[Prokaryotic expression, purification and preparation of polyclonal antibody for human UNC5CL]

Abstract

Aim: 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.

Conclusion: The rabbit polyclonal antibody against human UNC5CL is prepared. It can react with UNC5CL specifically and provide basis for further function study of UNC5CL.