T7噬菌体表达载体的组氨酸改造及应用

Constrution and Application of T7 Phage Vector with a Histidine Tag

利用基因重组技术,对已有载体T7 Select10-3b进行改造,得到被组氨酸标签标记(His-Tag)的新型表达载体,并利用该载体构建cDNA文库,为后期开放阅读框的筛选提供更为简便有效的方法。以T7 Select10-3b载体为出发材料,将复性得到组氨酸标签表达序列插入其多克隆位点处。再利用包装蛋白包装重组DNA,得到有侵染活性的重组T7噬菌体。用该载体构建乳腺癌cDNA文库,过镍柱,筛选开放阅读框(open reading frame,ORF),计算ORF重组率。测序结果显示该标签已经成功插入,化学发光免疫检测显示该标签在T7中表达。重组T7噬菌体文库的ORF筛选率由筛选前的5.6%提高到82%。T7噬菌体中插入的His标签对提高cDNA文库中ORF的重组率具有重要意义。

A new type of histidine tag expression vector was obtained on the basis of the vector T7 Select10-3b through gone recombination technology. The recombined vector was used to construct cDNA library as an effective method of screening open reading frame （ ORF ） . A His-Tag was inserted into multiple clone site of vector T7. The sequence of His-Tag was obtained by renaturation. In order to get infectious T7 phage, T7 packaging extracts was utilized to package recombined DNA. CDNA library was Constructed using modified vector, and ORF was purified by Ni-nitrilotriacetate affjnity chromatography. DNA sequencing revealed that His-Tag was inserted into the expression vector accurately. Chemiluminescence immunoassay displayed that inserted His-Tag sequence could express accurate protein. Screening rate of ORF rised from 5.6% to 82%. It proved that vector T7 inserted His-Tag had important significance for ORF screening.