摘要
为提高胰高血糖素样肽-2(GLP-2)生产效率以适应畜牧生产应用的需求,本试验利用基因工程技术表达出定点诱变的p[Gly2]GLP-2。用甘氨酸取代GLP-2氨基末端第2位的丙氨酸后,再根据大肠杆菌的偏好性对p[Gly2]GLP-2序列进行优化并在目标肽序列N-端添加肠激酶识别位点,合成基因序列,通过KpnⅠ和XhoⅠ双酶切位点连接到pET-40b(+)构建原核重组表达载体p[Gly2]GLP-2-pET-40b(+),重组质粒转化大肠杆菌BL21(DE3)感受态细胞,诱导表达重组蛋白。优化后最适表达条件为:菌液D 600 nm值为0.6时,用0.2 mmol/L IPTG在37℃诱导培养5 h;最终得到p[Gly2]GLP-2重组蛋白表达量较高的基因工程菌株。通过镍离子亲和层析柱纯化及分梯度洗脱获得纯度较高的p[Gly2]GLP-2融合蛋白,本结果为后续p[Gly2]GLP-2功能性的研究及其在畜牧生产的应用奠定了基础。
In order to improve the production efficiency of GLP-2 to meet the application needs of animal husbandry,the porcine derived[Gly2]GLP-2(p[Gly2]GLP-2)was induced by site directed mutagenesis using genetic engineering technology.After replacing alanine at the second amino end of GLP-2 with glycine residue,the codon sequence of p[Gly2]GLP-2 was optimized according to the codon preference of Escherichia coli,and enterokinase recognition sites were added to the N end of the target peptide sequence to synthesize the gene sequence.Prokaryotic recombinant expression vector p[Gly2]GLP-2-pET-40b(+)was constructed by connecting KpnⅠand XhoⅠdigestion sites to pET-40b(+).The recombinant plasmid was transformed in E.coli BL21(DE3)competent cell to induced the expression of recombinant protein.The optimum expression parameters were that 0.2 mmol/L IPTG was used to induce the expression of p[Gly2]GLP-2-pET-40b(+)recombinant plasmid for 5 h when the bacterial solution D 600 nm value was 0.6,and the recombinant strain with high expression of p[Gly2]GLP-2 was finally obtained.High purity p[Gly2]GLP-2 fusion protein was obtained by purification with nickel ion affinity chromatography column and gradient elution,which laid a foundation for further functional research and application in animal husbandry.
作者
韩斐
赵睿骁
王刚
江明锋
HAN Fei;ZHAO Ruixiao;WANG Gang;JIANG Mingfeng(Institute of Qinghai-Tibet Plateau,Southwest Minzu University,Chengdu 610041,China;National Engineering Research Center for Biomaterials,Sichuan University,Chengdu 610064,China)
出处
《中国畜牧兽医》
CAS
北大核心
2019年第11期3137-3143,共7页
China Animal Husbandry & Veterinary Medicine
基金
西南民族大学研究生创新型项目(CX2018SZ47)
四川省国际科技创新合作/港澳台科技创新合作项目“牦牛复胃关键发育阶段分子机制研究及胃溶菌酶开发”(2019YFH0035)
