46 results about "Thionin" patented technology

Streptavidin-metallothionein chimeric proteins with biological recognition specificity in which the streptavidin moiety provides high affinity biotin binding and the metallothionein moiety provides a high affinity metal binding. The binding affinity of the streptavidin-metallothionein chimeric protein both for biotin and heavy metal ions allows specific incorporation into, conjugation with, or labelling of any biological material containing biotin with various heavy metal ions.

The present invention is one gene engineering process of preparing metallothionein in high yield. The process incldues constituting the expression plasmid or its mutant gene, transferring and fermentation culture in engineering colibacillus, induction and adding metal ion before further culture, collecting thallus; ultrasonic crushing thallus, centrifugation, static adsorption with affinity chromatographic column of glutathione-agarose gel 4B, incision with thrombase on the column, column chromatograhic desalting, gradient salt elution and collecting destination protein. The present inventioncan obtain destination protein with purity over 95 %. Using the said method to prepare 6 mutant proteins of monkey metallothionein I can obtain yield and purity similar to that in wild recombinant protein.

The present invention relates to a method for producing a human I type metal sulfur protein (Human metallothionein-I, hMT-I), in particular to the fusion expression by adopting the small molecule ubinquitin-related modifier mature peptide (Small Ubiquitin-related Modifier, SUMO) and hMT-I, and the fusion protein and the ubinquitin-related modifier protease 1 (Ubiquitin-like protease 1) are co-expressed in a prokaryotic organism. In the fermentation and expression process, the ubiquitin associative modifier gene protease 1 can hydrolyze the fusion protein which consists of the ubinquitin-related modifier mature peptide and the hMT-I, to produce the dissoluble hMT-I.

The invention belongs to the soil environment management field in the environmental engineering and relates to the construction and application of a new genetically engineered microorganism capable of restoring the soil polluted by heavy metal cadmium. The preparation method of the genetically engineered microorganism comprises the following steps: monkey metallothionein alpha structure domain tetramer is fused in the ice-nucleating protein N-end of Xanthomona campestris ATCC33913 to be used as a main functional unit for constructing a broad host range expression plasmid; then cadmium-specific promoter is used for expression in Pseudomonas putida X4 which can resist various heavy metals efficiently, thus realizing the surface display of monkey metallothionein alpha structure domain tetramer in the bacterium and obtaining genetically engineered microorganism CH01 which can adsorb heavy metal cadmium efficiently and specifically. The engineered microorganism can remarkably adsorb heavy metal cadmium in the presence of various heavy metals; and the result of the symbiotic water culture with wheat, cowpea and corn shows that the engineered microorganism CH01 can colonize in the rhizosphere of plant and fast adsorb cadmium ions. The invention also discloses a construction method of the genetically engineered microorganism CH01 of which preservation number is CGMCC No.M209320.

The invention discloses an enzyme-linked immunosorbent assay method for detecting cadmium-metallothionein in the flesh of a shell. The method comprises the following steps of: inducing the shell to synthesize the cadmium-metallothionein by using cadmium chloride, inducing a rabbit to synthesize an antibody by using purified metallothionein, separating and purifying the antibody, using an electrophoretically pure rabbit anti-shell metallothionein antibody as a primary antibody and a goat anti-rabbit enzyme labelled antibody as a secondary antibody, and assaying the cadmium-metallothionein content of the shell by using the indirect competitive enzyme-linked immunosorbent assay method. The method has the advantages of scientific and rational principle and relatively mature technical process and is used for assaying the cadmium-metallothionein content of the flesh of the shell so as to indirectly reflect the cadmium pollution of the flesh of the shell and provide a fast and sensitive detection method for assaying the heavy metal cadmium in the flesh of the shell.

This invention provides a method for expressing human metallothionein (MT) by using plant oil. The method comprises: designing and synthesizing human MT gene according to the preference of plant codon, fusing human MT gene with plant oil protein gene, constructing an expression vector with the fused genes, converting recipient plant by using the expression vector to obtain converted plant, and efficiently expressing human MT by using the seed of the converted plant. Activity experiments show that purified recombinant MT has comparable activity with human origin MT. The method utilizes plant bioreactor to produce MT, thus can avoid possible attack by animal pathogen and toxin in E. coli. The method can largely simplify the separation and purification process of human MT, and lower the production cost, this is advantageous for MT industrialization.

