71 results about "Colibiogen" patented technology

The invention discloses preparation of novel duck reovirus recombinant sigma B protein antigen, and application of the novel duck reovirus recombinant sigma B protein antigen in detection of a novel duck reovirus antibody. With RNA (ribonucleic acid) of the novel duck reovirus (NDRV) as a template, a nucleotide complete sequence of an NDRV S3 gene coding region can be amplified through a reverse-translation polymerase-chain reaction, is directionally subcloned to a fusion expression vector of the prokaryotic expression vector pET-30a-C(+) of escherichia coli, and transformed into the host cell of the Escherichia coli BL21 (DE3), and is subjected to inducible expression with IPTG (isopropyl-beta-d-thiogalactoside), purification of Ni-NTA Agarose, inclusion body refolding to obtain the expressed NDRV alpha B recombinant protein antigen. The expression mode of the protein is fusion protein (His-alpha B) with molecular weight being about 44kU; and immunoblotting assays prove that the protein has immunoreactivity similar to natural protein..

The invention provides a recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein, as well as an encoding gene and expressing, purifying and inclusion body renaturing methods of the fusion protein. The IL2 realizes a very important immunomodulatory effect in a disease generation process, but has the defects of high elimination speed in blood plasma and high industrialization cost. The invention provides the recombinant porcine IL2-Fc fusion protein by utilizing a prokaryotic expression system of escherichia coli in order to obtain a large amount of porcine IL2 with a longer half-life period, wherein a porcine IL2 part is all sequences of a porcine IL2 extracellular region, an Fc segment part comprises a hinge region, a CH2 region and a CH3 region of an antibody, and the porcine IL2 part and the Fc segment part are directly fused. The recombinant porcine IL2-Fc fusion protein provided by the invention saves most of biological activity of IL-2, greatly prolongs the half-life period and provides the guarantee for low-cost large-scale expression and the industrialization.

The invention discloses PCV2 virus-like particles as well as a preparation method thereof and splitting and VLP assembly buffer liquor. Based on an autonomous optimization design, a PCV2 nucleocapsid protein gene which is suitable for efficiently expressing in a prokaryotic expression system is artificially synthesized, a full-length gene sequence of the PCV2 nucleocapsid protein gene is expressed by an escherichia coli prokaryotic expression system, and the virus-like particles are efficiently and autonomously assembled by utilizing soluble nucleocapsid protein of the full-length gene sequence under a special condition. An innovation point of the invention is that PCV2VLPs are obtained by utilizing the prokaryotic expression system instead of adopting the conventional method for obtaining VLPs through an eukaryotic expression system; the method is low in cost, simple and efficient, and suitable for large-scale industrial application; moreover, an innovative buffer liquor formula integrating double functions, which not only can promote thallus splitting, but also can be suitable for self-assembling of VLPs, is also applied; besides, the PCV2 virus-like particles obtained in the invention are very highly similar with wild type virus in outline and good in immunogenicity, and can be applied to developing a subunit vaccine and a drug delivery carrier with utilization potentiality of porcine circovirus.

The invention discloses a novel glucose-resistant beta-glucosidase gene and an application thereof. The novel glucose-resistant beta-glucosidase gene has a nucleotide sequence shown in SEQ ID NO.1, and has an amino acid sequence shown in SEQ ID NO.2. The invention also discloses a preparation method of the novel glucose-resistant beta-glucosidase gene and a recombinant plasmid pET32a-Bgl2238 containing the novel glucose-resistant beta-glucosidase gene; the invention further discloses a recombinant beta-glucosidase and a preparation method thereof; the recombinant beta-glucosidase gene is over-expressed in an escherichia coli prokaryotic expression system, the recombinant beta-glucosidase is subjected to high-efficiency solution expression in an escherichia coli expression system. The invention also discloses an application of the recombinant beta-glucosidase in hydrolysis of polydatin in polygonum cuspidatum; the recombinant beta-glucosidase obtained by the method is indicated to haverelatively high catalytic activity and glucose tolerance activity and have great industrialized production and application prospects.

