58 results about "Plasmid recombination" patented technology

The invention discloses a CRISPR / Cas9 system-containing targeted knockout vector and adenovirus and applications thereof. The targeted knockout vector is prepared through the following steps: after a pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid is subjected to enzyme digestion and filling-in by using EcoRI and SacII, connecting the pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid with a pAdTrack-CMV plasmid subjected to enzyme digestion and filling-in by using BstXI; after the obtained product is linearized by using BbsI, connecting the obtained product to a specific target sequence of a desired gene; and after the obtained object is linearized by using PmeI and dephosphorylated by using CIAP alkaline phosphatase, recombining the obtained product with a pAdEasy-1 plasmid. The targeted knockout vector can mutate gene sequences in target sequence areas, and the mutation rate is high, up to 30.6-45.8%, therefore, the targeted knockout vector can be used for gene site-directed mutation, and lays a foundation for gene therapy.

The invention provides aminopeptidase for catalytic synthesis of carnosine, and a preparation method and application thereof. The aminopeptidase has the amino acid sequence shown by SEQ ID NO:2. For the preparation of biological enzymes, firstly, a gene engineering strain of the biological enzymes is built; the biological enzyme gene fragment obtained through full gene synthesis is subjected to linearized plasmid recombination by using restriction enzymes SalI. The built gene-engineered strain is suitable for high-density culture; the exoenzyme is biologically synthesized; solid-liquid separation is performed; ultrafiltration concentration is performed to obtain enzyme liquid; histidine and beta-alanine can be catalyzed in normal-temperature and normal-pressure water solution to efficiently synthesize carnosine; the reaction environment is friendly; the production cost is low; the conversion time is short; the process operation is simple; the reaction system impurity content is low; the post treatment is easy; the wide prospects of large-scale industrial application are realized.

A plasmid, recombinant engineering bacteria and a preparation method of hyaluronic acid with uniform molecular weight belong to the technical field of gene engineering. A first expression plasmid containing a Pasteurella multocida hyaluronan synthase gene (SEQ NO.1 in the sequence table) and a second recombinant expression plasmid containing hyaluronic acid precursor synthesis related genes (SEQ NO.2, SEQ NO.3, SEQ NO.4 and SEQ NO.5 in the sequence table) cloned from Bacillus subtilis are introduced into gram positive safe microorganisms, and an expression regulation and control system consisting of the two recombinant expression plasmids respectively controls hyaluronan synthase and hyaluronic acid precursor synthesis related enzyme, so as to obtain hyaluronic acid with uniform molecular weight by a biosynthesis method. The method provided by the invention not only improves the capability of the host strains to synthesize hyaluronic acid, but also can obtain several varieties of sufficient pure hyaluronic acid with uniform molecular weight.

The invention provides fengycin, a fusion gene encoding lipopeptide compounds, recombinant plasmid, recombinant bacteria and applications, and belongs to the technical field of plant disease control.The fengycin is produced by Bacillus velezensis HN-Q-8; and the accession number of the Bacillus velezensis HN-Q-8 is CGMCC No. 19554. The Bacillus velezensis HN-Q-8 has efficient biocontrol effects.In the inhibitory substances produced by the Bacillus velezensis HN-Q-8, the fengycin plays a principal role of bacteriostasis. The fengycin has obvious inhibition effects on potato black scurf.

The invention discloses an application of a glycosyltransferase gene, namely an application of the glycosyltransferase gene in improving the synthesis of lactobacillus plantarum exopolysaccharides; wherein the glycosyltransferase gene is a gene orf1595 or a gene cps4I, the nucleotide sequence of the gene orf1595 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the gene cps4I is as shownin SEQ ID NO: 2; an orf1595 or cps4I gene and a temperature-sensitive plasmid are recombined to construct a knockout vector, the knockout vector is introduced into a food-borne lactobacillus plantarum competent cell, and then a gene knockout strain is constructed through homologous recombination; a phenol sulfuric acid method is adopted to determine the exopolysaccharide production capacity of the strain, it is found that the exopolysaccharide production capacity of the knocked-out strain is lower than that of a wild strain, orf1595 or cps4I genes play a key role in exopolysaccharide synthesis, and the method has huge potential in the fields of exopolysaccharide biosynthesis research and application.

The invention discloses a new application of a dihydropterin aldolase gene folB, namely an application of the dihydropterin aldolase gene folB in improvement of folic acid synthesis of lactobacillus plantarum, and the nucleotide sequence of the dihydropterin aldolase gene folB is shown as SEQ ID NO: 1; the method comprises the following steps: recombining a folB gene and a pMG36e plasmid to construct an overexpression vector, and introducing the overexpression vector into a food-borne lactobacillus plantarum competent cell to obtain a folB gene overexpression strain BDB; an LC-MS method is adopted to measure the folic acid producing capacity of the strain, it is found that compared with a wild type strain, the folic acid producing amount of the BDB strain is remarkably increased, the folB gene plays a key role in folic acid synthesis, and the method has huge potential in the folic acid biosynthesis research and application field.

