摘要
目的鉴定、构建并高效表达从土壤中筛选到的一株耐热型、高活性的嘧啶核苷磷酸化酶(PyNPase)。方法以MY02总DNA为模板,通过简并PCR、特异PCR引物、筛选和DNA测序,在5’引物和3’引物中分别引入NcoI和XbaI酶切位点,将pynp基因克隆至分泌表达载体pBAD/gIII A中,转化至E.coliTOP 10F’,以L-阿拉伯糖诱导表达,并对诱导剂浓度、诱导时间等进行优化。结果成功构建了重组表达载体pBAD/gIII A-pynp,通过磷酸化和转化反应,鉴定此酶为PyNPase。结论优化PyNPase的纯化条件与复性条件,可获得大量的PyNPase。
OBJECTIVE To identify and construct and overexpress a heat resistant and high active Pyrimidine Nucleotide Phosphorylase（PyNPase） isolated form soil. METHODS PyNPase gene cloned with the degenerate PCR method, based on which a pair of special primers were designed according to PyNPase nucleotide sequence. And Nco Ⅰ, Xba Ⅰ recognising sites were introduced into 5＇ - and 3＇ - primers. An expression vector containing a （ His - tag） 6 was constructed by inserting pynp gene into pBAD/gⅢ A. Then transferred into E. coli TOP10F＇. L - arabinose induced the gene expression and L - arabinose concentration and induction time were optimized. RESULTS Recombinant expression plasmid pBAD/gⅢ A -pynp was constructed successfully. According to phosphorolytic and transferred reaction, the enzyme was PyNPase. CONCLUSION A mass of PyNPase could be achieved by optimizing purifying condition and refolding condition.
出处
《华西药学杂志》
CAS
CSCD
北大核心
2007年第6期600-604,共5页
West China Journal of Pharmaceutical Sciences