The invention provides a helicobacter pylori multiepitope vaccine. The active ingredient of the helicobacter pylori multiepitope vaccine is a piece of polypeptide; and the helicobacter pylori multiepitope vaccine mainly comprises a multi-copy body of Th and B cell antigen epitopes of helicobacter pylori urease A and B bi-subunit and a mucosal immune adjuvant of a cholera toxin B subunit. The preparation method mainly comprises the following steps of: synthesizing an artificial gene by using a gene synthesis technology, wherein the artificial gene comprises a gene sequence of the multi-copy body of the Th and B cell antigen epitopes of the helicobacter pylori urease A and B bi-subunit; coupling the artificial gene with the gene sequence of the cholera toxin B subunit to form a fusion gene;and expressing the fusion gene by using an escherichia coli prokaryotic expression system, and performing protein purification to obtain the helicobacter pylori multiepitope vaccine. The helicobacterpylori multiepitope vaccine can induce an organism to generate T cell immune response and high-titred specific antibody humoral immune response aiming at the urease A and B bi-subunit, and can be used for preventing and treating helicobacter pylori infection related diseases.

The invention discloses a method for preparing GLP-1 or GLP-1analogue polypeptides by using escherichia coli to express a tandem sequence. A general formula of a recombinant tandem protein of the GLP-1 or the GLP-1 analogue designed by the invention is X-(Y-Z)n, wherein X is a leader peptide, Y is a trypsin and CPB enzyme double restriction enzyme cutting site or a Kex2 enzyme and CPB enzyme double restriction enzyme cutting site and auxiliary sequence, Z is the GLP-1 or the GLP-1 analogue, and n is a number of tandem repetition. An inclusion body formed by performing fermentation induction expression on the recombinant tandem protein of the GLP-1 or the GLP-1 analogue through an escherichia coli prokaryotic expression system can be completely dissolved without a denaturing agent, and thedissolved solution can be subjected to trypsin and CPB enzyme double digestion or Kex2 enzyme and CPB enzyme double digestion to obtain the GLP-1 or the GLP-1 analogue. The expression level of the GLP-1 or the GLP-1 analogue obtained by the method is high, and the yield of the GLP-1 target protein after enzyme digestion and the purity after enzyme digestion are high.

The invention discloses the application of a recombinant human cystathionine Beta-synthase (rhCBS) in the preparation of drugs for curing or preventing hyperhomocysteinemia and related diabetes and cardiovascular diseases and a preparation method of the rhCBS. The preparation method of the invention mainly adopts an E.coli prokaryotic expression system which is optimized by expression conditions to obtain highly active rhCBS. The rhCBS can significantly reduce the HCY concentration in a cell-culture medium and improve the survival rate of myocardial cells and endothelial cells at the high HCY level. In an overall animal model with hyperhomocysteinemia, compared with traditional folic acid, VitB6 and VitB12 for curing the hyperhomocysteinemia, the rhCBS can greatly reduce the HCY level in plasma of a rat fed by homomethionin and alleviate the damage to cardiac muscle and vessels and has extremely obvious curative effects on the hyperhomocysteinemia. In addition, the rhCBS can be applied to the preparation of drugs for curing or preventing the hyperhomocysteinemia and related cardiovascular diseases and has broad market prospect.

The invention discloses a gene for coding a human ribonuclease 4-based recombinant protein. The nucleotide sequence of the gene is represented by SEQ ID NO:2, and the amino acid sequence of the human ribonuclease 4-based recombinant protein is represented by SEQ ID NO:1. The invention also discloses a recombinant expression, separation and purification method of the human ribonuclease 4 protein. The method comprises the following steps: optimizing the codon of the RNASE 4 encoding gene to make the optimized codon suitable for prokaryotic expression of Escherichia coli; constructing an RNASE 4 protein prokaryotic expression plasmid pET-11a-RNASE 4; inducing Escherichia coli BL21(DE3 / pET-11a-RNASE4) to express the recombinant RNASE4 protein; and separating the recombinant RNASE4 protein, and purifying the recombinant RNASE4 protein. The protein is used for preparing auxiliary medicines for treating neurodegenerative diseases.

