55 results about "MCherry" patented technology

The invention discloses a DNA fragment and an application thereof in construction of a recombinant influenza virus expressing a red fluorescent protein gene. An mCherry fluorescent gene is inserted behind an NS1 gene of an H9N2 influenza virus strain, wherein the complete NS gene is formed by sequentially linking an NS1 gene, the mCHerry fluorescent gene and an NEP gene; the gene and the NEP geneare connected by a PTV-1 2A sequence which can be recognized and cut by protease, so a fluorescent protein-labeled NS fusion gene is constructed and has the nucleotide shown in SEQ ID NO:1. The constructed recombinant influenza virus can produce live virus expressing red fluorescent proteins, is relatively stable in the process of virus passage, does not affect the function of the virus itself, has the growth curve and the titer similar to those of a wild virus strain, can be used for subsequent research, and is of great significance on development, screening and production of anti-influenza virus drugs.

The invention discloses a hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes and a construction method thereof. The method comprises the steps that firstly, a hepatoma cell line for overexpressed Chd1l and green fluorescent proteins is constructed; secondly, a carrier for conditionally-induced expressed Cas9 is transferred into the hepatoma cell line; finally, a pLVX-mCherry-hU6-Chd1l-SgRNA carrier with red fluorescence and targeted Chd1l cutting activity is constructed and transferred to the cell line, and the hepatoma cell line for conditionally-induced knockoutoverexpressed Chd1l is obtained at last. The cell line has the advantages of being the hepatoma cell line for overexpressed Chd1l before the induction of doxycycline (Dox) and being the hepatoma cellline for expressed knockout Chd1l after induction. The cell line has good contrast, the simultaneous culture of multiple cell lines can be reduced, the scientific research work is simplified, and thecell line is an ideal tool for gene function research.

The invention relates to a construction method for circuit amplification of a cadmium ion whole-cell biosensor. According to the research, the cadmium whole-cell biosensor p2T7RNAPmut-68 is constructed in a mode of adding a T7RNAPmut (amino acid at the 40th site mutates into a termination codon) amplification module for the first time. The sensitivity and specificity of the biosensor are improved by amplifying a circuit through a T7RNAPmut (the 40th amino acid mutates into a termination codon) amplification module, so that the defects of a traditional cadmium ion biosensor in detection are overcome. Plasmid pCDFDuet-2 and pGN68 are used as carriers, and the cadmium ion whole-cell biosensor is obtained by utilizing a cadmium specific binding protein CadR, a reporter element mCherry, a cadO operon and a T7RNAPmut amplification module. The invention provides the construction method of a whole-cell biosensor sensitive to cadmium ions. The whole-cell biosensor sensitive to cadmium ions has wide adaptability.

The invention discloses an optically-controlled expression vector for insect cells and application. An applicant successfully constructs a positive plasmid pMIB-C120-mCherry-GFP-EL222 and a control plasmid pMIB-C120-mCherry-GFP on an insect cell expression vector pMIB / V5-HisA based on a VP-EL222 optically-controlled transcription activating system; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is transfected with Sf9 cells, a green fluorescent protein expresses while a red fluorescent protein does not express; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is irradiated and stimulated by blue light, the red fluorescent protein expresses; after the control plasmid pMIB-C120-mCherry-GF is transfected with the Sf9 cells, the green fluorescent protein expresses while the red fluorescent protein does not express; after the control plasmid pMIB-C120-mCherry-GF is irradiated and stimulated by the blue light, the red fluorescent protein still does not express due to no EL222 transcription factor. An optically-controlled inducible expression system is successfully applied to the insect cells for the first time, and the application range of the technology is widened. According to a verification method constructed by the invention, the plasmids also provide a flexible and easy-to-use method for using an optically-controlled induced expression technology.

The invention discloses a non-resistant plantarum lactobacillus anchor expression vector pLQ2a, the base sequence of which is shown in SEQ ID NO.1; it is a biosafety exogenous protein expression system, with asd deletion strain E. coli 6212 / Δasd and alr missing L. plantarum NC8 / Δalr is the host bacterium, which will be derived from Lactococcus lactis ( Lactococcus lactis subsp.cremoris strain JM4) The P23 promoter on the genome acts as the promoter of alr gene expression ， Using pYA4545, pYA4545‑Mcherry, pLP‑1261 Inv, pSIP409PgsA', pSIP409PgsA'‑IL‑10, pSIP409PgsA'‑EGFP as base vectors to construct nutrient complementation screening markers in Escherichia coli with PgsA' and LP‑1261 as dual anchor models ‑Lactobacillus has no anti-anchoring expression vector pLQ2a, and EGFP and Mcherry are used as reporter genes for screening and anchoring expression verification; it solves the problem of low conversion efficiency of Lactobacillus plantarum and it is difficult to screen positive recombinants, and constructs the expression vector pLQ2 both in E. coli 6212 / Δalr replicate expression, but also in L. plantarum Expressed in NC8 / Δalr.

