梅花鹿S100A4基因的克隆及原核表达.docVIP

梅花鹿S100A4基因的克隆及原核表达 张英，李莉，郝林琳，刘松财，于浩 作者简介：张英（1979－），女，实验师，主要从事动物生物化学与分子生物学研究，E-mail:zhangying636@126.com 通讯作者：于浩（1977－），男，副教授，E-mail:yuhao1977@ 吉林大学 畜牧兽医学院　吉林长春　130062 （第一作者联系电话 单位名称：吉林大学畜牧兽医学院 详细通讯地址：吉林省长春市绿园区西安大路5333号畜牧兽医学院 电子邮箱：zhangying636@126.com） (通讯作者联系电话:0431 单位名称：吉林大学畜牧兽医学院 详细通讯地址：吉林省长春市绿园区西安大路5333号畜牧兽医学院 电子邮箱：yuhao1977@） 摘要：目的 为确定梅花鹿S100A4蛋白在鹿茸发生中的作用机制，大量制备并获得纯化的S100A4蛋白。 方法 本研究从梅花鹿锯茸血细胞中提取总RNA并进行反转录，用设计的特异性扩增梅花鹿的S100A4基因，将PCR产物克隆到pMD18-T载体并测序。而后将扩增的梅花鹿S100A4基因片段和原核表达质粒pET-28a(+)分别用EcoRI和HindⅢ进行双酶切并连接，再将重组质粒转入BL21（DE3）表达菌株进行诱导表达。 结果 S100A4基因序列同源性分析结果显示，梅花鹿S100A4基因与牛S100A4基因同源性最高（98.4%）。酶切鉴定结果显示，本研究成功构建了表达梅花鹿S100A4基因的重组质粒pET28a-S100A4，SDS电泳结果表明S100A4蛋白获得成功表达。 结论 在大肠杆菌中成功表达了梅花鹿S100A4基因，为下一步研究S100A4蛋白在鹿茸生长过程中的作用机制奠定了基础。 关键词：梅花鹿；S100A4基因；克隆；表达 Cloning and Prokaryotic Expression of S100A4 Protein of Sika Deer Zhang Ying, Li Li, Hao Lin-lin, Liu Song-cai, Yu Hao College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062 Abstract: Objective In order to understand the regulatory mechanism of S100 calcium binding protein A4(S100A4) in antler generation of sika deer. Methods we cloned S100A4 gene from total RNA of Sika deer’s antler blood using reverse transcription polymerase chain reaction (RT-PCR). And then pET-28a(+) plasmids and S100A4 fragment were digested with restriction enzymes EcoRI and HindⅢ respectively, and then linked together to construct the recombinant expressing plasmids pET28a-S100A4. And the recombinant plasmids were transformed into competent BL21(DE3) cells, and expression of protein was induced by the IPTG. Results The sequence analysis showed that the S100A4 nucleotide identity was 85.6%～98.4% among Sika deer and other five animals (share highest homology with Cattle). SDSresults showed that the S100A4 protein was successfully expressed in E. Coli.. Conclusions This experiment successfully produced Sika’s S100A4 molecule by prokaryotic expression system, which build the foundation for the