The invention provides a recombinant porcine interferon IFN beta1-Fc fusion protein as well as an encoding gene and expression, purification and inclusion body renaturation methods thereof, belonging to the biological genetic engineering field. The IFNbeta can enhance the immunity of pig and has a good application prospect in the veterinary medicine industry. However, natural porcine IFNbeta1 is less in expression quantity and is insufficient for research and development and application and has the deficiency of quick plasma clearance rate. The invention provides the recombinant porcine interferon IFN beta1-Fc fusion protein applicable to a coliform bacteria prokaryotic expression system. Part of the porcine IFNbeta1 is all sequences in an ectoenzyme area of the porcine IFNbeta1. The Fc section comprises a hinge region, a CH2 region and a CH3 region of an antibody. The porcine IFNbeta1 and the Fc section are directly fused. The fusion protein provided by the invention not only maintains the biological activity of the original protein IFNbeta1 to a great extent, but also extremely prolongs the half-life period of the original protein IFNbeta1, thereby providing the opportunity of industrialized development of the fusion protein.

The invention belongs to the technical field of biological engineering, and discloses fusion expression of chicken interferon genes lambda and alpha of an interferon fusion preparation, a production method and clinical application thereof. According to the interferon fusion preparation of biological engineering, total RNA (Ribonucleic Acid) of CEF cells is extracted, a specific primer is designed, the chicken interferon genes lambda and alpha are cloned and are fused by using a complementary hydrophobic flexible amino acid connector, and thus the complete chicken interferon fusion genes lambda and alpha can be obtained; the chicken interferon fusion genes lambda and alpha are cloned to a 19-T carrier, is massively cloned and expressed successfully, and is further connected with a pET-32a carrier for massive expression; a product is identified to ensure that the product is expressed in an inclusion body, and then modification, purification and renaturation are implemented; the anti-virus activity of the product is detected, and the clinical application effect of the product is evaluated. Prokaryotic expression plasmid pET32a-chIFN-lambda+alpha of the recombinant chicken interferon genes lambda and alpha is successfully established, and 1:1 fusion expression of the chicken interferon genes lambda and alpha on an escherichia coli prokaryotic expression system can be achieved.

The invention provides recombinant porcine interleukin (IL) 4-Fc fusion protein, a coding gene and an expression, purification and inclusion body renaturation method thereof, which belong to the field of the biological genetic engineering. Porcine IL4 can be used for treating porcine chronic infectious diseases and parasitic diseases and can be further popularized and applied to preventing and treating diseases such as porcine anaphylactic reaction and the like which are related to immunity. However, the porcine IL4 has the defects of high clearing speed in plasma and high industrialization cost. The invention provides long-acting recombinant porcine IL4-Fc fusion protein by adopting an escherichia coli prokaryotic expression system, wherein the porcine IL4 part includes the whole sequence of a porcine IL4 extracellular region, the Fc segment part includes a hinge region of antibodies, a CH2 region and a CH3 region, and the porcine IL4 part and the Fc segment part are directly fused. According to the recombinant porcine IL4-Fc fusion protein provided by the invention, the biological activity of the IL4 is improved, the half-life period of the IL4 is greatly prolonged, and the guarantee is provided for the low-cost mass expression and the industrialization of the expression.

The invention relates to a PCV2d (porcine circovirus type 2) virus-like particle vaccine and a preparation method thereof. By means of epidemiological analysis for PCV2d strains of China, comparison of amino acid sequence information of a large quantity of collected strains as well as phylogenetic tree analysis, a Cap gene of a currently epidemic PCV2d strain in China is selected, PCV2d Cap protein is effectively expressed by an Escherichia coli prokaryotic expression system through codon sequence optimization, PCV2d virus-like particles are prepared successfully by purification and assembly in an in-vitro assembly and dialysis buffer solution, and form, size and concentration of the virus-like particles are not affected when the virus-like particles are placed for 6 months in a storage buffer solution at 4 DEG C and subzero 20 DEG C; the prepared PCV2d virus-like particle vaccine immunizes 21-day-old piglets, and a PCV2d challenge test proves that the vaccine has a good protecting effect for the piglets.

