摘要
从脑垂体中提取总RNA,用RT-PCR方法克隆大鳞副泥鳅生长激素基因(pGH)cDNA。结果表明,克隆到的pGH的开放阅读框包括633 bp,编码210个氨基酸,其中包括22个氨基酸的信号肽和188个氨基酸的成熟肽,GenBank注册号为DQ350432。把pGH成熟肽的cDNA插入真核表达载体pPIC3.5K,PCR技术、酶切和测序证明重组子中确实定向插入了pGH成熟肽片段;将重组的pPIC3.5K-pGH用SalⅠ酶切后,转化巴斯德毕赤酵母GS115,PCR技术筛选证明pGH已经整合到酵母染色体上。从而成功克隆大鳞副泥鳅生长激素基因cDNA,并构建了胞内真核表达载体pPIC3.5K-pGH。
pGH（Paramisgurnus dabryanus growth hormone） cDNA was cloned from total RNA of pituitary gland by RT-PCR. The sequence included an ORF of 633 bp which encoded a precursor of 210aa comprising a 22aa signal peptide and a 188aa mature protein. The accession number in GenBank was DQ350432. The mature protein pGH cDNA was inserted into pPIC3.5K and was proved after PCR selecting, digesting and sequencing. The recombined pPIC3.5K-pGH was digested by Sail and transferred into Pichia pastoris strain GS115 by electrophoration. PCR selecting results showed that pGH gene had been integrated into the yeast chromosomes. This study completed the clone cDNA of pGH and construction of eukaryotic cell expression vector of pPIC3.5K-pGH, and provided the theoretical basis for the expression and function research of pGH gene in cell.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2012年第12期121-126,共6页
Journal of Northeast Agricultural University
基金
国家自然科学基金(30771666)
关键词
大鳞副泥鳅
生长激素
基因克隆
真核表达载体
Paramisgurnus dabryanus
growth hormone
gene cloning
eukaryotic expression vector
