编码结构域Tt APuX25基因片段的分离与克隆

目的]将Tt apux25基因片段克隆到表达载体pET21a(t)中。方法]以热产硫化氢高温厌氧杆菌菌液为模板,根据编码结构域Tt APuX25的基因片段Tt apux25设计1对特异引物进行PCR扩增。将Tt apux25克隆到表达载体pET21a(+)中,筛选阳性克隆并对其进行测序鉴定。结果]通过PCR扩增获得一个250～500bp的扩增片段。对获得的阳性克隆进行筛选和测序鉴定,发现所得阳性克隆为表达载体pET21a(+)中插入有序列正确的Tt apux25片段。阳性克隆中的重组质粒被命名为pEX25,并将其转化进表达宿主菌大肠杆菌Tuner感受态细胞中。结论]该方法直接将Tt apux25基因片段克隆到表达载体pET21a(+)中,操作简便并取得了较好的效果。

Isolation and Cloning of the Gene Fragment Encoding Tt APuX25 Domain

Objective] The research aimed to clone gene fragment Tt apux25 into expression vector pET21a(t).Method] With the bacterial liquid of Thermoanaerobacter thermohydrosulfuricus as templates,a pair of specific primers were designed according to the gene fragment Tt apux25 that encoded Tt apux25 domain for PCR amplification.Tt apux25 was cloned into the expression vector pET21a(+),then the positive clone was screened out and it was sequenced and identified.Result] One fragment of 250～500 bp was obtained from PCR amplification.Through screening and sequencing the obtained positive clone,it was found that expression vector pET21a(+)contained the inserted fragment at corret site,and moreover,the sequence of the inserted fragment was the same as that of Tt apux25 adopted in Gene Bank.The recombinant plasmid in the positive clone was named as pEX25 and it was transformed into Tuner competent cell in the expression host bacteria E.coli.Conclusion] Gene fragment Tt apux25 was directly cloned into the expression vector pET21a(+) by this method.So this method was simple to operate and obtained better effects.