87 results about "Isopropyl Thiogalactoside" patented technology

The invention discloses a method to prepare engineering bacteria of 5-glycyl propionic acid and methods for using them, which comprises the following steps: incorporating radial agrobacteriocin; setting the preserved number at CGMCC No. 1938; activating engineering bacteria with LB medium flat; getting monoclonal of the engineering bacteria; seeding to shake; culturing; getting first-grade seed; seeding the first-grade in shake; culturing; getting second-grade seed; seeding the second-grade seed in fermenter; culturing; cooling and evoking with isopropyl-beta-D-sulfo-galactose-glycoside; culturing continually; proceeding supplementing material culture. This engineering bacteria possesses higher ALA synthase expression, simple craft, low cost and good industrial prospect.

The invention discloses recombinant oral protein TAT-GH of tilapia, a preparation method for the recombinant oral protein TAT-GH and application of the recombinant oral protein TAT-GH. The method comprises the following steps of: extracting total messenger ribonucleic acid (mRNA) from a pituitary gland of male tilapia, performing reverse transcription-polymerase chain reaction (RT-PCR) to obtain complementary deoxyribonucleic acid (cDNA), performing PCR amplification to obtain a growth hormone (GH) gene of the tilapia, and amplifying a TAT-GH gene by a PCR overlap extension method; constructing a recombinant plasmid pET-32a(+)-TAT-GH; and transforming Escherichia coli BL21 (DE3) by using the recombinant plasmid pET-32a(+)-TAT-GH, efficiently expressing fusion protein TAT-GH in the form of an inclusion body under the induction of isopropyl thiogalactoside (IPTG), and purifying and renaturing to obtain active protein. The protein can effectively promote the growth and development of fries of the tilapia after being fed to the fries of the tilapia.

The invention discloses a type B hepatitis virus X protein transduction system expression vector and its construction, by constructing restructured plasmid pTAT-GFP-X through the X gene of the human hepatitis B virus and pTAT-GFP vector, wherein the constructed pTAT-GFP-X restructured plasmid can express the fusion protein containing X protein of hepatitis B viruse, and a vast amount of expression fusion proteins can be evoked from isopropylthiog-alactoside (IPTG), purification can be carried out through 6 histidines (His6), the TAT polypeptide has the structure capable of traversing cell membrane.

The invention relates to the field of molecular biology, and in particular to a galectin-3 binding protein, and preparation and application thereof. The galectin-3 binding protein is shown as an amino acid sequence SEQ ID No.1 in the sequence table. The preparation method is as below: using Cynoglossus semilaevis cDNA as a template; conducting PCR amplification by primers F1 and R1; connecting PCR products to an expression vector to obtain a plasmid pETG3BP; conversing BL21DE3 to obtain a transformant BL21/pETG3BP; inducting by isopropyl-beta-D-thiogalactoside; and purifying the recombinant protein with affinity chromatography column to obtain the galectin-3 binding protein. The galectin-3 binding protein has significant binding ability to various bacteria.

The invention discloses a method for producing lactulose by means of a recombinant bacillus subtilis, and belongs to the technical field of bioengineering technology. The recombinant bacillus subtilis is used as the production strain, the recombinant bacillus subtilis includes cellobiose epimerase capable of catalyzing lactose for producing the lactulose, and induction expression is performed through the lactose or isopropyl-beta-D-isopropylthiogalactoside (IPTG) so as to use the lactose or whey as a substrate for a biological catalysis reaction. Bacillus subtilis wet cells or a fermentation solution is used for the catalysis reaction, the method is simple and easy to implement, and enzyme activity losses and cost waste in the enzyme separation and purification process are avoided; the food-grade bacillus subtilis is used as the production strain, the method is safe and reliable, and the effective references are provided for industrial and environment-friendly lactulose production.

The invention belongs to the technical field of biology, and relates to a method for preparing virus analogs of nervous necrosis viruses. The method comprises the following steps of: (1) performing expression of the virus analogs of the nervous necrosis viruses on escherichia coli of pQE30 plasmid vectors coded with nervous necrosis virus capsid protein genes under the pronucleus condition, wherein the expression is performed under the following conditions of: inoculating 1 mass percent of escherichia coli solution of which OD600 is equal to 1.5 into a culture medium, culturing the escherichia coli at 37 DEG C under 250rpm till the OD600 is 0.3 to 0.5, adding isopropyl-beta-D-thiogalactoside into the escherichia coli solution till the final concentration of the isopropyl-beta-D-thiogalactoside is 900muM, transferring the solution, and continuously culturing the escherichia coli for 3 to 4 hours at the temperature of between 25 and 30 DEG C at the rotational speed of 200rpm to finish the expression; and (2) after the expression is finished, breaking, separating and purifying the strains to obtain the virus analogs of the nervous necrosis viruses, wherein the plasmid coded genes also can be nervous necrosis virus capsid protein genes containing histidine tags, and the expression product of the genes can be purified by affinity chromatography. The method provided by the invention can obtain high virus analog expression amount; and the chromatography and purification method is simple and convenient and has low costs in required apparatuses and reagents.

