RT-PCR法扩增的人溶菌酶cDNA的克隆及其核苷酸顺序分析

The Cloning, Sequencing and Analysis of Chinese Human Lysozyme Gene cDNA Amplified with RT-PCR from Human Placental Total RNA

本文以人胎盘全RNA为底物进行逆转录-聚合酶链反应(RT-PCR),制备出了人溶菌酶的cDNA片段。在限制性内切酶Sma Ⅰ存在的连接体系内,将此cDNA克隆入载体pUC12的Sma Ⅰ位点。用重组质粒双链DNA的末端终止法测定了其全部的核苷酸顺序,证明其全长为444bp,编码了18个氨基酸的信号肽和130个氨基酸的成熟蛋白组成的溶菌酶的前体蛋白,并证明已成功地在此cDNA的3′末端导入了两个终止密码子及一个限制性内切酶Sal Ⅰ的识别位点。由中国人溶菌酶cDNA推导出的氨基酸顺序与有关报道不同,有5个氨基酸的改变。表达蛋白的研究工作正在进行中。

The cDNA encoding Chinese human lysozyme was prepared by RT-PCR method from the human placental total RNA. The cDNA fragment was ligated into Sma Ⅰ site of pUC12 in the presence of restriction enzyme Sma Ⅰ. The nucleotide sequence of the 444bp cDNA fragment coding signal peptide and mature protein was determined by the dideoxynucleotide chain-terminator method, and two stop codons and a Sal Ⅰ recognation site was successfully introduced into the 3' end of the cDNA. The deduced amino acid sequence for the protein is changed in five sites, and it would be an allele of the lysozyme gene.