[Preparation and identification of polyclonal antiserum against heat shock protein 72].
OBJECTIVE
To express fusion protein of histamine (His) and human heat shock protein 72 (hHSP72) in Escherichia coli (E. coli), and to prepare hHSP72 antiserum in rabbit.


METHODS
hHSP72 gene was inserted into pPROEX-1. The recombinant vector was identified by restriction endonuclease digestion analysis and sequence. Fusion protein His-hHSP72 was expressed in E. coli under isopropyl-beta-D-thiogalactoside (IPTG) induction. The rabbit antibody against His-hHSP72 was prepared by using purified His-hHSP72 protein as immunogen, and the specificity and sensitivity of polyclonal antibody were identified by Western blot.


RESULTS
The restriction endonuclease digestion analysis of recombinant plasmid and sequence demonstrated that the hHSP72 gene had been exactly inserted into pPROEX-1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the relative molecular mass of the fusion protein was about 73 ku. Western blot result proved that the rabbit polyclonal antiserum could fuse with over 20 ng hHSP72 protein when diluted to 1:100,000.


CONCLUSIONS
The polyclonal antibody against hHSP72 can be prepared in E. coli, it is a new reagent with high specificity and sensitivity for the research of hHSP72.
Huang
Fan
F
Department of Neuroscience, The First Hospital Affiliated of Sun Yat-sen University, Guangzhou 510080, Guangdong, China.
Xu
Kang
K
Wu
Xing-gang
XG
Ma
Zhong-fu
ZF
Lin
Zheng
Z
D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D050884:HSP72 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D008297:Male; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014162:Transfection
D050884:HSP72 Heat-Shock Proteins; D007106:Immune Sera; D011993:Recombinant Fusion Proteins