Cloning And Sequencing Of Human α-defensin-1(HNP-1) Gene And Construction Of Its Eukaryotic Expression Vector

Objective: To construt an eukaryotic expression vector containing the gene encoding human α-defensin-l(HNP-l).Methods: Isolating the human peripheral blood and acquiring polymorphonuclear cells. Total RNA was extracted from polymorphonuclear cells in human peripheral blood and complementary DNA was made by reverse transcription as template for polymerase chain reaction. PCR product of expected size was then insert into pGEM-T cloning vector. Recombinant pGEM-T-HNP-1 was transduced into Escherichia coli DH5a competent cells. PCR and restriction analysis were performed to identify the clone containing the DNA fragment, followed by sequencing. Then the recombinant plasmid was digested with restrictive endonuclease EcoR I and BamH I ,and subcloned into plasmid pcDNA3.1(-) to construct recombinant plasmid pcDNA3.1-HNP-1.Results: We obtained a 303bp DNA fragment which is identical to the HNP-1 cDNA sequence reported in the GeneBank; PCR, restrictive endonuclease digesting andDNA sequencing confirmed the recombinant eukaryotic plasmid vector which contained HNP-1 gene had been constructed correctly.Conclusions: An eukaryotic expression vector pcDNA3.1-HNP-1 was constructed successfully, as a base for expression of HNP-1 gene in the eukaryocyte.