水牛垂体催乳素cDNA的克隆与序列分析

Cloning and sequence analysis of the pituitary prolactin cDNA of buffalo

根据GenBank中报道的几种哺乳动物催乳素(PRL)基因序列,设计1对特异性引物。从广西本地水牛脑垂体中提取总RNA,利用RT-PCR技术扩增水牛PRL cDNA全序列,并连接到pMD18-T载体,经PCR和酶切鉴定的阳性克隆进行测序,测定结果表明:该cDNA开放阅读框(ORF),长度为690bp,编码氨基酸为229个,其中前19个氨基酸为信号肽。用NCBI上的Blast功能将测序结果同GenBank已发表的序列进行比对,具有很高的同源性,其中与牛属动物同源性最高,达98.4%,证明所克隆的序列为水牛PRL cDNA全序列,已提交到GenBank(登陆号:EF054878)。本研究成功克隆水牛PRL cDNA全序列,为下一步构建原核表达载体、表达体外蛋白,以及进一步研究其生物学功能奠定了基础。

A pair of primers with specificity to amplify PRL gene was designed and synthesized using conservative domain of mammalian PRL cDNA reported in GenBank. Total RNA was extracted from pituitary gland of Guangxi local buffaloes and the sequence of PRL cDNA was amplified and cloned using pMD 18-T vector. Through PCR and enzyme di- gestion, the sequence analysis of positive cloning indicated that the cDNA contained an ORF of 690 bp which codes 229 amino acids, of which the first 19 amino acids were signal peptide. By comparing with published prolactin gene sequence in GenBank with Blast function, the sequence showed highest identity with PRL gene, and the sequence homology of buffalo to cattle was found 98.4%. This proved that the cloned sequence was complete buffalo PRL gene sequence, and that has been submitted to GenBank （GenBank accession No. EF054878）.