The invention discloses a cell nucleus DNA (Deoxyribose Nucleic Acid) staining method. The cell nucleus DNA staining method is characterized by comprising the following steps: 1, fixation: sequentially placing sample slices into a BS fixing solution at the temperature of 30-40 DEG C to fix for 20-30 minutes, and rinsing; 2, hydrolyzation: completely fixing the sample slices, hydrolyzing in 5N hydrochloric acid at the temperature of 30-40 DEG C for 20-30 minutes, and rinsing; 3, staining: after the hydrolyzation is completed, staining in a Thionin staining solution at the temperature of 30-40 DEG C for 30-40 minutes, and rinsing; 4, after the staining is completed, dewatering by adopting fast gradient ethanol, and mounting. In conclusion, compared with other Feulgen staining methods, the cell nucleus DNA cell nucleus DNA has the obvious advantages of practicability, stability, efficiency, cost saving, repeatability, compatibility of automated equipment and the like and can meet the requirement of clinical DNA quantitative cytology on fast diagnosis.

The invention discloses a ramie metallothionein gene and recombinant protein and an application thereof. The ramie metallothionein gene is a nucleotide sequence shown as the sequence table SEQ ID NO.1, and the ramie metallothionein is an amino acid sequence shown as the sequence table SEQ ID NO.2. A new metallothionein gene is obtained from ramie which is a herbaceous plant through in-vivo cloning, a recombinant expression engineering strain is constructed, and the recombinant metallothionein is obtained through expression and purification. The recombinant protein contains 12 cysteines (Cys),is abundant in mercapto groups that can chelate with cadmium and other heavy metal ions, and has wide application prospects in the fields of preparation of a heavy metal chelating antidote, chemical or medicine or heavy metal polluted cultivated land and pollution treatment of agricultural non-point sources.

The invention discloses a gene engineering applying method of elsholtzia metal sulphur protein gene EhMT1 in Haizhou, wherein the GenBank entry number of cDNA sequence of EhMT1 is DQ059081 and the coded amino acid sequence is AAY57923; EhMT1 participates the accumulation of Haizhou elsholtzia root; the analysis of mRNA expression is that the Haizhou elsholtzia is induced by excessive copper; EhMT1 can be goal gene leading-in plant, which improves the copper resistance of plant and the accumulation of copper.

The invention belongs to the bio-pharmaceuticals field, in particular to a method for preparing metallothionein by taking the liver of sturgeon as raw material. The method mainly comprises steps: sturgeons fed with feed added with inducer-zinc acetate by humans are slaughtered to take livers thereof; liver homogenate buffer solution is used for homogenizing and centrifuging, glucan and iminodiacetic acid mixture chromatography column chromatography collects chromatography liquid, and then the metallothionein is obtained by vacuum cooling drying. The liver of sturgeon is rich in metallothionein and is 100 times of liver content of other animals. The method is used for extracting the metallothionein, which can reduce the cost by 30%, increases yield by 40% and quality, and the purity the metallothionein reaches more than 99%. The metallothionein is expensive, has obvious physiological function and extensive use. Extracting metallothionein by using the liver of sturgeon has wide market prospect.

The invention provides a gene cloning method, expressing method and application of psychrophilic yeast metallothionein. Metallothionein genes are extracted, a primer F1 and a primer R1 are designed, subjected to PCR amplification and then connected with an expression carrier, and a recombinant expression carrier is constructed; the recombinant expression carrier is converted into an expression strain, and a recombination expression transformant is constructed; the expressing and purifying method of recombinant protein is built. The nucleotide sequence and the coded amino acid sequence of the metallothionein genes are shown in the sequence table SEQ ID NO:1 and the sequence table SEQ ID NO:2. The novel metallothionein obtained through cloning comprises 16 pieces of cysteine, multiple metal and heavy metal ions can be chelated through rich sulfydryl, and the novel metallothionein has broad prospects in the medicine field, the food health caring field, the cosmetic additive field, the genetic engineering reagent field, the chemical engineering field, the environmental protection field, the animal field, the agriculture field and the like.