The invention discloses a novel high-temperature-resisting lipase gene which is named as lip34 and which has a nucleotide sequence shown as SEQ ID NO.1. Also, the invention discloses a preparation method of the novel high-temperature-resisting lipase gene, a recombinant plasmid pET32a-lip34 containing the novel high-temperature-resisting lipase gene, and an expression vector thereof. The invention further discloses a high-temperature-resisting recombinant lipase and a preparation method thereof. The novel high-temperature-resisting lipase gene lip34 is excessively expressed in an escherichia coli prokaryotic expression system, and the production period of lipase is substantially shortened. The recombinant lipase obtained with the method has relatively high catalytic activity and thermal stability, and has good industrialized production and application prospects.

The invention provides a porcine interferon alpha and an application thereof. The porcine interferon alpha is synthesized through selecting high-expression codon design according to the codon bias of the of a Escherichia coli prokaryotic expression system, the amino acid sequence of the porcine interferon alpha is represented by SEQ ID NO. 1, and the nucleotide sequence for encoding the porcine interferon alpha is represented by SEQ ID NO. 2. A method for preparing the porcine interferon alpha has the advantages of simplicity, low cost, easiness in industrial production, high expression level of the porcine interferon alpha, thorough denaturation in the inclusion body treatment process, and high protein renaturation rate porcine. The porcine interferon alpha prepared in the invention has high titer, can inhibit 100TCID50 vesicular stomatitis virus, has a specific activity reaching up to 10<8>, has good bioactive functions, can be used to prepare medicines for preventing and treating the swine virus infection and enhancing the immune function, and has a broad market application prospect.

The invention discloses tandem duck Alpha and Nu interferon genes and a preparation method and application thereof and belongs to gene fusion expression in the biotechnical field. A nucleotide sequence of the tandem duck Alpha and Nu interferon genes is shown as in SEQ ID No. 1. The invention also provides the preparation method of the tandem duck Alpha and Nu interferon gene, a prokaryotic expression plasmid Alpha and Nu interferon prokaryotic expression plasmid pET32a-IFNAlpha-linker-IFNNuexpressing the tandem duck Alpha and Nu interferon gene and its preparation method, and application of a fusion protein expressed by the tandem duck Alpha and Nu interferon gene in the preparation of duck antiviral drugs. Recombinant duck Alpha and Nu interferon prokaryotic expression plasmid pET32a-IFNAlpha-linker-IFNNu is established successfully herein, the duck IFN-Alpha and IFN-Nu genes are tandemly expressed on an Escherichia coli prokaryotic expression system, and it is also possible to ensure that the two interferon genes are expressed in a ratio of 1:1.

The invention provides a recombination porcine IFN (interferon) alpha1-Fc fusion protein as well as a coding gene and expression, purification and inclusion body renaturation methods of the recombination porcine IFN alpha1-Fc fusion protein. IFN alpha1 is the most common interferon and has remarkable anti-virus, anti-tumor, hematopoietic cell proliferation inhibition, immune adjustment functions and the like. But natural IFN alpha1 is expressed in an organism in a very small amount, lots of IFN alpha1 is difficult to directly extract in vivo for clinical research and application, and the defect of fast clearing speed of the IFN alpha 1 in blood plasma exists. Therefore, the invention provides the recombination porcine IFN alpha1-Fc fusion protein suitable for an escherichia coli prokaryotic expression system, wherein a porcine IFN alpha1 part is an entire sequence of a porcine IFN alpha1 extracellular region, an Fc fragment part comprises a hinge region, a CH2 region and a CH3 region of an antibody, and the porcine IFN alpha1 part and the Fc fragment part are directly fused. The fusion protein provided by the invention maintains the most of biological activity of the IFN alpha 1, the half-life period of the fusion protein is greatly prolonged, and conditions are provided for the industrialization of the fusion protein.

