摘要
细胞外脂肪酸结合蛋白(Extracellular Fatty Acid-Binding Protein,Ex-FABP)是一种鸡胚发育过程中表达于肥大软骨、肌肉纤维和血粒细胞中的脂蛋白,大小约为21 ku。试验通过构建Ex-FABP原核表达载体,以实现Ex-FABP蛋白的大量表达,并对其进行纯化及鉴定;以艾维茵肉鸡胫骨软骨总RNA为模板,RT-PCR扩增Ex-FABP基因,连接pGEM-T克隆载体后,再与载体pET-28a连接并转化,获得含pET-28a-Ex-FABP重组表达质粒的Escherichia coli BL21(DE3)表达菌株;使用IPTG诱导表达蛋白,SDS-PAGE鉴定表达情况,利用镍柱亲和层析法纯化蛋白,对纯化蛋白进行Western Blot鉴定。结果表明,试验成功构建重组表达载体pET-28a-Ex-FABP;表达产物经SDS-PAGE和Western Blot鉴定,结果显示,目的蛋白在24 ku附近出现特异性条带,表明表达蛋白正确。试验成功表达了大量纯度比较高的Ex-FABP蛋白,可为后续的动物试验以及家禽疾病的研究提供理论依据。
Extracellular Fatty Acid-binding Protein(Ex-FABP)is a 21 ku lipocalin,expressed during chicken embryo development in hypertrophic cartilage,in muscle fibres and in blood granulocyte.In this experiment,protein expression,purification and identification of Ex-FABP were achieved by constructing Ex-FABP prokaryotic expression vector.Using the total RNAs of the tibial cartilage of Avian broilers as templates,Ex-FABP gene was amplified by RT-PCR and ligated into the cloning vector pGEM-T,then ligated and transformed with the prokaryotic expression vector pET-28a.Finally,the Escherichia coli BL21(DE3)expression strain containing pET-28a-Ex-FABP recombinant expression plasmid was obtained.The expression of Ex-FABP protein was induced by IPTG(Isopropylβ-D-Thiogalactoside)and then identified by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis).The protein was purified by nickel column affinity chromatography and then identified by Western Blot.The results showed that the recombinant expression vector pET-28a-Ex-FABP was successfully constructed.The results of SDS-PAGE and Western Blot showed that the purified target protein showed a single distinct band around 24 ku,indicating the correctness of the expressed protein.The experiment successfully expressed high-purity Ex-FABP protein,which provided a basis for subsequent animal experiments and further research in the field of poultry.
作者
田志雄
李娜娜
卢晓晓
郭敏
张宁
李欣
张鼎
赵宇军
闫芳
高荣琨
宁官保
田文霞
TIAN Zhixiong;LI Nana;LU Xiaoxiao;GUO Min;ZHANG Ning;LI Xin;ZHANG Ding;ZHAO Yujun;YAN Fang;GAO Rongkun;NING Guanbao;TIAN Wenxia(College of Animal Science and Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Wenxian Agricultureand Forestry Bureau,Wenxian 454850,China;Jincheng Science and Technology Bureau,Jincheng 048000,China)
出处
《山西农业科学》
2020年第6期855-859,888,共6页
Journal of Shanxi Agricultural Sciences
基金
国家重点研发计划项目(2016YFD0500800)
晋中市重点科技创新平台(P171002-3)
山西省回国留学人员科研资助项目(2017-073)
国家自然基金项目(31072179)。
关键词
细胞外脂肪酸结合蛋白
基因克隆
原核表达
蛋白纯化
Extracellular Fatty Acid-binding Protein
gene cloning
prokaryotic expression
protein purification
