[The construction of recombinant mouse interleukin 4 prokaryotic expressing plasmid and the expression and purification of target protein].
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.


CONCLUSIONS
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.
Luo
Xiao-Lei
XL
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Ren
Xiao-Xiao
XX
Huang
Nong-Yu
NY
Qiu
Ji
J
Li
Qian
Q
Zhou
Xi-Kun
XK
Li
Jiong
J
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D015847:Interleukin-4