以抗體阻斷纖維芽細胞生長激素受體活化之研究

This Fibroblast growth factors and their
receptors are an important set of signal
transduction system in animals. the
association between this system and human
diseases was understood recently.
Achondroplasia, a common type of
dominantly inherited drawfism, was found to
be linked to FGFR3, and patients were found
to have the FGFR3 mutations. Initiated by
this finding, the FGF receptors (FGFR1,
FGFR2 & FGFR3) was found to be related to
several human bone dysplastic diseases
including hypochondroplasia, thanatophoric
dysplasia, Apert and Crouzon syndromes.
The mutations causing achondroplasia
and thanatophoric dysplasia were stimulating
mutations. Therefore, it is possible to modify
or to block the activation of the mutated
receptors by antibody or other methods. We
got FGFR3 cDNA frome Dr. Hayman. We
cloned a portion of the cDNA responsive for
the extracellular domain of the receptor into
2
pRSET vector. The expressed recombinant
protein was used to immunized rabbit for
FGFR3 antibody. This antibody detected a
130 kDa protein in both human skin
fibroblasts lysate and in many cell lines by
western blot analysis. However, this antibody
was unable to capture FGFR3 protein in
immunoprecipitation (IP) experiment. Since
IP is the indispensable tools in the study of
membranous receptors, either for the
expression, function or activation. We tried
to raise monoclonal antibody with this
pRSET protein. We got some clones at the
initial stage of screening, but the potencies of
these clones dropped rapidly during
continuing culture.
In order to solve the problem, we tried
to express other fragments of FGFR3. It is
pretty difficult to express any of the extramembranous
domains of FGFR3, but the
intracellular domain was smoothly expressed.
With the expressed intracellular domain, a
second antibody was raised. However, this
antibody detected more than one protein in
western blot analysis.
The difficulty in this study may arise
from the character of FGFR3. It is a
membranous receptor with intense
glycosylation. E coli expressed protein may
not reflect the structure of FGFR3, and thus
difficult to capture the receptor in IP study.
The intracellular domain, the tyrosine kinase
domain, in the way is homologous to other
tyrosine kinase proteins. However, we got a
lot of experience in this study which will help
us in future studies.