[专业论文]SUMO表达系统高效_可溶性表达重组鼠源IL_1_.pdf

! 684 ! 20 10 26 doi : 10. 3969/ j. issn. 1000484X. 2010 . 08. 00 3 SUMO IL1 ( 东北农业大学生命科学学院生物制药教研室, 哈尔滨 150030) Q78 A 1000484X(2010) [ ] : LPS IL1, , IL1 , IL1: LPS RNA, cDNA , , GenBank mIL1, PCR, m IL1, pHisSUMO ex press SUMO , pHisSUMO expressmIL1Rosetta ( DE3) , IPTG mIL1/ SUMO , Ni NTA Agarose , SDSPAGE Western blot , SUMO protease1 SUMO , m IL1MTT L929 : DNA GenBank , SDSPAGE 37 kD, 17 kD, , Western blot mIL1 95% , MTT L929 , : mIL1 [ ] mIL1 SUMO MTT Efficient expression of t e soluble murine IL1wit SUMO expression system HA O JianQuan, REN GuiP ing , LI U ShengWei, F UJunH ua, G UX ueJia, YA O WenBing , LI N ing , LI DeShan Lfi e Science Center of Northeast Agricultural University , Dep artment of B iolog ical Pharmacy , H arbin 150030, China [Abstract] Objective:To clone mou se interleukin1from thymus that stimulated by LPS , and obtain the soluble and biologically active mouse IL1 protein by prokaryotic expression f or further study of the IL1 protein. Met ods:Total RNA was isolated from mouse thymus th at stimulated by LPS, and reversetranscripted into cDNA,w hich was used as the template for PCR reaction . The nested PCR prim ers f or cloning mIL1w ere designed according to the published sequ ence. The cDNA coding for mIL1w as cloned and constru cted into the prokaryotic ex pressing vector pHisSUMO expressmIL1. The vector was transform ed into E coli R osetta ( DE3) , the mIL1/ SUMO fusion protein was in duced by IPTG and purif ied by NiNTA Agarose. The expressing product was evalu ated by SDSPAGE and Western blot . After purif ication, the SUMO tag w as removed by protease1 cleavage to obtain the mature mIL1protein. The biological activity of the target protein was determine