Prokaryotic Expression Of Human Rb-PTCH In Hedgehog Signalling And Preparation Of Its Antiser Um

AIM: To clone human pancreatic cancer gene PTCH, construction and expression of PET22b/PTCH plasmid. The protein was used to immunize rabbit to prepare antiserum. which was then used to detect the expression of PTCH in pancreatic cancer . Methods: The total RNA was isolated from human pancreatic cancer strain SW₁990 The PTCH gene was synthesized and amplified from the total RNA by RT-PCR. The resulting product was cloned into PET22b vector. The PET22b / PTCH construct was then transformed into E.coliBL21-CodonPlus ?-RP, and the gene was sequenced.The fusion protein was expressed by IPTG and verified by Western blot method.The expressed protein was purified with a His.Bind column .An antiserum against PTCH was perpetrated by immunizing rabbit with purified PTCH.The expression of PTCH in 20 patients with pancreatic cancer was detected by immunohistochemistry. Results: A human pancreatic cancer gene with a reading frame of 789 bp was successfully cloned from human pancreatic cancer strain SW₁₉₉₀, which had the same sequence as that of PTCH gene in Genbank. The expression of PET22b/PTCH was proved by Western blot.A specific antiserum against PTCH was proved by Western blot. The expression of PTCH in pancreatic cancer was 55.o%. Conclusion: Human pancreatic cancer gene PTCH was successfully cloned and expressed in E.coli. antiserum of PTCH has been successfully established ,which provide an useful tool in the studies of Hedgehog signal in pancreatic cancer.