The invention relates to a K99-987P-F41 recombinant protein, wherein the amino acid sequence thereof is shown by SEQ ID No.2. The invention also provides a coding gene of the recombinant protein, a preparation method and an application in preparing an enterotoxigenic escherichia coli (ETEC) vaccine. The three-section target genes ETEC adhesins K99, 987P and F41 are serially connected and sub-cloned to an escherichia coli prokaryotic expression vector pET30a(+), and can be used for preparing an escherichia coli K99-987-F41 series connection expression subunit vaccine; multiple antibodies can be obtained by immunizing once, and the vaccine is convenient to use and overcomes the shortcomings of a monovalent vaccine; meanwhile, since a few surface proteins are contained, many unrelated antigenic determinants of bacteria and side reaction caused by crude extraction or semi-purification preparation are eliminated; and the K99-987P-F41 recombinant protein has the characteristics of good safety and good stability, can overcome the shortcomings of whole-cell inactivated vaccines, and has the advantage of high specificity of subunit vaccines.

The invention discloses a controllable escherichia coli acid-resistant system based on an ion channel and application thereof. The original glutamine-mediated acid-resistant system of escherichia coli is based, and the survival rate of escherichia coli is enabled to be related to the expression quantity of ammonium ion channel proteins and the concentration of ammonium ions in a culture medium under an extreme acidic condition with glutamine by knocking out glutamate decarboxylase GadA and GadB in the original system, so that the acid resistance of escherichia coli can be controlled by regulating the concentration of extracellular ammonium ions and / or the expression quantity of intracellular ammonium ion channel proteins, thus controlling the survival of escherichia coli under the acidic condition. The acid-resistant system can also be used as a mode system for measuring the substrate and functions of the ion channel proteins.

The invention discloses an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for bovine coronavirus. The ELISA detection kit is characterized in that purified BCoV-CD isolate recombinant pET-32a-N protein is used as coating antigen, reaction conditions are optimized, and an indirect ELISA diagnosis method for detecting bovine-serum characteristic N protein antibodies. The ELISA detection kit disclosed by the invention has the beneficial effects that escherichia coli is adopted for prokaryotic expression, the raw-material sources are wide, the preparation is easy, the standardization iseasy, and the production and detection processes are safer and more reliable, so that the ELISA detection kit is suitable for being promoted and used in basic units.

The invention belongs to the biotechnical field, in particular relating to a method for high-efficient expression of Mycobacterium tuberculosis heat shock protein Hsp16.3 in colon bacillus. The method clones coding genes of the Hsp16.3 protein into a colon bacillus prokaryotic expression vector, transforms colon bacillus host bacteria and obtains the Hsp16.3 protein through IPTG inducement. The expression product is recombinant proteins with 6 His tags on the N end and with a molecular weight of approximate 19kD, and pure Hsp16.3 protein is obtained through further purification through affinity chromatography.

The invention discloses an enterovirus PCR (Polymerase Chain Reaction) quality control serum armored RNA and a preparation method. An expression vector of the armored RNA is a pET32a-MS2-CP-ENV-2B plasmid; the expression vector, namely the pET32a-MS2-CP-ENV-2B plasmid, is transferred into escherichia coli prokaryotes expression strain BL21 to induce expression; after the expression vector is ultrasonically disrupted, precipitates are centrifugally collected to obtain virus-like particles of the enterovirus PCR quality control serum armored RNA; and in the expression vector, namely the pET32a-MS2-CP-ENV-2B plasmid, the nucleotide sequence of the MS2-CP is as shown in SEQ ID NO:1 in the sequence list, and the nucleotide sequence of the ENV-2B is as shown in SEQ ID NO:2 in the sequence list.During storage, the armored RNA cannot be degraded by RNase enzyme and is an enterovirus PCR quality control serum with stable performance. The preparation method of the armored RNA is easy to operate, safe and effective.

