人整合素β_3亚基毕赤酵母表达载体的构建及鉴定

Construction and Identification of Human Integrin β_3 in Pichia Expression System

目前研究表明整合素β3亚基是多种病毒的细胞表面受体,然而整合素β3亚基与不同病毒受体结合蛋白VAP相互作用的分子机制还不完全清楚。为此,设计引物,采用RT-PCR方法扩增人β3亚基包膜外区约2.1kb片段,克隆入毕赤酵母表达载体pPIC3.5K中。双酶切鉴定挑取阳性克隆pPIC3.5K-β3,测序证实目的基因序列正确。将pPIC3.5K-β3转化毕赤酵母工程菌GS115,在浓度为3.0mg/mL的G418选择培养基上,筛选出含有多拷贝目的基因的阳性菌株,PCR可扩增出约2.1kb的目的条带。阳性菌株经1%甲醇诱导表达,Western-blot可在酵母菌体中检测到约80kDa的目的蛋白条带。上述结果表明,成功构建了人整合素β3亚基毕赤酵母表达载体,筛选获得多拷贝阳性菌株并成功表达,为进一步研究人整合素β3在病毒感染中的作用及其单克隆抗体的制备奠定基础。

The β3 integrin is the receptor of some viruses, but the whole interaction between β3 integrin and different VAPs were not known completely. The primers were designed and the β3 integrin fragment （ a 2.1 kb segment of extracellular region） was amplified by RT-PCR, and then the fragment was cloned into Pichia vector pPIC3.5K. The recombinant pPIC3.5K-β3 was identified correctly by BamH I and EcoR I digested respectively and transfected into Pichia engineering strain GSll5. The positive strains which contain multicopy β3 genes were screened out from 3.0 mg/mL G418 selective medium by PCR. The positive strains were induced by methanol and an 80 kDa band could be detected by Western-blot. The results above suggested that the Pichia expression vectors, pPIC3.5K-β3, has been successfully constructed and the expressed protein could be detected distinctly and peculiarly. It builds a basis for the further study of action during viral infection of β3 integrin and of the monoclonal antibody preparation.