The present invention concerns a method for modifying the growth characteristics of plants by modulating expression in a plant of a nucleic acid sequence encoding a metallothionein and/or modulating activity in a plant of a metallothionein. The invention also relates to transgenic plants having modified growth characteristics, which plants have modulated expression of a nucleic acid encoding a metallothionein.

The invention provides a fusion protein of a human epidermal growth factor and metallothionein, which comprises a first part and a second part, wherein the first part is the human epidermal growth factor, the second part is the human metallothionein, and the human metallothionein is positioned at the C terminal of the human epidermal growth factor. The invention further provides an application of the fusion protein in the preparation of drugs for treating burns or scalds or wounds or ulcers and the application of the fusion protein in the preparation of skin nursing drugs. Compared with the original human epidermal growth factor and the metallothionein, the fusion protein can increase the stability, reduce the clearance rate, reduce the degradation, bring benefits and convenience to the use of the EGF-MT fusion protein and further reduce the using dosage and the medication frequency. Simultaneously, the formed fusion protein is a bifunctional molecule which has double effects of EGF and MT.

The invention discloses a method for a detecting peanut metallothionein mRNA expression level by real-time quantitive PCR, comprising the following steps: 1) connecting metallothionein gene obtained from peanut tissues with vectors, and constructing a recombinant plasmid as a standard substance for real-time quantitive PCR; 2) extracting the recombinant plasmid containing target genes, calculating a molecule copy number, diluting the recombined plasmid into different times to serve as a standard substance template for real-time quantitive PCR reaction; and 3) extracting the total RNA of required tissues, performing reverse transcription on the total RNA to be changed into cDNA serving as a real-time quantitive PCR template, carrying out real-time quantitive PCR to obtain the copy number of an initial template, and detecting and analyzing the mRNA expression level thereof.

The invention discloses a yeast strain for displaying metallothionein on a cell surface and application of the same in heavy metal adsorption. The yeast strain for displaying metallothionein on the cell surface is obtained by integrating a box gene for displaying metallothionein on the cell surface and a constitutive promoter P<PGK1> gene into the chromosome of a host stain via an integrative vector; and the yeast strain can continuously and stably express metallothionein on the cell surface. The recombinant Saccharomyces cerevisiae strain provided by the invention has good adsorption capability and tolerance to a plurality of heavy metals like Cd<2+>, Cu<2+> and Cr<6+>; e.g., the strain needs 12 h for complete reduction of Cr<6+> with a concentration of 50 mg/L, 6 h faster compared with an original strain, and has a reduction rate 1.5 times of the reduction rate of the original strain; the removal rates of the strain to Cu<2+>, total Cr and Cd<2+> are increased by 13.35%, 10.13% and 19.39% respectively compared with the original strain; so the strain has great application potential in the field of treatment of heavy metals.

The invention discloses a human metallothionein-4 fusion protein expression vector. The upstream of an expression vector cloning region comprises human heart-type fatty acid-binding protein (hFABP6), and the downstream comprises the human metallothionein-4 (MT-4). The invention also reveals an application method of the expression vector, and the method comprises the following basic steps: construction of an expression bacterial strain, amplification, inducible expression and purification of fusion proteins. The hFABP6-MT4 fusion protein expression vector has the following characteristics (1) high-efficiency expression of MT4 proteins; (2) soluble expression.

The invention discloses an expression vector which comprises at least one regulation unit. Each regulation unit comprises an enhancer and a promoter, wherein the enhancer is an hrx enhancer or functional fragment thereof or sequence variant, and the promoter is an MT promoter or functional fragment thereof or sequence variant, or is an Act5C promoter or functional fragment thereof or sequence variant. The hrx enhancers are hr5 enhancers in Lepidoptera bar viruses AcMNPV, hr3 enhancers in Bombyx mori or hr3 enhancers in AcMNPV. The MT promoters are drosophila cell metallothionein promoters which are induced promoters and induced by metal ions. The Act5C promoters are Actin 5C promoters in drosophila cells. The expression vector can perform efficient expression in drosophila S2 cells. The recombinant protein expression vector with the Lepidoptera bar virus gene hr5 enhancers is built and capable of performing efficient expression in the diptera insect drosophila S2 cells, and overcomes the technical bias that bar viruses are not spread in dipsters due to species isolation in the prior art.