The invention discloses a preparation method of endothelial vesicles of Escherichia coli for anti-tumor drugs with endogenous high expression of nucleic acids. According to the preparation method of the endothelial vesicles of Escherichia coli, a 'tRNA scaffolding means' is adopted so as to stably insert microRNA into a vector; the vector is transformed into Escherichia coli, thereby allowing highendogenous expression of microRNA with cancer-cell-killing effects; epicellular and periplasmic components of the Escherichia coli are removed by using lysozyme, thereby obtaining protoplast of Escherichia coli; the protoplast is filtered by using polycarbonate membrane, thereby having the protoplast broken; and then, purification is carried out by adopting a ultracentrifugation means so as to have endothelial vesicles of the protoplast separated, thereby obtaining the endothelial vesicles of Escherichia coli with high expression of miRNA and low toxicity. The preparation method provided by the invention is easy to operate, low in production cost, feasible for large-scale fermentation and preparation, low in toxicity, and highly efficient. As a novel pharmaceutical carrier, being appliedin anti-tumor drug preparation, the endothelial vesicles of Escherichia coli are capable of significantly inhibiting development of non-small cell lung cancer; and thus, the endothelial vesicles of Escherichia coli have broad application prospects in the field of pharmaceutical carriers.

The invention provides a novel fasciola hepatica multi-epitope vaccine. An active ingredient of the novel fasciola hepatica multi-epitope vaccine is a polypeptide. The polypeptide is mainly formed by fasciola hepatica sphingolipid activated mucin-like protein-2 Th, a multicopy body of B-cell epitope and a mucosal immunologic adjuvant cholera toxin B subunit. An artificial gene is mainly synthesized through the gene synthesis technology and comprises Th of sphingolipid activated mucin-like protein-2 and a gene sequence of the multicopy body of the B-cell epitope, then the artificial gene is coupled with the gene sequence of the cholera toxin B subunit, and a fusion gene is formed. An escherichia coli prokaryotic expression system is utilized for expressing the fusion gene, and the fasciola hepatica multi-epitope vaccine is obtained after protein purification. The fasciola hepatica multi-epitope vaccine can induce an organism to generate sphingolipid activated protein T cell immune responses and high-titer specific antibody humoral immune responses, and can be used for preventing and treating fasciola hepatica infection related diseases.

The invention discloses an establishing method for a drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor. The establishing method comprises the following steps: 1, expression and purification of helicase protein: artificially synthesizing Japanese encephalitis virus NS3 gene, using NcoI and XhoI to respectively digest helicase segments; connecting the digested helicase segments with escherichia coli prokaryotic expression vector over the night to obtain a helicase protein with a sequence shown as SEQ ID No. 1; collecting liquid supernatant of the expression protein, using Ni2+ affinity chromatography to purify the helicase protein, using a BCA process to measure the protein concentration; 2, designing helicase DNA substrate; 3, adding helicase protein 3uM, compound to be sieved and reaction buffer solution to black 96 holes for mixing and incubating, detecting fluorescence intensity in the detecting system by a fluorescence detector, not adding a control group of the substrate; and 4, calculating the inhibition ratio of the compound. The establishing method is simple and easy to carry out, has high flexibility, low cost, and less samples, can monitor the reaction process of helicase, and has a great meaning in developing the Japanese encephalitis virus medicine in the future.

The invention relates to a method for producing a Delta sleeping peptide by utilizing an escherichia coli prokaryotic expression system. The adopted technical scheme is as follows: carrying out enzyme digestion on genes dsip of the obtained Delta sleeping peptide, then inserting into a vector plasmid pET28a, and constructing a plasmid pET28a-dsip; transferring the plasmid pET28a-dsip into escherichia coli BL21 competent cells, inoculating the obtained recombinant escherichia coli into an LB culture medium, using 1mM IPTG for inducing for 4-5h under the condition of 30 DEG C when the absorbance value of bacteria liquid is 0.6, and collecting bacterial cells in a centrifugal manner; using ultrasonic waves for breaking the bacterial cells, removing insoluble cell broken pieces and collecting supernatant liquid; and passing through an Ni-NTA affinity chromatography column twice, and obtaining the Delta sleeping peptide. The method can realize the mass expression of the Delta sleeping peptide through the escherichia coli expression system and solve the problem of impurity of a product formed by chemical synthesis and low content of active ingredients.

