[Preparation of anti-human integrin beta 3 monoclonal antibody and study on its antitumor activity].
OBJECTIVE
To prepare the monoclonal antibody (mAb) against human integrin beta 3, and explore its role in tumor therapy.


METHODS
Total RNA was isolated from human lymphocytes, and the DNA fragment encoding extra-cellular domain of human integrin beta 3 was amplified by RT-PCR. The integrin beta 3 gene was cloned into the prokaryotic expression vector pQE30, and the expression plasmid pQE30-beta 3 was transformed into E.coli M15. The expression of beta 3 protein was induced by IPTG, and the expressed beta 3 protein was purified by Ni-affinity agarose. BALB/c mice were immunized with the purified beta 3 protein, and mAb was prepared by hybridoma technique. The specificity of the mAbs was identified by Western blot. The mAbs were screened by their inhibitory effect on the tumor growth in vivo. The inhibition of the mAb to HUVEC cell growth was detected by MTT assay. The role of the mAb in inducing HUVEC apoptosis was analyzed by flow cytometry (FCM).


RESULTS
The extra-cellular gene fragment of human integrin beta 3 was amplified by RT-PCR, and the expression plasmid pQE30-beta 3 was constructed. Human integrin beta 3 protein was expressed and purified, and used for immunization. Eight clones of mAb against human integrin beta 3 were successfully prepared. The mAb 4F12 was found to inhibit tumor growth in vivo and reduce blood vessels in tumor. Furthermore, the mAb 4F12 could inhibit HUVEC growth and induce apoptosis in vitro.


CONCLUSIONS
The human integrin beta 3 protein was obtained and the mAbs against it have been prepared successfully. The mAb 4F12 can inhibit tumor growth in vivo and reduce blood vessels in tumor. One of the mechanisms of the antitumor effect is inhibition of growth and induction of apoptosis of endothelial cells of blood vessels in tumor.
Ma
Shu-hua
SH
Fudan University Shanghai Medical College, Shanghai 200032, China. mah_de@sina.com.cn
Gan
Yu
Y
Zhang
Yi
Y
Zhu
Ming-jie
MJ
Hu
Jing-ying
JY
Yang
Yao-qin
YQ
Zhao
Xin-tai
XT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D039661:Integrin beta3 / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014470:Umbilical Cord / Q000166:cytology
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D039661:Integrin beta3