[Prokaryotic expression and immunogenicity of hWAPL, a norel oncogene related to cervical cancer].
OBJECTIVE
To construct the prokaryotic expression plasmid of hWAPL, induce the expression of the protein and prepare polyclonal antibody.


METHODS
The hWAPL cDNA was amplified by RT-PCR from HeLa cells derived RNA and then cloned to pMD18-T according to A-T. The DNA fragment was isolated and linked to prokaryotic expression vector pET28a. The recombinant protein was induced by IPTG and identified by SDS-PAGE. Then it was injected into mice to prepare polyclonal antibody. The immunogenicity and specificity of the recombinant protein were identified by ELISA and Western blot.


RESULTS
The sequencing, PCR and endonucleases digestion results showed that the hWAPL fragment was correctly inserted into pMD18-T and pET28a vectors. SDS-PAGE showed 25,000 fusion hWAPL protein was expressed in BL21 cells. The titer of polyclonal antibody was 1:3 200 by indirect ELISA. The 25,000 fusion hWAPL protein was detected by SDS-PAGE and Western blot.


CONCLUSIONS
The hWAPL protein can be expressed by prokaryotic vector pET28a. Furthermore, the expression protein can be used as immunogen to generate antibodies with specificity.
Cao
Chun-xia
CX
Department of Microbiology, Medical College, Xi'an Jiaotong University, Xi'an 710061, China. chunxiacao@yahoo.com.cn
Ma
Jun
J
Xun
Meng
M
Xue
Xin
X
Chen
Ping
P
Chu
Yong-lie
YL
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*; D000097722:Cohesins
D000906:Antibodies; D002352:Carrier Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C488092:WAPL protein, human; D000097722:Cohesins