家蚕膜蛋白基因BmMFS的组织表达特征和重组蛋白的表达

Tissue Expression Profile and Recombinant Expression of Membrane Protein Gene BmMFS in the Silkworm,Bombyx mori

膜蛋白在生物体内承担细胞内外环境物质和能量的交换及信息传递,也是药物设计的靶标。在家蚕蛋白质数据库中筛选到一个含有6个跨膜区的膜蛋白,包含一个主要协助转运蛋白超家族(MFS)的保守结构域,将该蛋白质的编码基因命名为BmMFS。荧光定量PCR检测家蚕5龄幼虫不同组织中的BmMFS转录水平存在明显差异,在中肠中的转录水平最高,在气管中的转录水平最低。以家蚕蛹cDNA为模板扩增BmMFS片段,构建重组质粒pET-32a-Ph20-BmMFS,在Ph20和BmMFS序列之间引入凝血酶(thrombin)酶切位点;转化E.coli Rosetta,以1 mmol/L IPTG诱导融合基因表达,菌液调节pH初步纯化后进一步通过镍柱亲和层析纯化目的蛋白质,经Western blotting和质谱鉴定获得的融合蛋白为Ph20-BmMFS;进一步利用家蚕杆状病毒表达系统构建重组病毒(vPh20-BmMFS)并感染BmN细胞,Western blotting检测显示融合BmMFS蛋白成功表达。将家蚕膜蛋白基因与家蚕杆状病毒多角体蛋白基因片段Ph20融合表达,提高了膜蛋白在原核和真核表达系统中的表达量,利用该多角体蛋白能在较低pH条件下溶解的特性简化了融合蛋白纯化过程。

Membrane proteins play important roles in substance and energy exchange and signal transduction between the cell and its environment, and can be used as target for drug design. A membrane protein containing six transmem- brane domains was obtained from searching the protein database of silkworm. This protein has a conservative major facil- itator superfamily （MFS） domain and its coding gene is designated as BmMFS. Quantitative PCR analysis showed re- markable difference of BmMFS gene transcription between various tissues in the fifth instar larvae, having the highest lev- el in midgut and the lowest level in trachea. BmMFS fragment was amplified from silkworm pupal cDNA and was used to construct recombinant plasmid pET32a-Ph20-BmMFS, which contained a thrombin cleavage site between Ph20 andBmMFS. The recombinant plasmid was transformed into E. coil Rosetta and induced with 1 mmol/L IPTG for fusion protein expression. After primary purification by adjusting pH value of the bacterial solution, target protein was fur- ther purified by Ni-affinity chromatography, Western blotting and mass spectrometry verified that the obtained fusion protein was Ph20-BmMFS. Furthermore, recombinant baculovirus virus （vPh20-BmMFS） was constructed using silkworm baculovirus expression system and used toinfect silkworm BmN cells. Western blotting analysis showed that the fused BmMFS protein was successfully expressed in BmN cells. Fusion expression of silkworm membrane protein gene and Bombyx moil nucleopolyhedrovirus polyhedrin gene fragment Ph20 imporve the expression level of membrane protein in prokaryotic expression systeme and eukaryotic expression system. Meanwhile, protein purification procedure was simplified based on the dissolution characteristics of polyhedron under the condition of low pH.