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Published on: April 11, 2018
[The construction of recombinant mouse interleukin 4 prokaryotic expressing plasmid and the expression and
Xiao-Lei Luo1, Xiao-Xiao Ren, Nong-Yu Huang
1State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Objective:
To construct Recombinant Mouse Interleukin 4 prokaryotic expressing plasmid, express it in E. coli strain BL21 (DE3), purify and identify the expressed cytokine.
Methods:
The optimized mIL-4 cDNA fragment was cloned into the prokaryotic expressing vector pET-32a (+) to generate pET32/rmIL-4 and transformed into BL21 (DE3) cells. After induction, the expressed protein wasfound to be in the inclusion of E. coli cells. The induced product was purified through Q Sepharose Fast Flow Column and Gel Filtration Column under renaturing condition. The purified protein was identified by Western blot analysis, and the biologic activity was identified by the generation of mIL-4 dependence cell CTLL-2 and in vivo experiment of mouse psoriasis model.
Results:
The recombinant plasmid pET32/rmIL-4 has been constructed correctly. The inclusion body was washed with 3 mol/L guanidine hydrochloride and denaturized in 7 mol/L guanidine hydrochloride. Then, the denaturized protein was gradient dialysis in the condition of pH 9. 5. The protein we purified has the right immunology specificity and biologic activity.
Conclusion:
The recombinant mouse interleukin-4 with high purity and biologic activity was prepared in this study,which will become the basis for the further study of the biologic activity of IL-4.
