摘要
根据已发表的β-防御素5基因序列设计引物,采用RT-PCR技术扩增奶牛白细胞β-防御素5基因片段,构建原核表达载体并进行诱导表达.结果表明:将PCR产物插入pGEM-T easy载体后,经琼脂糖凝胶电泳、PCR、酶切及DNA测序证明构建的重组克隆载体完全正确,以该重组质粒为模板扩增目的基因的成熟肽片段,产物用EcoR I+NotⅠ双酶切并与原核表达载体PET28a连接,获得了预期的重组表达质粒.将重组表达质粒转化到BL21(DE3)宿主菌中,用异丙基--βD-硫代半乳糖苷诱导表达,经尿素-SDS-PAGE检测表明表达产物为6.4 kda的融合蛋白.
A pair of primers were designed and synthesized based on the published β-defensin 5 gene sequence of dairy cattle,and then β-defensin 5 gene was amplified using RT-PCR technique. Then, the PCR product was inserted into pGEM-T easy vector to construct the recombinant plasmid that was correct identified by agarose gel electrophoresis, PCR, enzymatic digestion and DNA sequencing. The mature peptide fragment of the target gene was amplified according to the recombinant plasmid template. After digested with EcoR I and Not I, the amplified product was ligated with PET28a expressive vector to form recombinant expressive plasmid. Sequence analysis showed that the inserted target gene was completely correct. Then, the recombinant plasmid was transformed into BL21 （DE3） and induced with IPTG to express the fusion proteins with relative molecular mass of 6.4 kda.
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2009年第1期7-10,共4页
Journal of Gansu Agricultural University
基金
国际科技合作计划项目(2005DFA30720)
关键词
奶牛
β-防御素5
基因克隆
原核表达
dairy cattle
β-defensin 5
gene clone
prokaryotie expression
