550 results about "Secretion expression" patented technology

The invention aims to provide a method for improving the expression amount of secretory foreign protein in pichia pastoris. Byintracellular co-expression of double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP in pichia pastoris, the expression amount of secretory foreign protein in pichia pastoris is effectively improved. According to the invention, double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP are transformed into pichia pastoris secreting and expressing exogenous xylanase to obtain recombinant pichia pastoris strains, thereby significantly increasing the expression amount of the exogenous xylanase. During co-expression of double genes of HAC1 and ERO1 in pichia pastoris, the secretion expression amount of exogenous xylanase is generally increased by 15%-25% compared with the initial level; and during the co-expression of three genes HAC1, ERO1 and BIP, the secretion expression amount of xylanase is increased by 45%-57% compared with the initial level.

The invention relates to a preparation method of virus-like particles (VLPs) of Chikungunya virus (CHIKV). The method comprises the steps of: modifying genetic elements of a structural protein encoding gene C-E3-E2-6K-E1 of CHIKV, cloning the modified genetic elements into the expression vector of an insect cell, then transfecting the obtained recombined expression vector and baculovirus linear DNA respectively to an SF9 insect cell and making the cell secrete and express CHIKV VLPs. Additionally, the invention also makes preliminary studies on the immune effects of CHIKV VLPs and applicationof CHIKV VLPs in virus specific antibody detection, thus laying a foundation for research and preparation of immunological detection reagents and even vaccines based on CHIKV VLPs.

The invention relates to a vaccine production technology in the technical field of biology, in particular to a CHO cell strain which is established by utilizing a gene engineering means and is used for expressing recombinant protein PigFC-pigSCFVE2, and a preparation method and application of the recombinant protein. The recombinant fusion protein PigFC-pigSCFVE2 provided by the invention is A1) or A2) shown as follows, wherein A1) is protein of which the amino acid sequence is as shown in SEQ ID No.2, and A2) is protein which is obtained by substituting, losing and / or adding one or several amino acid residues in the amino acid sequence of the protein of the A1) and has PigFC-pigSCFVE2 activity. A monoclonal cell strain which is obtained through the method and capable of carrying out secretory expression on PigFC-pigSCFVE2 is higher in fusion protein expression quantity, fusion protein obtained through affinity separation and purification of an antibody can be combined with a monoclonal antibody, animals can be immunized, the immunity of a generated neutralizing antibody is higher than that of a present market product, the fusion protein can be used for swine classical fever preventive vaccine, and the production cost and the immunity failure loss can be reduced.

The invention discloses a bacillus subtilis chitosanase as well as a preparation method and application thereof. The invention optimizes an encoding gene of bacillus subtilis chitosanase according to the preference of pichia pastoris codon. The optimized nucleotide sequence is shown as SEQ ID NO.2. A pichia pastoris expression system is further utilized to perform efficient secretory expression on the optimized chitosanase encoding gene, so as to obtain the bacillus subtilis chitosanase with the amino acid sequence as SEQ ID NO.1. The bacillus subtilis chitosanase obtained according to the invention has higher hydrolytic activity to chitosan substrates at different degrees of deacetylation; the crude enzyme generated through shake-flask fermentation has the hydrolysis capacity of degrading 5g of chitosan by 1mL crude enzyme (0.3mg of protein), about 150mg of non-specific commercial enzyme is required for degrading the same amount of chitosan, and the efficiency is theoretically increased by 500 times; and the bacillus subtilis chitosanase has excellent industrial application prospects.

The invention relates to a host cell and a method for the host cell applied in efficient secretion expression of a recombinant protein, which belongs to the filed of microbiological engineering and fermentation engineering. The invention provides a bacillus licheniformis host cell CBB3008 which is preserved in the China Center for Type Culture Collection with the preserving number of CCTCC NO: M 208236. The invention transforms an expression plasmid of an industrial enzyme which is obtained by a gene cloning technology into the host cell to synthesize an industrial enzyme preparation in the host cell efficiently, and then secretes the synthesized enzyme protein into a culture medium efficiently through a protein secretion system of the host cell, thus the invention can guide efficient secretion production of the industrial enzyme preparation. Therefore, the invention is helpful to reduce the fermentative production cost of the industrial enzyme preparation, simplify the fermentative production process and reduce the environmental pressure of the fermentation industry. A method for host cell screening and genetic improvement of the invention can also be used for other types of host cells, in particular for the breeding of the host cells of bacillus subtilis, bacillus megaterium, bacillus pumilus, bacillus starch solution and the like.