The invention relates to the field of microorganisms, in particular to recombinant strains, construction methods and applications thereof. The invention protects the engineered strain obtained by introducing the target gene into Corynebacterium or Brevibacterium by means of recombinant plasmid or insertion into chromosome. The result of the experiment of producing L-valine by shaking flask fermentation showed that the L-valine yield of the starting strain CGMCC 1.299 was 3.2g / L, while the L-valine yield of the engineering strain MHZ-1011 of the present invention was 6.5g / L. L, the yield was 103% higher than that of the starting strain, with a very significant difference (P<0.01). The experimental results of L-valine production by 50L tank fermentation show that: the L-valine yield of the starting strain CGMCC1.299 is 7.3g / L, while the L-valine yield of the engineering bacterium MHZ-1011 of the present invention is 18.4g / L, the yield was 152% higher than that of the starting strain, with a very significant difference (P<0.01).

The invention discloses recombinant escherichia coli for producing pentamethylene diamine and application of the recombinant escherichia coli. A ribulose diphosphate carboxylase gene in rhodospirillum, a ribose phosphate kinase gene and a lysine decarboxylase gene in spinach are synthesized from the beginning, PCR copy an escherichia coli molecular chaperone gene to construct a plasmid pTrc99a-cadA-cbbM and a plasmid pCWJ-prkA-GroEL / GroES, the double-plasmid recombinant bacteria grow well in a specific culture medium, and glucose is efficiently utilized. According to the recombinant escherichia coli, inducers arabinose and IPTG are respectively added in different time periods, the heterologous gene expression is stable, the reaction system is simple, the condition is mild, the period is short, few byproducts are produced, the method is clean and pollution-free, the method is a simple, rapid and efficient production way, and the fermentation result shows that the yield of the recombinant escherichia coli KACCPG is obviously improved.

The invention discloses an alginate lyase SHA-6 gene with a nucleotide sequence as shown in SEQ ID NO:1; the alginate lyase SHA-6 gene is recombined with pET-32a(+) plasmids for being converted into escherichia coli BL21 to obtain engineering bacteria of alginate lyase SHA-6; the bacterial strain is induced to express to generate a great number of inclusion body proteins SHA-6; the alginate lyaseSHA-6 inclusion body is subjected to protein purifying and renaturation, so that the inclusion body recovers activity; the re-natured alginate lyase has wide substrate specificity, polymannose aldehyde acid PolyM and polygulose aldehyde acid PolyG can be utilized as a substrate, and enzyme activity reaches 13.5U / mg, so that the alginate lyase SHA-6 gene is alginate lyase with a wide application prospect.

The invention relates to the field of wound repair materials, and provides a preparation method of a wound repair material with a cell growth regulation and control function, which comprises the following steps: S1, adding a high-molecular polymer and nano bioactive glass powder into a solvent to prepare an electrostatic spinning precursor solution; S2, sucking the electrostatic spinning precursorsolution prepared in the step S1 into an injector to carry out an electrostatic spinning process, and carrying out vacuum drying to obtain a polymer fiber film compounded with nano bioactive glass; S3, extracting a cell regulatory factor targeting gene segment, constructing a target gene plasmid, obtaining a plasmid DNA recombinant by using a gene recombination technology, and adding the plasmidDNA recombinant into the chitosan solution and the trehalose solution to obtain a mixed solution; and S4, coating the polymer fiber film obtained in the step S2 with the mixed solution obtained in thestep S3 to obtain the polymer fiber film. The wound repair material prepared by the invention has better histocompatibility, tensile strength, biological safety, antibacterial property and repair capability.

The invention discloses a preparation method and application of recombinant human leucocyte interleukin 27. The preparation method comprises the following steps: amplifying EBI3 segment and P28 segment of human leucocyte interleukin 27, and cloning onto a vector pFASTbac-dual; recombining the obtained vector transformation Escherichia coli DH10Bac and a Bacmid plasmid into a recombinant transposition plasmid rBacmid, transfecting rBacmid into sf9 insect cell under the mediation of liposome to obtain recombinant baculovirus; infecting the recombinant baculovirus into the sf9 insect cell; and carrying out sf9 insect cell culture, and purifying the supernatant through a nickel affinity chromatography column and a gel chromatography column to obtain the recombinant human leucocyte interleukin 27. The human leucocyte interleukin 27 has the function of resisting hepatitis B virus infection, and can be used for preparing medicines for resisting hepatitis B virus infection. The recombinant human leucocyte interleukin 27 obtained by the method is more approximate to a natural state, and has high bioactivity; and the method has the advantages of low production cost and high yield.

