[The expression, renaturation, purification and preliminary identification of human IgE Cepsilon3-Cepsilon4].
OBJECTIVE
To gain recombinant protein Cepsilon3-Cepsilon4 of IgE Fc (E34).


METHODS
We cloned the gene coding human IgE Cepsilon3-Cepsilon4 (E34) and constructed an expression vector pET28a(+)-E34. The target protein was expressed as inclusion body in E.coli BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hIgE mAb was identified by Western blot and ELISA.


RESULTS
The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hIgE mAb.


CONCLUSIONS
The expression vector pET28a(+)-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.
Qiao
Chun-xia
CX
Department of Molecular Immunology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. bioqcx@eyou.com
Lin
Zhou
Z
Hu
Mei-ru
MR
Liu
Rui
R
Qin
Wei-song
WS
Feng
Jian-nan
JN
Shen
Bei-fen
BF
D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D007073:Immunoglobulin E