The invention relates to a ZEN (zearalenone) degrading enzyme gene and a high-yield strain. The ZEN degrading enzyme gene comprises DNA molecules of (a), (b) or (c), wherein (a) has DNA molecules of a nucleotide sequence shown in SEQ ID NO.1; (b) hybridizes with the nucleotide sequence in (a) under a strict condition and encodes DNA molecules of protein with ZEN degrading enzyme activity; (c) has DNA molecules of a nucleotide sequence with 90% or higher of homology with the nucleotide sequence in (a) or (b). The invention establishes the high-yield strain for producing ZEN degrading enzymes through the sequences and further provides a high-density fermentation method. ZEN degrading enzymes can be subjected to efficient secretion expression through the high-yield strain with the high-density fermentation method, and ZEN can be quickly and efficiently degraded during fermentation.

The invention belongs to the technical field of microbial gene engineering, aims to solve the problem of difficult degradation of the PET (polyethylene terephthalate) plastics, and provides a PET hydrolytic enzyme expression unit, a DNA (deoxyribonucleic acid) recombined vector built based on the unit and an application of the vector, wherein the vector can effectively secrete colibacillus expressing PET hydrolase (PETase). The DNA recombined vector can effectively achieve secreting type constant expression of the PET hydrolytic enzyme in colibacillus host bacteria, nutrient solution of the vector has remarkable lipase biological activity, PET plastic films can be degraded in continuous culture process, a genetic engineering approach is provided for biodegradation of the PET plastics, andthe vector has a good application prospect.

The invention provides a method for producing FGF21. The invention first designs and synthetizes full-length FGF21 nucleotide sequence which is integrated with secreted signal peptide to be structured into an expression carrier capable of secreting and expressing FGF21 protein; the expression carrier is led into an appropriate host cell; under the guidance of the secreted signal peptide, FGF21 is expressed in cell periplasm, so that the purification procedure is convenient, the finished product ratio is improved and the mass production of rhFGF-21 becomes possible.

The invention discloses an optimized gene of recombinant glucose oxidase and an expression vector and application of the optimized gene. On the premise that the amino acid sequence of the glucose oxidase is not changed, the gene sequence of the glucose oxidase is optimized according to pichia pastoris preferred codons by comprehensively considering the influencing factors such as use frequency ofthe codons, adjustment of GC content, deletion of instable sequences, secondary mRNA structure and the like; and the nucleotide sequence of the optimized glucose oxidase gene is shown as SEQ ID NO.1.The invention further provides the expression vector and a recombinant host strain containing the optimized gene of the glucose oxidase. The optimized gene is transferred to the pichia pastoris for expression, and the test results show that: compared with the gene before optimization, the secreting expression quantity of the optimized gene in the pichia pastoris is remarkably improved. The application effect tests of the glucose oxidase show that the expressed recombinant glucose oxidase has the same using effect as a commercial enzyme preparation.

The invention discloses a Chinese hamster ovary (CHO) genetic engineering cell line for performing high-level secretory expression on AcAPc2 and relates to a CHO cell line for performing high-level secretory expression on the AcAPc2. The invention aims to solve the problems that the expression level of an exogenous gene product expressed by using CHO is low at present and the industrialized production cannot be realized. The CHO genetic engineering cell line for performing high-level secretory expression on the AcAPc2 is obtained by the following step of: screening dihydrofolate reductase-deficient CHO cells which are taken as host cells, and amplifying to obtain the cell line for performing stable and high-level expression on the AcAPc2. The CHO cell line can perform high-level expression on the AcAPc2; the expression level can be up to 10mg / L.72h; and the CHO cell line can be industrially produced. The cell line is used for resisting blood coagulation and treating tumor, septicaemiaand the like.