The invention relates to a negative selection marker for escherichia coli gene knock-in, namely, the virulent gene ccdB driven by a plac promoter, and plac-ccdB and the gentamicin resistance gene aacC1 form a gene cassette for gene knock-in. In the first step, DNA fragments containing homologous arms plac-ccdB and aacC1 are obtained through amplification, and electro-transformed into escherichia coli for expression of recombinase. Under gentamicin screening, recombinase catalyzes homologous recombination and the DNA fragments are integrated into escherichia coli genome. In the second step, same homologous arms are utilized for amplification of foreign DNA fragments, ccdB is expressed in the bacterial strains obtained in the first step during the electrotransformation for expression of recombinase under the screening of isopropyl-beta-d-isopropylthiogalactoside, the toxicity serves as the feature of the negative selection marker, recombinase catalyzes homologous recombination among homologous arms, and foreign DNA fragments replace a plac-ccdB-aacC1 gene cassette to realize gene knock-in.

The invention relates to a Corynebacteria inducible promoter, an expression vector comprising same, and an application thereof, wherein the expression vector is converted to corynebacterium glutamicumto obtain a 5-aminolevulinic acid producing bacterial strain, by that 5-aminolevulinic acid can be produced through a fermentation method. The expression vector constructed from the promoter, throughcontrollable expression of gene through inositol induction, can overcome problems of inhibition effect on growth of bacterial cells with and high production cost due to high price of an inducing agent, (isopropyl[beta]-D-thiogalactoside, IPTG). The expression vector has characters such as stability. By integrating the promoter with genome of a host or converting the expression vector into the host, corynebacterium glutamicum, the 5-aminolevulinic acid producing bacterial strain is acquired, yield of the 5-aminolevulinic acid reaching 24.2 g/L.

The invention relates to a hepatitis B virus multi-epitope fusion protein and a preparation method and application thereof. The fusion protein is obtained by inserting hepatitis B virus multi-epitope fusion peptide (with the sequence shown as SEQIDNo.1) formed by serially connecting HBsAg313-321, HBsAg335-343, Pol150-159, Pol455-463 and Padre epitopes through connecting peptide between amino acidat the 78th position and amino acid at the 79th position of a hepatitis B virus core protein; and the preparation method comprises the following steps of: constructing a hepatitis B virus multi-epitope fusion protein expression plasmid pET28-HBc-HP; performing isopropyl thiogalactoside (IPTG) inducing expression by using an Escherichia coli expression system; and purifying by using affinity chromatography. The fusion protein carries a plurality of supertype epitopes of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and polymerase, is viral particles, has the advantages of strong immunogenicity, wide applicable range and the like, and can be used for preparing therapeutic hepatitis B vaccines.

The invention discloses a fermenting technology for improving expression amount of fusion protein of recombinant human brain natriuretic peptide. The fermenting technology comprises the following steps of adding 18 to 20L of BHD culture medium, 65 to 80mL of glycerin, and ampicillin into a fermenting tank until the final concentration is 100mg/L; inoculating, adjusting the air ventilation amount until the dissolved oxygen content in the fermenting liquid is 30% to 50%, adjusting the pH (potential of hydrogen) value to 6.8 to 7.2, and culturing at the temperature of 37 DEG C; when OD600 is 10 to 15, adding isopropyl thiogalactoside until the final concentration is 0.5 to 1.0mmol/L, inducing, adding 150 to 300mL/A of supplementing liquid A into the fermenting tank, stopping fermenting culture after 3 to 6h, and harvesting bacteria. The fermenting technology has the advantages that the expression level of rhBNP fusion protein is obviously improved, the expression amount reaches up to 34.5% to 38.3%, and the good commercial development value and great success in commerce are obtained.

The invention discloses a PPA-linker-thanatin fusion protein of which the amino acid sequence is as shown in SEQ ID NO: 2. The invention also discloses a preparation method of the PPA-linker-thanatin fusion protein, comprising the following steps of: acquiring target genes of recombinant pinellia pedatisecta schott agglutinin and thanatin by a two-step PCR (Polymerase Chain Reaction) method, cloning the target genes onto an E.coli expression vector pET-28a-c(+), and acquiring the expressed protein by IPTG (isopropyl thiogalactoside) induction, wherein the expressed protein is the PPA-linker-thanatin fusion protein, namely the expression product of the pinellia pedatisecta schott agglutinin-thanatin. The expression product of the pinellia pedatisecta schott agglutinin-thanatin has biological activity and has inhibiting effect on the growth of tumor cells.

