人体β-防御素-3突变基因的克隆及在大肠杆菌中的表达

目的对人体β-防御素-3基因进行定点突变,并在E.coli中表达。方法从人正常皮肤组织中提取总RNA,经RT-PCR扩增编码β-防御素-3成熟肽的cDNA,测序正确后,设计含突变位点的引物,用PCR重叠延伸法对β-防御素-3成熟肽cDNA进行定点突变,将突变产物克隆至pUC18,并在E.coli中融合表达。结果测序结果表明,经RT-PCR所得的β-防御素-3成熟肽序列与GenBank中报道的序列完全一致。经过突变,β-防御素-3成熟肽第29位谷氨酰胺密码子由CAG突变为精氨酸密码子CGA,其余核苷酸序列均未发生变化。将突变β-防御素-3基因在E.coli中诱导表达后,可见突变β-防御素-3蛋白表达。结论已成功克隆并表达了β-防御素-3突变体基因。

Cloning of Site-directed Mutant of Human β-Defensin-3 Gene and Its Expression in E. coli

Objective To induce the site-directed mutation of human β-defensin-3(hBD-3) gene and express the mutant in E.coli.Methods Extract total RNA from normal human skin tissue,then amplify the cDNA encoding the mature peptide of β-defensin-3 by RT-PCR and identify by sequencing.Design a pair of primers containing mutation site and induce the site-directed mutation of the amplified cDNA by over-lap extension PCR.Clone the mutant into vector pUC18 and express in E.coli.Results The gene sequence of the amplified mature peptide of β-defensin-3 was completely consistent with that reported in GenBank.After site-directed mutation,the codon at site 29 of β-defensin-3 mature peptide changed from CAG encoding glutamine to CGA encoding arginine,however,no change of the rest nucleotide sequence was observed.The mutant of human β-defensin-3 was expressed in E.coli.Conclusion The site-directed mutant of human β-defensin-3 gene was successfully cloned and expressed.