The present invnetion relates to a biological medicine preparation for relieving heavy metal poisoning. It is a heavy metal antidote using human metallothionein-L (MTL) as main component. The MTL contains the polypeptide of amino acid sequence described by SEQ IP NO2, its nucleotide code sequence and the nucleotide sequence described by SEQ ID NO1 have at least 70% of homology. It can relieve heavy metal and semimetals poisoning, in the course of development it can regulate internal balance of zinc and copper, and make metallic metabolism and detoxification, and can eliminate free radical. There, this invention can be used as auxiliary medicine preparation for curing tumor, and also relates to its production method and application.

The invention belongs to the field of gene clone in molecular biology, and specifically relates to a hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and a cloning method thereof. The hyperaccumulator solanum nigrum metallothionein gene sequence is type 3 metallothionein gene, and the gene sequence is shown by a nucleotide sequence in SEQ ID NO. 1. The successive cloning and sequencing of the hyperaccumulator solanum nigrum type 3 metallothionein gene helps to establish a base for further analyzing the biological stress resistant capability under the influence of MT-L3, and screening and applying other functions of MT-L3.

The invention discloses a plant expression vector utilizing beer yeast MF-alpha to mediate recombinant protein secretion. The plant expression vector contains 35S constitutive promoter, beer yeast MF-alpha gene and exogenous gene. The MF-alpha-exogenous gene in the vector is expressed under control of the 35S constitutive promoter; after the plant expression vector is transferred into wild tobaccoand geranium through agrobacterium mediation, the exogenous gene under control of the 35S constitutive promoter can efficiently express protein coded by the exogenous gene and then secretes the protein out of plants. Experiment results show that yeat MF-alpha signal peptide mediates reporter gene Egfp protein to be secreted out of the plants; when metal sulfur protein MT3 gene of human is used toreplace Egfp gene, it is found that Cd2+ enrichment of transgenic plants is enhanced. Therefore, a genetic engineering operation method can be provided for further building MF-alpha to mediate otherrecombinant proteins to be secreted to the extracellular in the plants.

This invention belonging to metallothionin extraction field is about the extraction procedure of active zinc-sulfur protein from sea cucumber and abalone. The whole procedure involves feedstuff inducing, cleaning the raw material, squashing, filtering, homogenate, crystallization and extraction. With the procedure we reported here, most of sea cucumber and abalone'effective efficiency is remained, the purity of our product is up to 90%, and the procedure is easy to manipulate. We could extract 300g zinc-sulfur protein from 500g dried sea-cucumber or extract 300g active zinc-sulfur protein from 500g alive sea-cucumber.

The invention relates to food-grade lactococcus lactis with human metallothionein-I anchored on the surface and a preparation method and a use thereof. The preparation method comprises the following steps of 1, splicing a cA gene and an hMT gene by overlapping PCR, introducing linker between the two genes, and introducing an expression promotion label to an end N to obtain a recombinant cA-hMT gene, 2, connecting the recombinant cA-hMT gene and an expression carrier to obtain a connection product and transferring the connection product into a escherichia coli competent cell, and 3, carrying out inducible expression of the recombinant fusion protein by engineering bacteria obtained by the step 2, carrying out ultrasonication on the obtained engineering bacteria, then carrying out centrifugation and carrying out incubation of food-grade lactococcus lactis by the supernatant so that the fusion protein is anchored on the surface of the food-grade lactococcus lactis without protein purification. The prepared food-grade lactococcus lactis with human metallothionein-I anchored on the surface can enrich heavy metals. The preparation method has the advantages of high efficiency, safety, no toxicity, low cost and no protein purification.