The Feasibility Study Of Tobacco Mosaic Virus U2Strain Coat Protein Used To Build Functional Composite Materials

Biological molecules like proteins represent an ideal template for constructing complex material structures with molecular precision due to their inherent molecular recognition, self-assembly capabilities, genetic controllability and suitability for amplification. Virus is a simple biomolecule, consisting the outer coat protein and internal genome DNA or RNA. It is virtually a nano-structured particle whose capsid is formed by the regular gathering of genetically-coded coat protein subunit in a certain way. At present, the development of nano biological technology and the use of specific inorganic binding peptide have provided new ideas for exploiting new composite materials.In this essay tobacco mosaic virus U2strain, tobacco mild green mosaic virus (TMGMV) coat protein (CP) is used as template. The derivatives’123th Amino acid is changed from Alanine to Cysteine with C-terminal carrying the IrO2binding peptide. The recombination TMGMV-CP is constructed with PGEX4T-1prokaryocyte expression vector and the restructured plasmid is expressed in E.Coli.BL21(DE3). The123th amino acid residues after mutations, Cys is a kind of acid with-SH, which can be acylation reacted with-OH,-COOH,-SH and connected with functional molecules. As the link or bridge, Iridium oxide binding peptide can identify metal oxide specifically. In a word, after gene mutations, tobacco mild green mosaic virus can be used as the functional biological template.At first, study on the bio-inorganic interactions is conducted under the background of prokaryotic expression system. We design the primer and take technology of fixed-point mutations to obtain the recombinant plasmid through PCR amplification. The objective fragment with designed primers is constructed with PGEX4T-1prokaryotic expression vector and transformed into E.coli BL21(DE3). At last, the restructuring expression vector PGEX-4T-1/TMGMV-CP-IrO2can be obtained.Second, the recombinant protein is expressed after induced by IPTG. To get a mass of soluble recombinant protein, we tries some kinds of technology to optimization of recombinant protein expression conditions, analysis of protein expression form, carries on the exploration of the soluble express conditions. The result of experiment indicates that the expression product exists as inclusion body, and the optimized expression condition is0.1mmol/L IPTG and inducing6h at37℃. Meanwhile even we test more than36conditions, such as low temperature inducing, low concentration inducing stile can’t obtain the soluble protein, which indicates that the fusion protein GST/TMGMV-CP-IrO2just exists in form of inclusion body.Third, express the restructured plasmid under the optimal conditions, and harvest the inclusion bodies, renature in refolding buffer, concentrate till2ml, and purify by AKTATM purifier. The chromatogram of purification indicates that an absorption peak appeared after eluting45ml protein buffer. SDS-PAGE and Western Blotting analysis confirm that it’s the exact fusion protein what we want.Fourth, IrO2nanomaterial is combined with the purified protein to test the function. The result of experiment indicates that the GST protein showes little combination with IrO2nanomaterial while the fusion protein showes obvious combination with it. In another test, fusion protein is combined with IrO2nanomaterial and a little TiO2nanomaterial, which means, fusion protein can be combined with IrO2nanomaterial, although the specificity is not so strong.At last, the GST/TMGMV-CP-IrO2fusion protein is cut by a certain percentage of the enzyme of thrombin and we get the restructured TMGMV-CP-IrO2protein which can be used to construct the functional biological template.This topic research is based on rigid dense virus capsid proteins, and a functional biological template has been constructed. In this essay, we complete the designated mutation innovatively, restructure a functional inorganic combined with short peptide design capsid proteins to express, purification and the function identification. The thought, method and result are worth using for reference and have positive significance for developing complex material in energy, biological medicine and nano electronics and other areas.