The invention relates to genetically engineered organisms, especially to microbes like Escherichia coli with activity in catalysis of glucuronidation of flavonoids, and provides a genetically engineered bacterium used for biological catalysis of glucuronidation of flavonoids. The genetically engineered bacterium is prepared by coexpression of four genes respectively coding phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, uridine diphosphate glucose dehydrogenase and uridine diphosphate glucuronyltransferase in a cell and introduction of the functional enzyme genes into the cell through expression vectors. The genetically engineered bacterium induces expression of functional enzyme protein under the condition of addition of an inductive agent--isopropyl thiogalactoside (IPTG) and is directly used for biological catalysis of glucuronidation of flavonoids; and the advantages of good cell growth, a short fermentation period and low cost are obtained.

The invention relates to a genetic engineering kit for detecting environmental estrogen pollutants and application thereof, and belongs to the technical field of detection. The kit comprises the following components in part by volume: 2*10<4> parts of LB culture medium, 1 part of isopropyl-beta-D-thiogalactoside, 20 parts of chloroform, 10 parts of solution of lauryl sodium sulfate, 20 parts of o-nitrophenyl-beta-D-galactoside and 100 parts of solution of sodium carbonate. A detection process of the genetic engineering kit is simple, convenient and quick, and results can be obtained within 2 to 3 hours; the genetic engineering kit does not have special requirements on instruments, demand quantity of samples is fewer, detection stability is high, and the detection sensitivity can reach n g/L; and the genetic engineering kit improves the analytical rate while reducing the analytical cost greatly.

The invention provides a bran coat source peroxidase anti-tumor active fragment as well as a preparation method and application thereof. The preparation method of the fragment comprises the followingsteps: extracting mRNA of rice seedling tissues by a Trizol method, taking the mRNA as a template, performing enzymatic catalysis, and performing inverse transcription to obtain cRNA; obtaining a protein gene sequence in Genebank according to FMBP mass spectrum identification results of the rice anti-tumor active protein, respectively performing truncated screening according to different functional regions, determining a gene sequence of Ca<2+> binding sites as an anti-tumor effect structural domain, and designing a specific primer thereby; taking the rice cDNA as a template, and amplifying the FMBP-C gene sequence by a PCR (Polymerase Chain Reaction) method; respectively introducing BamH I and HindIII restriction enzyme sites at the upstream and downstream of the sequence, connecting thesites with a pMal-s vector, constructing a pMal-s-FMBP-C recombinant plasmid, transferring E.coli DH5alpha competent cells, and screening to obtain a positive transformant; performing induced expression on a target protein through IPTG (Isopropyl Thiogalactoside), performing affinity purification, desalting and concentrating, and detecting functions of resisting tumors and reversing multiple drugresistance of tumors by an MTT method. The results show that the active fragment has obvious activities of resisting tumors and reversing multiple drug resistance of the tumors.

The invention discloses a high density fermentation method of recombinant porcine interferon alpha 1(rPoIFN alpha 1) gene engineering bacteria, and belongs to the technical field of fermentation engineering. The high density fermentation method of the recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria is as follows: recombinant escherichia coli BL21/pET-32a-rPoIFN alpha 1 which can construct and express rPoIFN alpha 1 by itself is used as a production bacteria strain for fermentation at 37 DEG C for 2-3h, inducer isopropyl-beta-d-thiogalactoside (IPTG) with the concentration of 50 g / L-100 g / L is added into each liter fermentation solution by one time, fermentation is continued at 32 DEG C for 4-5 h, and then the fermentation process is completed. The high density fermentation method can realize the high-efficiency expression of the rPoIFN alpha 1, an objective protein expression amount accounts for more than 40% of the total bacteria protein, the potency can reach above 1*10<6>IU/ml, the protein is a soluble protein, denaturation and renaturation problems of inclusion body expression can be avoided, the production cycle is shortened, and the high density fermentation method provides a solid foundation for industrial production of the rPoIFN alpha 1.

The invention discloses high-stereoselectivity methionine adenosyltransferase as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting a genome of streptomyces xinghaiensis; by taking a genome as a DNA (Deoxyribose Nucleic Acid) template and adopting a PCR (Polymerase Chain Reaction) amplification technology, obtaining a gene sequence of methionine adenosyltransferase in the streptomyces xinghaiensis, namely a metK target fragment; carrying out double enzyme digestion on the metK target fragment and an expression vector pET-32a (+), and then connecting to construct a recombinant plasmid; transforming the recombinant plasmid into competent cells of escherichia coli BL21 by adopting a thermal activation method, and carrying out inducible expression by virtue of isopropyl-beta-D-thiogalactoside, so as to obtain SxMAT. According to the invention, high-stereoselectivity methionine adenosyltransferase is subjected to denaturation and renaturation later to obtain high-purity SxMAT, the SxMAT is good in stability when the temperature is 25-55 DEG C and the pH value is 8-10.5, the optical purity (ee) of the product is higher than 90%, the yield is 80%, and the in-vitro double-enzyme cascade reaction is constructed by combining the SxMAT and the fluorinase, so that the understanding of people on a fluorination pathway in streptomyces is facilitated, and the application of fluorinated natural products is derived.