[Prokaryotic expression and purification of RPS13 and preparation of polyclonal antibody against RPS13].
OBJECTIVE
To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody.


METHODS
RPS13 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the highly expressed cell of gastric multidrug cell SGC7901/VCR. After sequenced, the gene was cloned into the expression vector pET-28a(+) to construct RPS13 expression plasmid pET-28a(+)-RPS13. The expression plasmid was transformed into the E.coli BL21 and screened by ampiline, and then the E.coli BL21 containing the expression plasmid was induced by IPTG and the protein was purifed by Histag column. BALB/c mice were immunized by the immunogen His-RPS13. The titer was measured by ELISA and the polyclonal antibody was obtained.


RESULTS
The expression plasmid pET-28a(+)-RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E.coli BL21 by IPTG for 3 hours, purified by Histag column and confirmed by SDS-PAGE and Western blot. The polyclonal anti-His-RPS13 antibody was obtained by immunizing the mice with RPS13 protein. RPS13 could be specifically recognized by the antibody based on Western blot.


CONCLUSIONS
RPS13 has bene expressed and purified successfully and polyclonal anti-His-RPS13 antibody has been prepared. Our study can be used for the preparation of monoclonal anti-His-RPS13 antibody and for further research of the function of the RPS13 protein in tumor.
Guo
Xue-Yan
XY
National Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Shi
Yong-Quan
YQ
Zhai
Hui-Hong
HH
Sun
Li
L
Liu
Li-Li
LL
Han
Shuang
S
Lei
Ting
T
Fan
Dai-Ming
DM
D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013274:Stomach Neoplasms / Q000378:metabolism
D000906:Antibodies; C047013:RPS13 protein, human; D012269:Ribosomal Proteins