The Expression, Purification And Functional Preliminary Exploration Of Silkworm Membrane Magnesium Transpotrer Portein

Membrane protein undertake irreplaceable biological function in the activities of life,and ithas become the main target of molecular drug action,but separation and purification of naturalmembrane protein are very difficult,because it has some novel features different fromintracellular protein. Now fusion tags technology has been widely used to study the crystalstructure and biological function of membrane proteins. In2007, membrane protein MMgT genefamily was originally identified in Xenopus oocyte, and it had been demonstrated that MMgT isone of protein family of transporter magnesium. But it is unclear that whether MMgT geneexists in the silkworm.In this study,a protein-coding gene was obtained from a cDNA library of Bombyx morisilkworm chrysalis. Bioinformatics analysis showed it is associated with transporter Mg²⁺andnamed BmMMgT. Further analysis showed that BmMMgT is a single transmembrane protein.In order to research the function of BmMMgT in silkworm, BmMMgT was first cloned into theprokaryotic expression vector pET-32a （+） by recombinant DNA technology, the fusion proteinwas highly expressed by IPTG induction. Fusion protein was purified by nickel ion affinitychromatography and anion-exchange column, mass spectrometry analysis indicated that theproduct of fusion protein was correct. Next, Purified fusion protein was used to immunize NewZealand rabbit as an antigen for polyclonal antibody. Antibody titer measured by the indirectELISA method is more than1:25600. Unlabeled membrane protein BmMMgT was obtained bythe thrombin cleavage sites in the vector. To parse the high structure of the membrane proteinBmMMgT, in this study,we has not only screened crystallization conditions of the membraneprotein BmMMgT, but aslo has screened crystallization conditions of BmMMgT and Mn-SODthat had parsed its high structure fusion expression. Furthermore, the interaction betweenmembrane proteins and total pupa protein power was investigated by His-tag pull downtechnology,mass spectrometry analysis showed that it could be Silkworm neuropeptideprecursor.Finally, in the study preliminary explore on function of BmMMgT gene in the silkwormcell was executed. We took advantage of Bac-to-Bac system to construct recombinant viruse vBmMMgT and vBmMMgT was transfected in the BmN cells, Western blotting analysisshowed that the fusion protein BmMMgT was successfully expressed in the BmN cell. Theanalysis of real-time PCR showed that Mg²⁺has a significant effect on the mRNA transcriptionof BmMMgT gene in BmN cells, it showed the function of the gene BmMMgT was related withMg²⁺.These results provided a reliable basis for elucidating the function of BmMMgT gene inthe future.