The invention discloses a method for gene suppression in gluconobacterium oxydans, belonging to the technical fields of genetic engineering and bioengineering. In the present invention, the corresponding target gene crRNA is expressed in Gluconobacter oxydans. By verifying the inhibitory effect on the fluorescent expression of mCherry on the plasmid, it was shown that the recombinant Gluconobacter oxidans has a gene inhibitory effect, and by inhibiting other genes in the Gluconobacter oxydans genome, a similar inhibitory effect was achieved, further proving that This system suppresses the function of genes on the genome. A new method is provided for inhibiting the target gene in Gluconobacter oxydans, which can quickly and effectively inhibit the expression of the target gene, and improves the gene editing efficiency of Gluconobacter oxydans.

The invention discloses a method for stably expressing secretable human insulin in mesenchymal stem cells, comprising the following steps: (1) constructing an mCherry-2A-insulin-His fusion gene lentiviral expression vector; ‑2A‑insulin‑His fusion gene lentiviral expression vector is expressed in human mesenchymal stem cells to obtain extracellular human insulin. After transfection of cells, a stable expression cell line is obtained by screening. The cells express the fusion polypeptide and under the action of self-cleaving polypeptide 2A, human insulin and mCherry red fluorescent gene are separated, and human insulin can be secreted outside the cell. This inventive method solves the current dilemma of expressing human insulin in mesenchymal stem cells, and has great practical significance and potential medical application value.

The invention discloses a technological system for preparing a corn dominant genic male sterility line from a p5126-ZmMs1 and mCherry gene construct and a corn seed cross breeding and producing application method thereof. The construct contains three gene expression cassettes: an expression cassette for causing corn dominant genic male sterility, an expression cassette for marking corn peel colorand a weed killer resistance expression cassette. When the construct is guided into a corn callus tissue, the corn dominant genic male sterility line seeds can be prepared; under normal light, the transgenic sterility line seeds have no obvious difference from the non-transgenic fertile seeds, and goods quality is not affected; however, under green excitation light, the transgenic sterility line seeds show red fluorescence, and the non-transgenic fertile seeds show normal corn color and have no fluorescence. The technological system disclosed by the invention has qualitative difference from acorn recessive male sterility technology (namely a corn multi-control sterility technology system) which has been already applied for authorization by the team and can be efficiently applied to corn sterility cross breeding and hybrid seed production.

The invention discloses a technological system for preparing a corn dominant genic male sterility line from a p5126-ZmMs1 and mCherry gene construct and a corn seed cross breeding and producing application method thereof. The construct contains three gene expression cassettes: an expression cassette for causing corn dominant genic male sterility, an expression cassette for marking corn peel colorand a weed killer resistance expression cassette. When the construct is guided into a corn callus tissue, the corn dominant genic male sterility line seeds can be prepared; under normal light, the transgenic sterility line seeds have no obvious difference from the non-transgenic fertile seeds, and goods quality is not affected; however, under green excitation light, the transgenic sterility line seeds show red fluorescence, and the non-transgenic fertile seeds show normal corn color and have no fluorescence. The technological system disclosed by the invention has qualitative difference from acorn recessive male sterility technology (namely a corn multi-control sterility technology system) which has been already applied for authorization by the team and can be efficiently applied to corn sterility cross breeding and hybrid seed production.

The present invention discloses a preparation method of an anti-RET mutant protein monoclonal antibody variable region sequence. Mutation is performed at the C634 position of a RET protein to synthesize a polypeptide sequence, and the polypeptide and a carrier protein KLH are respectively coupled and combined with freund's adjuvant for emulsification to prepare an emulsified antigen, mutants are separately used to construct expression sequences to a purple fluorescent protein mcherry amino terminus to be combined with an expression vector to prepare a detection agent, mice are immunized with the emulsified antigen and mouse myeloma SP2 / 0 cells and spleen cells of the immunized mice are subjected to cell fusion, and are cultured with a semi-solid culture medium and the detection agent, by observing formation of clone balls and color changes of the culture medium, positive clones are selected into new 6-well plates and subjected to expanded culture by a changed liquid culture medium, each cell line is then transferred to a shake flask for culture, a supernatant of the culture medium is collected, antibodies are purified, and the antibodies are sequenced to obtain the variable regionsequence.

The invention belongs to the technical field of gene engineering, and discloses a method for analyzing protein interaction in prokaryotes based on a Yeast Endoplasmic reticulum Sequestration System (YESS). The method includes the steps: constructing an expression plasmid pESD-PPI-GFP-mCherry capable of performing Golden Gate assembling, with gfp and mCherry fluorescent genes, based on pESD-PPI plasmid; selecting 5 mutually interacted protein pairs from the existing data of Zymomonas mobilis on a UniProt database and the protein pairs which are not reported to have interaction as positive and negative contrasts for studying protein interactions of proteins in Zymomonas mobilis; and quantifying the analysis results. The method for analyzing protein interactions in prokaryotes can achieve high-throughput and quantitative study on protein interaction in Zymomonas mobilis. The method for analyzing protein interactions in prokaryotes based on YESS has many advantages of being simple, being easy to operate, being highly efficient, being sensitive and the like.