Method for preparing two-dimensional nanometer structure from tobacco mosaic virus capsid protein mutant
A technology of tobacco mosaic virus and capsid protein, applied in botanical equipment and methods, biochemical equipment and methods, viral peptides, etc., can solve the problem of complex synthesis steps of two-dimensional nanostructures and poor biocompatibility of two-dimensional nanostructures , the reaction conditions are not mild enough, etc., to achieve the effect of improved in vitro self-assembly ability, good biocompatibility, and simple steps
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[0042] Specifically, the above-mentioned method for preparing a two-dimensional nanostructure based on tubular virus particles mainly includes the following three steps:
[0043] The first is step S1, introducing cysteine into the capsid protein of tobacco mosaic virus: mutate the 103rd amino acid sequence of the capsid protein monomer of tobacco mosaic virus into cysteine, construct a vector, and Expression is realized in vivo, and the transformed virus capsid protein monomer is obtained by anion chromatography column purification.
[0044] Specifically, in this step, the 103rd position of the amino acid sequence of the capsid protein monomer of tobacco mosaic virus is mutated into cysteine, which is used as a TMV capsid protein mutant (modified monomer), which is known in the above In the case of the mutant amino acid sequence and the cDNA complete gene sequence of the wild-type TMV capsid protein monomer, those of ordinary skill in the art can easily obtain the coding gen...
Embodiment 1
[0054] Construction of expression plasmid of TMV capsid protein mutant and its expression and purification in Escherichia coli
[0055] Step 1: According to the cDNA synthesis of the wild-type TMV capsid protein monomer, the nucleotide sequence is the mutant coding gene shown in SEQIDNo.3, which is transferred into the plasmid vector PET32a to construct the plasmid vector PET32a-T103C-TMVcp;
[0056] Step 2: After the correctness of the mutant gene sequence was confirmed by DNA sequencing, the plasmid vector PET32a-T103C-TMVcp was transferred into E.coliBL21 competent cells by the calcium chloride method, and single clones were picked from the plate after being plated for 12 hours. Put into 5mL LB test tube culture medium, add ampicillin (final concentration 100μg / mL), incubate overnight at 37°C and 180r / min; Penicillin (final concentration 100 μg / mL), cultured at 37°C with constant temperature and shaking at 180 r / min for about 2.5 hours (OD600 between 0.4 and 0.6), added IPT...
Embodiment 2
[0060] Generation of tubular virus particle templates
[0061] The high-purity T103C-TMVcp obtained in Example 1 was dialyzed in the assembly buffer assembly buffer (pH7, 400mM phosphate buffer), incubated at room temperature for 5 days, and self-assembled; the separation and purification of self-assembly was carried out by sucrose density gradient centrifugation tubular virus particle template.
[0062] Wherein, the preparation steps of sucrose density gradient:
[0063] a) Prepare PB buffer solution (pH7.0, 150mM phosphate) and 50wt% sucrose solution (prepared with PB buffer solution) earlier, and then make the mass / mass ratio of 10%, 15%, 20% with the two as mother liquor %, 25%, 30%, 35%, 40%, 45%, 50% sucrose solution;
[0064] b) After the sample volume is reserved at the top of the transparent ultracentrifuge tube, the remaining length is divided into 9 parts of equal volume, and marked with a marker pen;
[0065] c) Fix the ultracentrifuge tube on a suitable centrif...
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