Prokaryotic Expression And Polyclonal Antibody Preparation Of N Terminal Protein Of SHH Ligand In Hedgehog Signal Path In Human

[Aim] Prepare the polyclonal antibody of SHH in hedgehog signal path in human pancreatic cancer, and then measure the expression of SHH in pancreatic cancer tissue. [Methods] The total RNA was isolated from human pancreatic cancer strain SW₁₉₉₀ After being synthesized and amplified from the total RNA by the method of RT-PCR, the SHH-N terminal gene was cloned into pET22b vector, which was then transformed into E coli. BL21 (DE3) to express SHH recombination protein with the induction of IPTG. The protein was purified by affinity chromatography through a His. Bind column. Then the purified protein was immunized to KM mouse to produce polyclonal antibody. By using this kind of self-made antibody, SHH expression was detected in pancreatic cancer by immunohistochemistry.[Results] Vector pET22b-SHH-N was constructed successfully, and the interest protein was induced to express. The SHH polyclonal antibody was made with high titer and large output. After being proved by Western blot, this kind of antibody was used in assessing pancreatic cancer specimen with high quality of immunohistochemistry staining.[Conclusion] Recombinant plasmid pET22b-SHH-N was constructed and expressed protein successfully. The SHH polyclonal antibody was made, which provide a useful tool in the further study of Hedgehog signal path in human pancreatic cancer.