[Prokaryotic expression, purification and preparation of polyclonal antibody for human UNC5CL].
OBJECTIVE
To construct the prokaryotic expression vector of UNC5CL, purify the fusion protein of GST-UNC5CL, prepare the polyclonal antibody of UNC5CL and characterize the reactivity and specificity of the polyclonal antibody.


METHODS
The human UNC5CL nucleotide sequence encoding amino acid from 280 to 518 was amplified and cloned into pGEX-4T-2 vector, then transformed into E.coli BL21, in which the fusion protein GST-UNC5CL (aa280-518) was induced and purified by Glutathione Sepharose-4B. Then the purified protein immunized the rabbits and anti-serum was collected. The anti-serum was further detected by ELISA, Western blot and immunohistochemistry.


RESULTS
The prokaryotic expression vector of pGEX-4T-2-UNC5CL(aa280-518) was constructed successfully. It can be induced by IPTG and express a fusion protein of GST-UNC5CL (aa280-518) with M(r); 5 2000. The ELISA, Western blot and Immunohistochemistry demonstrated the rabbit antiserum obtained by immunized by the purified fusion protein can detect the target protein.


CONCLUSIONS
The rabbit polyclonal antibody against human UNC5CL is prepared. It can react with UNC5CL specifically and provide basis for further function study of UNC5CL.
Department of Immunology, Peking University Health Science Center, Beijing, China.
Dan
D
Lü
Xiao-ping
XP
Qian
Xiao-jun
XJ
Tian
Yu
Y
Zhang
Jun
J
Zhang
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008297:Male; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C486467:UNC5CL protein, human