Cloning of Human Lysozyme and Lactoferrin Genes and Expression in Methylotropic Yeast Pichia pastoris

• Main Authors: Yen-Long Sin, 辛彥龍
• Other Authors: Yan-Ming Horng
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/22920450032827920214

碩士 === 國立嘉義大學 === 動物科學系碩士班 === 92 === Cloning of Human Lysozyme and Lactoferrin Genes and Expression in Methylotropic Yeast Pichia pastoris Yen-Long Sin Abstract The purpose of the studies was to clone the human lysozyme(hLZ) and lactoferrin(hLF) genes, and to detect the gene expression in methylotropic yeast Pichia pastoris. The 392 bp fragment of hLZ cDNA was amplified by PCR from the template of human placenta cDNA with hLZ-Fns and -Rns primers. The hLZ cDNA fragment was inserted into the vector pCDNA3.1/ V5/His-TOPO to generate recombinant plasmid pCMVhLZ. The inserted fragment was then cut with restriction enzymes, isolated and ligated into pPICZαA vector. The recombinant plasmid was named as pPICZαAhLZ392. Which was introduced into the methylotropic yeast Pichia pastoris by electroporation. The hLZ mRNA transcripted in yeasts induced by methanol could be detected. The Western blotting was carried out to detect the hLZ protein expression. A 14 kDa band of hLZ protein in the gel was found. The hLZ protein purified from the medium for activity assay. The results showed that the enzyme activity of tested sample was higher than the negative control. According to this study that was proven the recombinant hLZ. The 2076 bp fragment of hLF cDNA was amplified by PCR from the template of human mammary gland cDNA with hLF-Fns and -Rns primers. The hLF cDNA fragment was inserted into the vector pCDNA3.1/V5/His-TOPO to generate recombinant plasmid pCMVhLF. The inserted fragment was then cut with restriction enzymes, isolated and ligated into pPICZαA vector. The recombinant plasmid was named as pPICZαAhLF. Which was introduced into the methylotropic yeast Pichia pastoris by electroporation. The hLF mRNA transcripted in yeasts induced by methanol could be detected. The Western blotting was carried out to detect the hLF protein expression. A 80 kDa band of hLF protein in the gel was found. According to this study that was proven the recombinant hLF.