Construction of recombinant plasmid and prokaryotic expression in E. coli and biological activity analysis of human placenta arresten gene.
BACKGROUND
The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten.


METHODS
The arresten gene was obtained from a healthy puerperal's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5alpha, BL21 and BL21 (DE3) by the CaCl2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purified, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM).


RESULTS
The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities.


CONCLUSIONS
The successful construction of recombinant plasmid pBV220/arr and the effective expression in E.coli have laid a foundation for further study of its anti-angiogenic function and may pave the way for future anti-tumor application.
Department of Toxicology, Shanxi Medical Universiy, Taiyuan 030001, China. zhengzheng200101@yahoo.com.cn
Jin-Ping
JP
Zheng
Hai-Ying
HY
Tang
Xian-Jiu
XJ
Chen
Bao-Feng
BF
Yu
Jun
J
Xie
Tang-Chun
TC
Wu
D019393:Arrestin / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D010920:Placenta / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D011247:Pregnancy; D011387:Prokaryotic Cells / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D019393:Arrestin; D011994:Recombinant Proteins; D012313:RNA