编码鸡CD8α链无信号肽基因片段的克隆与表达  

Cloning and expression of the gene fragment encoding chicken CD8α chain without signal peptide

作　　者：谢国化[1] 鲍鸣[1] 仲大莲[2] 余为一[1] 李槿年[1] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国科学技术大学生命科学学院,合肥230026

出　　处：《安徽农业大学学报》2006年第1期56-60,共5页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(30270974)资助

摘　　要：应用RT-PCR技术,通过自行设计的1对引物以ConA诱导的鸡胸腺淋巴细胞RNA为模板,扩增出编码鸡CD8α链的无信号肽基因片段,大小为651 bp。该片段经酶切初步鉴定后,被插入pMD18-T载体进行测序,发现与已发表的鸡CD8α链基因序列的同源性达98%。进一步将该片段插入原核表达质粒,构建pGEX-4T-1-CD8α,并转化大肠杆菌BL21。经IPTG诱导培养并提取表达蛋白质。在SDS-PAGE电泳图谱中可见大小约为50 000的蛋白带,表明所克隆的编码鸡CD8α链无信号肽基因片段在原核细胞中得到表达。The total RNA was prepared from ConA stimulated thymocytes of a 5-week-old chicken according to the instructions of the trizol reagent. The CD8 alpha chain eDNA without its singal peptide sequence was amplified by reverse transcription polymerase chain reaction （RT-PCR） using a pair of gene specific primers. The obtained 651 bp fragment was identified by restriction enzyme digestion, the fragment was cloned to the pMD18-T vector and sequenced （the sequence has been deposited in GenBank with accession number of DQ202314）. The sequence showed 98% homology with that of published chicken CD8 alpha chain. Subsequently, the verified fragment was digested with EcoR I and Sal I and ligated to the pGEX-4T-1 vector using T4 DNA ligase. The recombinant prokaryotic expression plasmid, namely pGEX-4T-1-CD8α, was transformed into Escherichia coli strain BL21 （ DE3 ） and expressed as a GST fusion protein. The products were analyzed by SDS-polyacrylamide gel electrophoresis and a protein about 50 000 was observed. These suggest that the cloned gene encoding chicken CD8 alpha chain without signal peptide was expressed in prokaryotic system.

关 键 词：CD8Α 鸡 免隆 原核表达 

分 类 号：S831.2[农业科学—畜牧学] S852.4[农业科学—畜牧兽医]