Construction of cloning vector of c-myc-TFF2 fusion gene by PCR based gene assembly / 西安交通大学学报(医学版)

ABSTRACT

Objective To assemble fusion gene of c-myc tagged human trefoil factor family 2(hTFF2)(in vitro) and construct a cloning vector of this fusion gene.Methods Based on amino acid sequence of hTFF2 and codon of lactococcus lactis,cDNA of htff2 sequence was designed and extended at their 5' ends with a sequence encoding the c-myc tag;and the sequence of fusion gene c-myc-htff2 was designed.According to restriction enzyme sites of pBluescript Ⅱ sk(+),the SalⅠ and BamHⅠ were arranged at 5′ and 3′ ends of the fusion gene respectively.Instructed by DNAWORKS program,the fusion gene sequence of c-myc-htff2 was designed as 14 oligonucleotides that overlapped each other.The target gene fragment of cmyc-htff2 fusion gene was obtained by the means of polymerase chain reaction(PCR) based gene assembly.Then,the c-myc-htff2 was subcloned into vector of pBluescript II sk(+) for identification of the fusion gene by restricting enzyme excision and DNA sequencing.Results Assembly of c-myc-TFF2 fusion gene(in vitro) and construction of pBS-TFF2 had been completed successfully;and DNA sequencing showed that the sequence of synthetic gene accorded with the expectation.Conclusion With the help of DNAWORKS program,the design and assembly of oligonucleotides (in vitro) is effective to synthesize a target gene,and the cloning vector of c-myc-htff2 fusion gene has been successfully constructed.