of recombinant grass carp interleukin-6, recombinant grass carp IL-6 protein can be obtained by Pichia pastoris eukaryotic expression system, but the obtained recombinant grass carp IL-6 protein is highly glycosylated, and only 1000ng / mL of this protein can be up-regulated Expre...

Examples

Embodiment

[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...