The invention belongs to the technical field of food industry biology and relates to a bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof. The vector utilizes a bacillus subtilis vector pHB201 as a framework, the pHB201 enzyme cutting site is modified first through an enzyme cutting method to obtain a pHB-hc vector, composition type strong promoter P43, composition type strong promoter PamyE, molecular chaperone PrsA of an Sec path and neutral protease signal peptide SnprB are obtained by cloning in bacillus subtilis 168 genome through a polymerase chain reaction (PCR), and a bacillus subtilis secretion type expression vector pHBSR is obtained in construction. Lipoxygenase gene obtained from anabaenagenome DNA in amplification is inserted into autonomously constructed expression vector pHBSR and then electrically converted into a bacillus subtilis host WB800 to achieve secreting expression of the recombination lipoxygenase, and the largest enzyme activity in fermentation liquid can reach 76U / ml.

A method of secretory expression of lysostaphin in Escherichia coli at high level, which comprises constructing a expression vector by cloning a sequence encoding a signal peptide which is suitable for secretory expression in Escherichia coli before part or whole gene sequence which encodes mature lysostaphin, and ligating the cloned sequence with a promoter; and transforming Escherichia coli with the expression vector, culturing and fermenting, and then isolating lysostaphin from the supernatant of the fermentation broth. The advantage of secretory expression is that the expression product can exist in the medium in an active form, and thus does not need the process for renaturation of the inclusion body; it is more easily to purify from the supernatant of the fermentation broth with high rate of recovery; and there is less contamination from the host's proteins.

The invention discloses a preparation method of multi-copy high expressed recombined plectasin by pichia pastoris. The method comprises the following steps: a plectasin expressing gene sequence is designed according to preference performance to codon translated by pichia pastoris; the optimized plectasin gene is fused on an alpha-factor signal peptide C terminus of an expression vector pPICZalphaA to construct a single-copy expression vector, the vector comprises a plectasin expression cassette containing a start signal element alcohol oxygen dehydrogenase strong promoter (AOX), alpha-factor signal peptide gene and a plectasin gene fused in C terminus, a stop signal element AOX (TT) and the like. A complementation principle of restriction endonuclease Bg1II and BamHI cohesive end is used to obtain plectasin gene-containing recombinant plasmid of different copy cascade expression cassettes, pichia pastoris is electrotransformed and secreted and expressed plectasin with high efficiency under the methanol induction. The expression level and plectasin gene copy number exist a linear relation. The constructed multi-copy high expressed yeast cells can be used for raising the output and reducing the cost, and is adapted to large scale production of plectasin.

The invention provides novel antimicrobial peptide MP1106 (Seq ID No.1) designed based on Plectasin, and provides a high-efficiency expression of the novel antimicrobial peptide MP1106 (Seq ID No.1) in pichia pastoris. Through the preference of yeast codon for optimization of antibacterial peptide MP1106 encoding gene and construction of recombinant plasmid, the recombinant plasmid is transformed into the pichia pastoris, secretory expression of high density fermentation protein in a 5 L fermentation tank can be achieved, the total protein level reaches 2.134g / L, and purified recombinant MP1106 yield reaches 831mg / L. In addition, recombinant MP1106 cation-exchange chromatography one-step purification method is established, and novel antimicrobial peptide MP1106 in-vitro properties show that the novel antimicrobial peptide MP1106 has strong inhibitory activity against staphylococcus aureus, is free of cell toxicity and stable in high temperature, alkaline environment, serum incubation conditions, and is resistant to papain, pepsase and the like. The novel antimicrobial peptide MP1106 is superior in properties, can realize large-scale industrial production, and is potential to be developed into a novel anti staphylococcus aureus preparation.

