92results about How to "High protein expression" patented technology

The invention discloses a high-yield reactor, and a production method and an application thereof. The high-yield reactor comprises a bioreactor and an ATF perfusion apparatus connected with the bioreactor. The protein production method using the high-yield reactor comprises the following steps: 1, connecting the ATF perfusion apparatus with the bioreactor, and completing an offline disinfection or online disinfection program; 2, inoculating seed cells to the bioreactor to a work volume according to the density, and sampling and detecting the number and biochemical indexes of the cells each 24h; 3, starting the ATF apparatus program for perfusion culture when the density of living cells increases to a certain density; and 4, collecting a culture solution in the bioreactor or filtered through hollow fibers in the ATF perfusion apparatus, and purifying to obtain required protein products. The high-yield reactor has the advantages of great shortening of the technological exploitation time of monoclonal antibody or fusion protein expression, production cost reduction, benefiting for the project registering and reporting and the project period shortening, and acceleration of the accomplishment application speeds of the biopharmacy industry.

The invention provides a recombination baculovirus for expressing Africa swine fever CD2V protein in an SF9 cell. The CD2V protein can be recombined and expressed in the SF9 cell; the amino acid sequence of the CD2V protein is shown in SEQ ID NO:4; the sequence of the nucleotide fragment is shown in SEQ ID NO:3. The recombination baculovirus is used for preparing the Africa swine fever CD2V protein in an insect cell. The insect cell Sf9 is used for recombining and expressing the Africa swine fever CD2V protein, the protein expression amount is high, purification is easy, the recombination baculovirus is used for preparing and identifying a diagnosis product, and a solid foundation is laid for producing Africa swine fever subunit vaccines and diagnosis reagents.

The present invention discloses an E. coli engineering bacteria producing 1,5-pentanediamine through a whole cell catalysis and its application. The engineering bacteria according to the present invention, is Escherichia coli (E. coli) strain B or its derivative strains with the overexpression of a lysine decarboxylase gene and a proper expression of a lysine-cadaverine antiporter gene cadB. The engineering bacteria according to the present invention is the engineering bacteria producing 1,5-pentanediamine through the whole cell catalysis constructed from Escherichia coli B derivative strains, which has an overexpression of a lysine decarboxylase gene cadA and a proper expression of the lysine-cadaverine antiporter gene cadB. The present invention further discloses a method of producing a 1,5-pentanediamine catalyzed by the engineering bacteria, the yield and production intensity of 1,5-pentanediamine in bio-based production could be significantly improved through the method, hence it could be applied to mass production and convenient for extending applications.

The invention discloses a serum-free medium for HEK293 cells. The serum-free medium for the HEK293 cells is prepared from following multiple components or comprises following multiple components: aluminium chloride, barium acetate, biotin, cadmium chloride, calcium pantothenate, choline chloride, cobalt chloride, copper chloride, glucose, cholamine, ferric nitrate, folic acid, glutathione, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, inositol, arginine, asparaginate, aspartic acid, cysteine, glutamic acid and the like. Compared with the existing culture medium, the serum-free medium forthe HEK293 cells can increase the expression quantity obviously and more antibodies or proteins can be obtained in a shorter time.

The invention relates to a human papilloma virus (HPV) resistant trivalent vaccine as well as a preparation method and application thereof. Concretely, a kozak sequence capable of greatly improving the HPVL1 protein expression level is found through screening and analyzing a great number of kozak sequences, and genes 16, 18 and 58 of a main capsid protein L1 (HPV L1) of the HPV are redesigned, the sequences of the genes 16, 18 and 58 are optimized, a secondary structure of an mRNA (Messenger Ribonucleic Acid) causing influence to translation is eliminated, and then, a high-expression HPV L1 gene expression cassette is constructed on the basis. The expression cassette is particularly suitable for the expression of pichia pastoris and has the characteristics of high protein expression quantity and good stability.