The invention provides a novel alveolar echinococcosis subunit vaccine. A polypeptide serves as an active ingredient of the alveolar echinococcosis subunit vaccine, which is mainly composed of a surface antigen Emy162 of alveolar echinococcosis and a mucosal immune adjuvant, namely cholera toxin B subunit (CTB). According to the invention, the gene sequence of the alveolar echinococcosis surface antigen Emy162 is synthesized by virtue of a gene synthesis technology, and then the gene is coupled with the gene sequence of the cholera toxin B subunit, so that a fusion gene is formed. The fusion gene is expressed by virtue of an escherichia coli prokaryotic expression system, and protein is purified, so that the alveolar echinococcosis subunit vaccine is obtained. The alveolar echinococcosis subunit vaccine is capable of inducing a body to generate alveolar echinococcosis targeted T cell and B cell immune response and high-titer specific antibody humoral immune response, and the subunit vaccine can be used for preventing and treating alveolar echinococcosis infection related diseases.

The invention discloses a novel non-ribosomal peptide synthetase gene named NRPS114. The nucleotide sequence of the non-ribosomal peptide synthetase gene is shown in SEQ ID NO 1. Besides, the invention further discloses a preparation method of the non-ribosomal peptide synthetase gene, a plasmid pCC1FOS<TM> Fosmid-NRPS114 containing the non-ribosomal peptide synthetase gene and an expression vector of a recombinant plasmid pET28a-A-2his of five adenylylation structural domains in the non-ribosomal peptide synthetase gene. Additionally, the invention further discloses the five adenylylation structural domains and a preparation method thereof. Recombinant adenylylation structural domains are obtained by means of over-expression of the adenylylation structural domains A1, A2, A3, A4 and A5 in an escherichia coli prokaryotic expression system, and the non-ribosomal peptide synthetase gene has high activity enabling amino acid to undergo adenylylation, provides more genetic manipulation materials for study of combinatorial biology and has a huge application prospect in research and development of new drugs.

The invention provides cloning of chicken CR2 gene, expression and purification of protein as well as preparation of a polyclonal antibody of protein. An amino acid sequence and a gene sequence of ChCR2 protein are provided, a ChCR2 prokaryotic expression vector containing different labels is constructed, an Escherichia coli prokaryotic expression system is used for expressing ChCR2 protein, and the protein is purified. The purified protein can prepare the polyclonal antibody with higher titer. The ChCR2 gene plays important roles in a chicken immune system. CR2 can link congenital complementmedicated immune response with pathogens and foreign antigens and combine with C3d covalently attached to a target to produce adaptive immune response, and caused cell signaling phenomenon can greatlyreduce threshold of B cell activation. B cells are important in the immune system, so that the invention finds that ChCR2 is of great significance in both fundamental research and clinical application.

The present invention relates to gene engineering, and is especially magnesium ion regulated new expression vector. The magnesium ion regulated new expression vector pYS contains the promoter sequence Pmgt of magnesium ion transport protein gene mgtC / B. The expression vector of the present invention is controlled by Mg2+ concentration in the culture medium and has no need of adding exogenous inducer. The present invention provides new way for the improvement and development of colibacillus pronucleus expression system.

The invention relates to a method for efficiently synthesizing L-theanine by using gamma-glutamyl transpeptidase produced by Escherichia coli. The method comprises the following steps of: a) filtering a raw fermentation liquor of Escherichia coli by using an ultrafiltration membrane with the aperture of 0.001-0.01um; b) adding an ethylamine solution into an enzyme and bacterium trapped fluid to regulate the pH to be 10-11, and standing for 2 to 3 hours to obtain an enzyme concentrated solution; and c) adding the enzyme concentrated solution obtained in the step b) into an aqueous solution containing L-glutamine to obtain an enzyme reaction system, adding an ethylamine solution into the enzyme reaction system to control the pH of the enzyme reaction system to be 9-10.5, and controlling the enzymatic conversion temperature to be 30-35DEG C. The method is easy to implement and low in time and energy consumption, avoids the introduction of impurities and the deactivation of gamma-glutamyl transpeptidase, avoids the corrosion of a reactor due to the introduction of hydrochloric acid, improves the yield and purity of the L-theanine and reduces cost.