The invention discloses an engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application, and belongs to the genetic engineering field. A trypsin gene obtained through in-vitro amplification is fused with a leading short peptide YVEF, and is wholly connected to a Pichia pastoris GS115 chromosome to construct engineered Saccharomyces cerevisiae efficiently secreting and expressing recombinant trypsin, and the recombinant trypsin having an improved stability is obtained after purification, and the heat stabilities of the recombinant trypsin at 40DEG C, 50DEG C and 60DEG C are 1.77, 2.6 and 31 times wild trypsin respectively, so the problem of the low stability of trypsin is solved. The production of trypsin through applying the engineered Saccharomyces cerevisiae has the advantages of high output, simple technology, intelligible heredity and application background of engineered Saccharomyces cerevisiae, and convenient industrial application.

The invention provides a bacillus subtilis xylose induced secretion expression vector pXYL-amyQ. The expression vector is constructed by using bacillus subtilis plasmids pHT43 as starting plasmids, removing original promoters and a repressor gene sequence thereof and inserting the bacillus subtilis plasmids into an expression box of bacillus subtilis xylose promoters and a signal peptide sequence. The xylose promoter expression box comprises a xylose induced promoter sequence and a repressor gene sequence thereof. The constructed expression vector pXYL-amyQ can secrete expression homogenous and heterogenous proteins in bacillus subtilis. The host bacillus subtilis of the vector is a generally recognized as safe (GRAS) host, and the adopted xylose serving as an inducer is nontoxic, so the expression vector has biological safety and plays an important role in protein expression, particularly enzyme and antibody preparation.

The invention provides a nitrilase gene coding nitrilase, a recombinant vector containing the gene, a recombinant gene engineering bacteria obtained by converting the recombinant vector and application thereof in preparing recombinant nitrilase. The nitrilase gene can be connected with an expression vector for construction to obtain endoenzyme expression recombinant plasmid containing the gene or secretion expression recombinant plasmid, and then the endoenzyme expression recombinant plasmid containing the gene or the secretion expression recombinant plasmid is respectively and correspondingly converted to a colibacillus bacterial strain to obtain recombinant colibacillus; the recombinant colibacillus contains recombinant nitrilase and can recombine colibacillus into an enzyme resource for biological catalysis and conversion. The recombinant nitrilase serves as the enzyme for conversion, and racemisation mandelonitrile, acrylonitrile, iminodiacetonitrile or 2,2-dimethylcyclopropane carbonitrile and the like serve as a substrate for converting to react and prepare corresponding R-mandelic acid, crylic acid, iminodiacetic acid or chiral 2,2-dimethylcyclopropane formic acid and the like.

The invention discloses a preparation method for insulin aspart through recombinant expression by using yeast, and concretely relates to a preparation method for insulin aspart through recombinant expression by using pichia yeast. Concretely, the method comprises the following technological process: effectively secreting and expressing human aspart proinsulin, performing lysyl endopeptidase single enzyme digestion to obtain insulin aspart deleting B30, coupling with a threoninate, and performing deprotection, anti-phase purification and crystallization. The method is relatively suitable for industrialized preparation of recombinant insulin aspart.

The invention discloses a porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application. An antigen fusion gene shown as SEQ ID No. 1. is obtained by connecting an antigen locus of porcine epidemic diarrhea virus (PEDV) S glycoprotein and a cell wall anchoring sequence. The invention further constructs a secretion expression vector containing the antigen fusion gene, and secretion expression vector is converted into the bacillus megaterium to express recombinant protein on the cell wall or surface of the bacillus megaterium. Immunoblotting experiments indicate that the expressed recombinant protein can react with PEDV immune serum and has the same antigenicity as PEDV natural antigens. Immunofluorescent tests of live bacteria which are subjected to induced expression indicate that the expressed recombinant protein is positioned to the surfaces of the bacteria. Experiment results indicate that the recombinant protein can be prepared into safe and effective mucous immune live vaccines for preventing and treating porcine epidemic diarrhea.

