[Expression, purification and activity analysis of BCG HSP70.].
OBJECTIVE
To get BCG HSP70 protein with excellent biologic activity through E.coli. expression and purification.


METHODS
BCG HSP70 gene was amplified by PCR and inserted into vector pMD18-T. After confirmed by sequencing, the gene was subcloned into expression vector pET28a. Recombinant pET28a/HSP70 was transformed into E.coli. BL21(DE3). Recombinant BCG HSP70 protein was expressed with IPTG induction and the purified protein was then identified by SDS-PAGE and Western blot. And its effect on the proliferation of mouse splenocytes was observed.


RESULTS
Gene encoding BCG HSP70 which was identical with that published in GenBank was successfully obtained by PCR. SDS-PAGE analysis showed a protein with relative molecular mass of 70 000 was expressed. When the purified protein was detected by Western blot analysis, a specific protein with a molecular mass of 70 000 could be visualized. The purity of the purified protein was about 96.5%. The purified protein could stimulate the proliferation of mouse splenocytes significantly.


CONCLUSIONS
BCG HSP70 is expressed and purified successfully, which would lay a foundation for further research on BCG HSP70 and BCG.
Li
He
H
Department of Immunology, Norman Bethune College of Medical Sciences, Jilin University, Changchun 130021, China.
Cao
Zhao
Z
Zhang
Pei-Yin
PY
Wei
Hong-Fei
HF
Wang
Hua
H
Sun
Lu-Guo
LG
Wang
Li-Ying
LY
Yu
Yong-Li
YL
D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D009163:Mycobacterium bovis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins