[Expression of mouse PDL-1 extracellular region and its antibody preparation].
OBJECTIVE
To prepare mouse PDL-1 membrane extracellular region (mPDL-1) and its antibody for further study of the biological activity of PDL-1.


METHODS
The extracellular region gene fragment of PDL-1 was amplified by RT-PCR and then was cloned into pET28a(+) prokaryotic expressing vector. The recombinant protein mPDL-1 was induced by IPTG in E.coli BL21 (DE3) and the expressed protein was detected by Western blot. The purified protein was used to immune rabbits to prepare polyclonal antibody and the specificity and the titer of the antibody were detected with ELISA, immunofluorescence assay and FCM.


RESULTS
The plasmid pET28a(+)/mPDL-1 was successfully constructed and mPDL-1 protein was expressed in E.coli BL21(DE3) with high efficiency. Western blot showed that the recombinant protein was characterized with His antibody. Rabbit immunized with the purified protein produced high titer of antibody. Immunofluorescence assay displayed that the PDL-1 protein highly expressed in B16 melanoma cells was specifically combined with the antibody.


CONCLUSIONS
Recombinant mPDL-1 is expressed and purified with high antigenicity. The preparation of recombinant mPDL-1 and its polyclonal antibody lay the foundation for further research on mPDL-1 bioactivities.
Qin
Xiao-lin
XL
Institute of Molecular Biology, Three Gorges University, Yichang 443002, China. qinxiaolin200810@163.com
Liu
Chao-qi
CQ
Yang
Fan
F
Lu
Bai-rui
BR
Li
Bin
B
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D018122:B7-1 Antigen / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D060890:B7-H1 Antigen; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D008297:Male; D008546:Melanoma, Experimental / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits
D000906:Antibodies; D018122:B7-1 Antigen; D060890:B7-H1 Antigen; C498919:Cd274 protein, mouse; D008562:Membrane Glycoproteins; D010455:Peptides