The invention provides a method for culturing microzyme of secretory expression proteolytic enzyme. The method is characterized in that an inorganic salt culture medium is adopted for fermenting and culturing a recombinant methylotrophic yeast. Fermentation and culture comprise the growth period of the microzyme and the secretory expression period of the proteolytic enzyme. In the secretory expression period of the proteolytic enzyme, the feed flow of methanol can be controlled in real time according to the variation of oxygen dissolved in fermentation liquid, and the PH value of the fermentation liquid is adjusted with ammonia, thus expressing the proteolytic enzyme at high efficiency. Compared with the prior art, the method for culturing the microzyme of the secretory expression proteolytic enzyme has the characteristics of low culture cost, convenient and accurate reaction control mode and high expression activity of the proteolytic enzyme.

The invention discloses expression equipment for expressing exogenous protein by secretion in trichoderma reesei cells. The expression equipment comprises the following elements from 5' to 3': (1) an exo-glucan cellobiose hydrolase II promoter of trichoderma reesei; (2) a secretively-expressed signal peptide; (3) a polyclonal locus sequence; and (4) an exo-glucan cellobiose hydrolase II terminator of the trichoderma reesei. Exogenous genes are inserted into the expression equipment, agrobacterium tumefaciens is converted by a T-deoxyribonucleic acid (DNA) binary vector and is jointed with thetrichoderma reesei to obtain trichoderma reesei genetic engineering bacteria which can express heterologous genes from animals, plants, fungi and the like efficiently by secretion, and a large amountof exogenous protein is obtained from the trichoderma reesei genetic engineering bacteria. The trichoderma reesei has an extensive culture condition, and is suitable for solid culture and liquid submerged fermentation; and mycotoxin and antibiotics cannot be generated under the zymogenic condition, and the generated extracellular protein is easy to separate and purify and low in cost.

The invention discloses recombinant engineering bacteria for efficiently expressing human growth hormone (hGH), a construction method and an application and provides an escherichia coli operon for expressing recombinant hGH, an expression plasmid containing an operon sequence and engineering bacteria QJSW-SZ01 (CGMCC No:7258) used for secretory expression of the recombinant hGH and obtained by transformation of the expression plasmid containing the operon. The recombinant engineering bacteria are characterized in that a coding sequence of a signal peptide of an escherichia coli heat-stable enterotoxin is changed, and rare codons of escherichia coli in the coding sequence are mutated so as to avoid formation of a secondary structure; meanwhile, an amino acid is altered on the terminal of the coding sequence so that the coding sequence is more beneficial to guidance of secretory expression of hGH; the signal peptide, an escherichia coli alkaline phosphatsae promoter (phoA promoter) and an escherichia coli T7 terminator are combined to be used as an expression control element so that the recombinant hGH is efficiently secretory-expressed in the escherichia coli in a soluble form. The recombinant engineering bacteria lay the foundation of finally developing a low-cost hGH pharmaceutical product.

The invention discloses a recombinant bacterium capable of high efficiently secreting and expressing natto kinase, and the recombinant bacterium can prominently increases the output of natto kinase. The recombinant bacterium is a recombinant pichia pastoris containing at least two copies of a natto kinase Pro-NK gene and a Haclp regulatory factor gene. After the recombinant bacteria carry out fermentation for 96 hours in a shake flask, the enzyme activity of the natto kinase can reach 470 IU / mL.

The invention discloses a fully-humanized anti-human interleukin 17A single-chain antibody, relates to the technical field of genetically engineered antibodies and specifically relates to a fully-humanized antibody fragment which is genetically engineered, screened, expressed and provided with special affinity with interleukin 17A (IL-17A). The genetically engineered antibody fragment is connected with a heavy chain region and a light chain region end to end by virtue of a soft connecting fragment. By constructing a great-capacity natural phage antibody library, an antibody fragment with special adsorption capacity on IL-17A is obtained by biological elutriation. The antibody fragment is constructed into a full-length antibody, so that the reaction of IL-17A on interleukin 6(IL-6) released by a human fibrosarcoma cell HT1080 can be inhibited by virtue of eukaryotic secretory expression and affinity purification. The antibody fragment disclosed by the invention can be used for detecting and treating rheumatoid arthritis.

