[Expression of human HMGB1 A box in E. coli and its inhibitory effects on monocytes].
OBJECTIVE
To clone human high mobility guoup box1 A box (HMGB1 A box) and express it in escherichia coli effectly, investigate the inhibit effection of the purpose protern to the activation of monocytes stimulated by immunocomplex.


METHODS
According to human HMGB1 gene order which was optimized by our laboratory the PCR primer was designed which containing restriction enzyme cutting site. The HMGB1 A box gene was cloned following the whole gene synthesis template of human HMGB1, then the PCR product was inserted into clone vector pMD19-T. The positive colone was identified by colony PCR, zymography analysis and DNA sequencing. Recombinant colne vector was digested by restriction enzymes Nde I and Xho I and separated by agarose gel electrophoresis, then the fragment was inserted into the corresponding sites of expression vector pQE-T7-2. The positive recombinant expression vector was identified by colony PCR and the recombinant strains was induced by IPTG, then the purpose protein was identified by SDS-PAGE and Western blot. The recombinant protein of human HMGB1 A box was purificated by Ni(2+)-NTA chromatography and the inhibit effection of the purpose protern to the activation of monocyte stimulated by immunocomplex was identified by RT-PCR.


RESULTS
We acquired expression strains of recombinant human HMGB1 A box, the target protein account for up to 40% of the whole protein of E.coli. Western blot showed recombinant protein can specificly reacted with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody.The purpose protein was found more than 90% after purified, and can effectively inhibit the production of BAFF, IFN-gamma and TNF-alpha in monocyte which were induced by IC.


CONCLUSIONS
A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.
Zhu
Min
M
Center Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China. zhuminmin456abc@163.com
Cui
Bin
B
Jiao
Yu-lian
YL
Wang
Lai-cheng
LC
Qu
Yun-yun
YY
Sun
Xin-ping
XP
Liu
Xiao-wen
XW
Xu
Jie
J
Zhao
Yue-Ran
YR
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D024243:HMGB1 Protein / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D024243:HMGB1 Protein; D011994:Recombinant Proteins