The invention discloses an SOD-ELP fusion protein and a preparation method thereof. The SOD-ELP fusion protein is composed of ELP20 or ELP40 and SOD, the SOD-ELP40 fusion protein is purified by adopting three rounds of ITC, so that the purification process of the protein is simple, economic and efficient, and the method can be used for industrially preparing the recombinant humanized copper-zinc superoxide dismutase with high specific activity on a large scale. The fusion protein not only retains the activity of the enzyme, but also has good heat resistance and biocompatibility, and greatly prolongs the simulated plasma half-life period of the enzyme. According to the invention, the purified SOD-ELP40 is further embedded into a lipidosome, so that a lipidosome with high encapsulation efficiency and a transdermal effect is obtained. The SOD-ELP40 prepared by the invention can be widely applied to the fields of cosmetics, foods, medicines and the like.

The invention discloses a human blood group antigen P1 pentasaccharide synthesis method. The method includes steps: adopting a one-pot multienzyme system for coupling galactose to trisaccharide as shown in a formula (III) through a beta1-4 glucosidic bond to synthesize tetrasaccharide as shown in a formula (IV); adopting the one-pot multienzyme system for coupling galactose to the tetrasaccharide as shown in the formula (IV) through an alpha1-4 glucosidic bond to synthesize pentasaccharide as shown in a formula (I), wherein in the formula (I), the formula (III) and the formula (IV), R1 refers to hydroxyl, azide substituted alkyl, alkynyl substituted alkyl, sulfydryl substituted alkyl, alpha- or beta- configuration substituted alkyl, alpha- or beta- configuration serine residue and alpha- or beta- configuration threonine residue. By integration of high regioselectivity and high efficiency of enzymatic synthesis, P1 antigen pentasaccharide is synthesized for the first time. Glycosyltransferase, glucose nucleoside generating enzymes and glucokinse adopted in the synthesis method are all derived from prokaryotes, and high protein expression quantity, high substrate adaptability and high catalytic efficiency are realized.

The invention discloses a porcine reproductive and respiratory syndrome-Ankara vaccinia virus genetic engineering vaccine, the preparation method is that: 1. the PCR amplification of GP5 gene is carried out from the natural porcine reproductive and respiratory syndrome virus; 2. the porcine preferred codon is used for replacing the codon in the natural GP5 gene, so as to carry out GP5 gene optimization and the synthesis of the optimized sequence GP5A; 3. the epitope A in the optimized GP5A gene is replaced by the epitope B of the GP5 gene, so as to obtain the GP5-DB gene; 4. the GP5 and GP5-DB genes are respectively cloned to the expression carrier JN-2; 5. the constructed JN-2-GP5 and the JN-2-GP5-DB plasmid are recombined with Ankara vaccinia virus, so as to obtain the porcine reproductive and respiratory syndrome GP5 and GP5-DB gene Ankara vaccinia virus recombinant vaccine. Experiments confirm that the invention has excellent immune protection in BALB/c mice and pigs.

The invention discloses a method for preparing an Apis cerana MRJP1 eucaryon expression product for promoting cell growth. The method is as follows: firstly, construction of a Bac-to-Bac/BmNPV rhabdovirus expression system is utilized for obtaining recombinant Bacmid-MRJP1 which is then transfected to silkworm cells; secondly, after recombinant viruses are obtained, silkworm larvas are injected; thirdly, after the silkworm larvas are transfected for 96 hours, haemolymph is collected; fourthly, after purification of an affinity column, the high-purity recombinant MRJP1 is obtained. As shown by SDS-PAGE and Western-blotting analysis, the molecular weight of the expression product is approximately 60kDa; molecules are mainly in the state of dissolution; the expression amount is obviously higher than prokaryotic expression. The method establishes certain foundation for industrialized production of the MRJP1 and is hopeful to be applied in the fields such as biomedicine and so on for serving the human health.

