中国麇鹿朊蛋白核心片段的原核克隆表达与纯化

从采取的新鲜麋鹿血液中提取麋鹿基因组DNA,通过PCR的方法从麋鹿基因组中扩增麋鹿PrP蛋白核心片段的DNA,并分别在引物的5’和3’端引入EcoR I和HindⅢ两个酶切位点,下游引物将原序列TATTAC突变为TAATAA变成两个终止密码子。通过两个酶切位点将麋鹿PrP蛋白核心片段DNA连接到pET-32a(+),转化DH5α,通过酶切鉴定和PCR鉴定,筛选出阳性克隆送菌液测序,从测序结果分析阳性克隆是否符合要求。测序正确后将核心片段基因连接到pET-32a(+),阳性质粒命名为MPrP-pET-32a(+),阳性质粒转化E.coli BL21(DE3),进行诱导表达、蛋白质纯化鉴定。

Cloning and Expression of PK Resistant Core of PrP from CHINA MILU in E.coil

Genome DNA was extracted from fresh blood of milu-deer and the gene encoding PrP protein was amplified by PCR.The EcoR I and Hind Ⅲ enzyme cutting sites were introduced into the forward primer and reverse primer.The product of PCR was cloned into PET-32a,the recombinant plasmid was transformed into DH5αand identified by enzyme cutting and PCR.The positive clone was selected and subjected to sequencing and analysis.The gene fragment was inserted into PET-32a,named MPrP-pET-32a(+) and transformed into E.coli BL21(DE3).Expression of the PrP protein was induced by IPTG,the protein was purified and confirmed by DAB.