鸡肿瘤坏死因子α基因片段的分子克隆及序列测定

Cloning with Polymerase Chain Reaction and Determining Nucleotide Sequence for Chicken Tumor Necrosis Factor α

应用LPS诱生的鸡培养白细胞提取总RNA,参考几种动物的TNFα保守序列设计一对引物,以提取的培养白细胞总RNA为模板,通过RT-PCR扩增目的基因片段,将PCR产物与PUC19连接,转化到大肠杆菌DH52进行分子克隆,对提取的重组质粒经PCR鉴定和酶切鉴定,表明重组的基因片段是目的基因片段,其长度为576bp。

White cells were isolated from the blood of chickens,and stimulated to generate tumor necrosis factor α (TNFa)-specific mRNA by lipopolysaccharide(LPS).Total RNA was prepared to clone the gene for the chick TNFα utilizing polymerase chain reaction (PCR).After adding the TNFa translation stop primer,a second conserved consensus primer, and reverse transcriptase to the propeptide region,we amplified the cDNA between the primers. The PCR products were separated by electrophoresis in a native agarose gel, and cloned.The results showed that the expected 570bp band between the two primers could be detected in both PCR reactions.There are 576bp in this band by determining the nu-cleotide sequence.