The invention discloses a recombination human CYP3A4 (cytochrome P450 3A4) /CPR (cytochrome P450 oxidoreductase) /cyt b5 (cytochrome b5) protein co-transfection co-expression method. Expression vectors with CYP3A4, CPR and cyt b5 cDNA (complementary deoxyribonucleic acid) fragments and screening plasmids are co-transfected into drosophila S2 cells, the expression vectors are integrated into a drosophila cell genome, the cells are screened with blasticidin to obtain stable monoclonal cell strains, and the stably transfected monoclonal cell strains are subjected to enlargement culture for induction expression of three kinds of protein including CYP3A4, CPR and cyt b5. According to the method, the recombination vectors are easy to build, high in stability and capable of expressing three kinds of protein simultaneously, and have the advantages of high expression quantity, high activity and the like. The method can be widely used for researches of drug metabolism in vitro, drug screening development and application and the like.

The invention relates to new application and a new action mechanism of verbascoside, in particular to application of verbascoside in the drugs for treating and preventing type II diabetic nephropathy,and the research on the protection function of the verbascoside for the type II diabetic nephropathy and the action mechanism thereof. Results show that the verbascoside can alleviate liver injury caused by high glucose and lower the serum creatinine, urea nitrogen and urinary microalbuminuria levels, the blood fat level (total cholesterol and triglyceride) and the fasting blood-glucose and bloodinsulin level of spontaneous diabetes db/db mice; TGF (Transforming Growth Factor)-beta 1 and the signal transduction protein Smad3 and Smad4 and alpha-SMA protein expression thereof in a kidney tissue are obviously lowered; meanwhile, the verbascoside can alleviate the liver injury caused by high glucose; HK-2 proliferation and EMT (pithelial-Mesenchymal Transition) formation can be inhibited. In conclusion, the verbascoside has an obvious protection function on the type II diabetic nephropathy, the action mechanism of the verbascoside is that the kidney protection function of the verbascoside is implemented through oxidative stress reaction regulation and control, TGF-beta/smad signaling channel inhibition and kidney fibrosis alleviation.

The invention provides ZnT8 recombinant protein and a preparing method and application thereof. The protein has the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ IDNO.2. By analyzing and screening a target segment, optimizing and encoding the sequence and a carrier, transfecting an Expi 293 eukaryocyte and stably expressing protein with antigenic activity, thepreparing method which can keep the bioactivity of natural protein and reduce the production cost is provided, and the method has wide application prospects and huge market value.

The present invention discloses a preparation method of cholera toxin B subunit protein having biological activity. According to the present invention, a lot of cholera toxin subunits are expressed in Escherichia coli by using a prokaryotic expression vector, and then in vitro efficient renaturation is performed to obtain a large number of the cholera toxin B subunit having biological activity; the operations can be performed in the general laboratory, and no pollution is generated; and the most important advantage is that the labeling with various fluorescence, isotopes and tracers can be performed according to the experimental needs.

The gene for Streptococcus pyogenes DNase B has been cloned and vectors incorporating the cloned DNA have been used to transform Escherichia coli, allowing efficient and rapid production of the DNase in E. coli without the necessity of growing large quantities of S. pyogenes. The enzyme can be produced with a leader peptide at its amino-terminus. An improved method for the purification of naturally occurring S. pyogenes DNase B enzyme is also provided. The DNase B enzyme produced, either by purification of naturally occurring enzyme or by recombinant DNA techniques, can be used to generate antibodies and can also be used in immunochemical assays to detect the presence of anti-DNase B antibodies in serum as a marker of infection by S. pyogenes.

The invention relates to enzymatic modules and an Sda carbohydrate antigen synthesis method, and relates to six enzymatic modules. One of the six enzymatic modules comprises bifidobacterium longum N-acetylglucosamine kinase, escherichia coli glyconucleoside generating enzyme and campylobacter jejuni [beta]1-4-N-acetylglucosamine transferase. According to the Sda carbohydrate antigen synthesis method, the campylobacter jejuni [beta]1-4-N-acetylglucosamine transferase is utilized to replace human-derived [beta]1-4-N-acetylglucosamine transferase to introduce GalNAc, and Sda carbohydrate antigenepitope is constructed; and Sda carbohydrate antigen and ABH antigen correlated hybridSda carbohydrate antigen is rapidly and efficiently synthesized by helicobacter pylori [alpha]1-2 fucosyltransferase,helicobacter pylori [alpha]1-3-N-acetylglucosamine transferase and human-derived [alpha]1-3-galactotransferase.

