Cloning and identification of PTCH gene involved in hedgehog signal pathway in human pancreatic cancer / 中华消化杂志

ABSTRACT

Objective To clone human pancreatic cancer gene PTCH,construct the recombinant(expression) plasmid PET22b/PTCH and express the fusion protein.Methods The PTCH gene was(amplified) by RT-PCR from the total RNA extracted from human pancreatic cancer strain SW1990. The amplified product was inserted into the vector PET22b to construct the recombinant expression plasmid PET22b/PTCH,which was transformed into E.coliBL21-CodonPlus~(TM)-RP and then identified by(sequence) analysis.The expression of fusion protein was induced with IPTG and verified by Western blot method.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.Conclusion Human(pancreatic) cancer gene PTCH was successfully cloned and constructed with PET22b plasmid.The(prepared) fusion protein lays the basis for further study.