一种低温表达型T载体及其应用

An expression T-vector and its application at low temperatures

摘要:

在现代生物学和生物技术研究中，通过基因重组表达获得目标蛋白已成为常规技术。因其培养简单、操作方便、遗传背景清楚、克隆表达技术成熟，大肠杆菌表达系统通常是人们表达重组蛋白的首选。但是在常规温度下进行基因的重组表达，动、植物和常温微生物的基因产物多数在数小时内变性沉淀；还有一些重组蛋白对宿主具有细胞毒性，难以得到重组表达。因此，我们构建了1种新型T载体——pEXC-T；它结合TA克隆技术和低温诱导表达功能，具有表达水平高、操作方便、目标蛋白得到分子伴侣保护和低温保存等特点。采用构建和优化的pEXC载体，P1抗原蛋白、溶血素PLO两种不稳定性蛋白在pEXC中都实现了高效的可溶性表达。低温表达系统pEXC的建立和发展为蛋白质的结构与功能的研究，以及抗原和药用蛋白的制备提供了便利的途径。

Abstract:

In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.