The invention provides a talaromyces marneffei mannan protein and application of the talaromyces marneffei mannan protein in preparation of a talaromyces marneffei antibody detection kit. The amino acid sequence of the mannan protein is as shown in SEQ ID No.1. The invention further provides a preparation method of the talaromyces marneffei mannan protein. The mannan protein can be combined with a talaromyces marneffei antibody and has good sensitivity and specificity, the sensitivity reaches 85.1%, and the specificity reaches 100%.

The invention discloses an engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and a construction method and application of the engineering bacterium. On the basis of a genetic engineering strain BLK07 (pET20b-pm1), a strain with improved enzyme activity of pm1 is screened out through site-saturation mutagenesis, and an N-terminal encoding sequence (NCS) is inserted intoan N terminal of a gene sequence of the pm1, so that the protein expression quantity of the pm1 is improved, the yield of the pyruvic acid is increased, the catalytic time is significantly shortened and the catalytic efficiency is improved. When the concentration of substrates D-alanine and L-alanine is 110 g/L, the cell concentration is 4 g/L and a catalyst system only carries out catalysis at 37DEG C and 220 rpm in a shake flask for 8 h, the yield of the pyruvic acid can reach a maximum value 46.5 g/L, the conversion rate of the L-alanine is 84.5% and the resolution rate of the D/L-alaninereaches 84.5%.

The invention relates to a fluorine label and a preparation method thereof, and a method for synthesizing an oligosaccharide chain by adopting an assisted enzyme method. The structural formula of the fluorine label is G-R(I) as shown in the formula I. G represents monosaccharide or oligosaccharide; and R is one of the following formulas II and III, wherein R1 is one of C6H13 and C8H17. When G represents lactose and R represents a C8F17 difluoro chain, the formula II is shown in the specification. The fluorine label has the advantages of being easy to remove and recycle. The glycosyl receptor is used for synthesizing various different oligosaccharide chains, so that the oligosaccharide chain with a determined structure is quickly and efficiently synthesized.

The invention relates to a cryptocaryon irritans recombinant protein vaccine of which the amino acid sequences are as follows: NWVEKTAAADWKGTFVVTSSSCLASCGWKIGTTVVIADKASDATKVTWVTWVGTAHTTDSTNVDVASGSCKYVSSVANAGTAGTAAEVLNNNDECVFATGMCTIMGQKQKSPAKVTFNRDTTLDTKPFQILYKQLAMVPKAQTTVQAAADQATDCDTKASLVDTTTDAKSIVGTLKLSKATCDKCSWDTTKDLKITQDATKKYMVTLAGTIKETTSGDCKNKLTASEACYVTKKDDKTFILVSCTTLDTTNKGIPIAITTANSKTTLTLTRTDSSSQACNVVGEIT. By adopting the vaccine, the defects that an immobilization antigen DNA (Deoxyribonucleic Acid) sequence is hard to express in bacteria, and an effective gene recombinant vaccine for preventing cryptocaryon irritans infections of seawater fishes is not available in the market, are overcome, and the vaccine has the advantages of expression in escherichia coli, immunogenicity and prevention of cryptocaryon irritans infections of seawater fishes.

The invention discloses a self-induced protein expression vector based on AI-2 quorum sensing and application. A promoter used by the self-induced expression vector is an LsrR promoter which can be activated by an AI-2 quorum sensing system of escherichia coli, and can be activated by AI-2 signal molecules generated by bacteria to automatically start expression of a target gene when the escherichia coli grows to a logarithmic phase. The promoter sequence of the LsrR is shown as SEQ ID NO.1. The invention provides a construction method of the novel expression vector and application of the novelexpression vector in protein expression. Compared with a pET expression vector needing IPTG induction, the self-induction pXWZ-1 expression vector constructed by introducing the LsrR promoter has theadvantages that an inducer does not need to be added, economy and safety are achieved, the protein expression quantity is high, few inclusion bodies are formed, and low-temperature induction is not needed.