The invention relates to lactic acid galactococcus food-grade secretion expression vector, and the preparation method and the application thereof, in particular to the lactic acid galactococcus food-grade secretion expression vector which comprises the following components: a thyA gene selection marker, replicons of plasmid pWV01, multiple cloning sites, promoter used for secretion expression, a ribosome bind site of Usp45, a signal peptide sequence of Usp45 and a partial Usp45 mature peptides coded sequence.

The invention provides a method for preparing a recombinant novel coronavirus Spike protein. The invention firstly provides application of the following polypeptides as a signal peptide in preparationof recombinant novel coronavirus Spike protein: polypeptides composed of the amino acid sequence as shown in SEQ ID No. 10. By adopting a specific signal peptide, an expression vector containing polynucleotide for encoding the recombinant novel coronavirus Spike protein is constructed, and mammalian cells are transfected to secrete and express the recombinant novel coronavirus Spike protein, so that the secretory expression level of the Spike protein in HEK293 cells can be remarkably improved.

The present invention is expression vector to secretion express foreign gene in colibacillus or bacillus and its construction, and belongs to the field of gene engineering technology. The expression vector pBL-WZX contains one promoter sequence with function in both colibacillus and bacillus, one signal peptide sequence with function in both colibacillus and bacillus, one polyclonal site for foreign gene to be cloned, one artificially transcription termination sequence, one genetic marker for cloning selection between colibacillus and bacillus, and one DNA sequence for foreign gene to complete integration and gene copy number proliferation in the chromosome DNA of bacillus. The expression vector pBL-WZX can induce the transcription and translation of foreign gene and secrete the gene product to outside cell, and has foreign gene secretion expression interventing level up to 1 mg / mL in colibacillus and 3.1 mg / mL in bacillus separately.

The invention relates to an indirect ELISA (enzyme-linked immunosorbent assay) method for detecting a porcine reproductive and respiratory syndrome antibody, which comprises the following steps: by using a pGEX-6p-1 prokaryotic expression vector, performing tandem repeat on two epitopes to improve the antigen activity of an expressed protein, thus constructing a gene engineering bacterium BLpGEX-6p-GP5 capable of realizing secretory expression of the GP5 protein dominant antigen epitopes, wherein one epitope is a linear conservative neutralizing epitope (epitope B) of a screened PRRSV (porcine reproductive and respiratory syndrome virus) GP5 protein, which can be identified by a monoclonal antibody and can also be identified by porcine anti-PRRSV neutralizing serum, and the other epitope is a high-variability immunodominant epitope (A); and purifying and renaturing the expressed recombinant protein, and coating an ELISA plate, thus establishing the indirect ELISA method for detecting a PRRSV antibody to detect the PRRSV antibody level in porcine serum. Results show that the method has the characteristics of favorable repetitiveness and high specificity and can be used for PRRSV serological search.

A Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode relates to a Chinese hamster ovary (CHO) cell line capable of efficiently expressing the AcAP5 in secretion mode. The cell line resolves the problem that at present, expression amount is low by using CHO to express exogenous gene products, and requirements of industrial production cannot be met. The Chinese hamster ovary genetic engineering cell line for the efficient secretion expression AcAP5 utilizes dihydrofolic acid reductase defect type Chinese hamster ovary cells as host cells, and the cell line capable of stably and efficiently expressing AcAP5 is obtained after the host cells are screened and amplified. The CHO cell line can efficiently express the AcAP5, can be produced in industrialization mode and is used for resisting blood coagulation and thrombus. Furthermore, expression amount can reach to 12 mg / L / 72h.

The invention relates to a method for the excretive expression of a recombinant human granulocyte-colony factor (rHG-CSF) in colon bacillus. In the invention, a carrier of periplasmic cavity excretive type expression recombinant proteins can be established by adding DsbA signal peptide (SEQ ID NO:1) at the N end of an exogenous protein and using an lambadaPL promoter, the DH5alpha competent cells of the colon bacillus are converted, and the expression of the exogenous protein in the periplasmic cavity of the colon bacillus is realized by temperature induction. The method has high expression quantity and high excreting efficiency, and is suitable for effectively and stably excreting and expressing various exogenous genes.