The invention relates to the field of gene engineering, in particular to a method for improving cellulase expression of trichoderma reesei by interfering a phosphatase gene. RNAi-mediated gene silencing is carried out on the phosphatase gene ymr1 related to the trichoderma reesei participating in intracellular protein degradation, and the operation improves the protein expression quantity of the trichoderma reesei and the enzyme activity of cellulase.

The invention discloses a chemically synthesized HSV1 virus gD glycoprotein extracellular region gene fragment and expression and an application thereof, and relates to the fields of genetic engineering technology, vaccine and diagnostic reagent. By computer analysis, the invention screens out the HSV1 virus gD glycoprotein extracellular region fragment containing strong antigen epitope, totally 284 amino acids, namely from the 1st to the 284th; codons which are preferred by both eukaryon and pronucleus organism are selected; and a brand new gene sequence of the antigen epitope is chemically synthesized; the genetic engineering technology is utilized to express the gene fragment and prepare the strong antigen epitope fragment and antiserum of the HSV1 virus gD glycoprotein. The expressed HSV1 virus gD glycoprotein can be applicable to the detection of vaccine, HSV1 virus antibody or antigen and immune preparation of HSV1 virus monoclonal antibody and polyclonal antibody and the like.

The invention relates to triptolide acrylate, and a preparation method and an application thereof, and discloses a novel triptolide derivative. The novel triptolide derivative is shown in a formula Iwhich is described in the specification. The invention also discloses a preparation method for the compound and a medical application of the compound in preparation of anti-cancer drugs. The triptolide acrylate and pharmaceutically acceptable salts thereof have anti-cancer activity, and can effectively inhibit tumor growth of animals through animal in-vivo experiments. A plurality of in-vitro experiment results prove that the protein expression quantity of p53 can be obviously increased; the apoptosis of tumor cells is promoted; the growth of the tumor cells is effectively inhibited; and the effect of inhibiting cancer cell metastasis is achieved. More importantly, the toxicity of the compound to normal cells is less than the toxicity of triptolide.

The invention belongs to the field of biotechnology, and particularly relates to a production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein. According to the invention, amplifying and expressing porcine epidemic diarrhea virus S1 protein through a poly-fiber paper carrier by using a cell bioreactor, culturing for 2-3 days by using a DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal calf serum in a cell culture stage, discarding all the culture medium, washing with PBS (Phosphate Buffer Solution) for 1-2 times to remove the serum, then completely replacing the culture medium with a serum-free culture medium and starting to produce and express the porcine epidemic diarrhea virus S1 protein; collecting part of the culture medium every 3-5 days, adding serum-free culture medium again, and supplementing glucose according to the sugar consumption of cells every day to keep the concentration of the glucose is kept at 4 g/L. By adopting the method provided by the invention, the expression level of cells is effectively improved, the manufacturing cost of vaccines is reduced, and a foundation is laid for realizing large-scale production of vaccines.

A T7 RNA polymerase mutant disclosed by the invention is a mutant obtained by mutating a mutant amino acid in a wild T7 RNA polymerase amino acid sequence as shown in SEQ ID No: 1, and the mutant amino acid is one or a combination of more of Y639T, M635V, R632Q, K631L, Y571T, R627E and K472S. The T7 RNA polymerase mutant has RNA polymerase activity based on a T7 promoter, and one or more modified nucleotides can be used as substrates to synthesize modified messenger ribonucleic acid. Compared with a wild type T7 RNA polymerase, the T7 RNA polymerase mutant has the advantage that the mRNA synthesis efficiency based on the modified nucleotide substrate is obviously enhanced.