The invention relates to an avian influenza vaccine and a preparation method thereof, in particular to the preparation of a recombinant plasmid and a recombinant virus in the avian influenza vaccine preparation. The invention provides a recombinant gene and a preparation method thereof, and the recombinant gene is formed by fusing a baculovirus envelope protein gp64 signal peptide gene sequence, a major antigen gene HA sequence of an avian influenza virus and a baculovirus envelope protein gp64 transmembrane gene sequence. The invention further provides a recombinant plasmid that is constructed by recombining the recombinant gene and a pBacPAK8 plasmid, the recombinant bombyx mori baculovirus BmHA that contains the recombinant gene and a virus preparation method. The recombinant bombyx mori baculovirus BmHA that contains the recombinant gene is preserved in China General Microbiological Culture Center of Microbial Culture Collection Management Committee with the preservation number of CGMCC No.2316. The invention further provides an expression protein of the recombinant gene. The recombinant bombyx mori baculovirus BmHA and the protein that are provided by the invention can be used for preparing the avian influenza vaccine. The methods of the invention are applicable to large-scale production, and have lowered cost, high yield and large application value of the produced avian influenza vaccine.

The invention relates to a recombinant plasmid DNA vaccine composition for treating Hepatitis B. The composition contains a recombinant vaccine plasmid which simultaneously carries Hepatitis B middle protein S2S coding gene and Hepatitis B Core protein coding gene as well as a recombinant adjuvant plasmid, wherein the adjuvant plasmid is the adjuvant plasmid of recombinant interleukin 2 or recombinant human interferon gamma.

The invention relates to the technical field of biology, and especially relates to recombinant plasmids, recombinant adenovirus plasmids, and applications of recombinant adenovirus. The invention provides obtained escherichia coli (preservation number CCTCC No.2013212) containing a double-gene co-expressed homologous recombinant plasmid pAd.Easy-1(RGD)-pAdTrack-CMV-ING4-polyA-promoter-PTEN and recombinant adenovirus Ad.(RGD)--IG4-PTEN, and the escherichia coli and recombinant adenovirus can be used for preparation of drugs for treating malignant tumor and / or leukemia and chemotherapy sensitizers.

The present invention features that the tobacco mRNA is first extracted and the tobacco mRNA library is established, and MCK1 (Maize calmodulin kinase gene) is then utilized as the probe for screening, and from the screened positive cloning sequencing the whole gene sequence is obtained. After cloning gene to FfastHTb donor plasmid, recombining plastocont DNA to convert Sf9 cell, collecting recombinant virus and fusing virus infected cell, the cell is collected after 72 hr and the fusion protein is purified. The transgenic plant exhibits later blossoming characteristic and longer growth period, and tobacco calmodulin dependent protein kinase gene may be used in tobacco.

The invention relates to the technical field of interferons and particularly relates to a preparation method of a recombinant human interferon alpha2b. The preparation method comprises the following steps: (1) constructing an expression vector of a recombinant human interferon alpha2b gene, and converting the expression vector into a competent cell; (2) constructing a recombinant plasmid of an escherichia coli disulfide bond isomerase C gene; (3) converting the recombinant plasmid into the competent cell, so as to obtain a double-plasmid recombinant expression strain; and (4) carrying out induced expression, renaturation and purification on the double-plasmid recombinant expression strain, so as to obtain the recombinant human interferon alpha2b. According to the preparation method of the recombinant human interferon alpha2b, the correct pairing of disulfide bonds cys1-cys98, so that a slow carrying component of the recombinant human interferon alpha2b is reduced, and furthermore, the stability and antiviral activity of the recombinant human interferon alpha2b are improved.

The invention relates to the technical field of biology, and aims at providing a method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells. The method comprisesthe steps of 1, extracting a target gene from glutamic acid decarboxylase, conducting PCR amplification on the target gene, and connecting the amplified target genes to plasmids for recombination to construct a cloning vector; 2, conducting enzyme digestion treatment on the cloning vector through a restriction enzyme to obtain target gene fragments, and connecting the target gene fragments into acerevisiae vector to generate recombined plasmids; 3, guiding the recombined plasmids into the saccharomyces cerevisiae cells through a lithium acetate quick-conversion method to obtain recombined saccharomyces cerevisiae cells; 4, culturing the recombined saccharomyces cerevisiae cells in a Na2HPO4 citrate buffer solution, and mixing L-glutamic acid and 5'-pyridoxal phosphate for a reaction to obtain the gamma-aminobutyric acid. The invention further provides the gamma-aminobutyric acid. The production speed of the saccharomyces cerevisiae cells is high, culture is simple, an exogenous gene expression system is complete, and the method is high in catalysis efficiency, low in cost and easy to control.

The invention provides an islet-targeted protein and a eukaryotic expression method thereof. The method comprises the following steps of (1) constructing plasmids containing a protein coding gene IGRP206-214-dtSCT; (2) recombining the plasmids into a chromosome of saccharomyces pastorianus; and (3) obtaining a target protein through methanol fermentation induction. A soluble IGRP206-214MHC compound is prepared through an eukaryotic expression system. Compared with the previously prepared protein for prokaryotic expression, the islet-targeted protein has galactosylated modification, and is closer to an in-vivo protein crystal structure, and the recognition rate of specific CTL on the protein and the bonding stability of TCR and an MHC molecule are improved, the endocytosis amount of viruses coupled to the protein is increased, the efficacy of the recombinant protein is improved and the recombinant protein is more stable in an external environment after galactosylated modification, and the storage period is prolonged.

The invention provides an expression and purification method of natural coronavirus main protease. The expression and purification method comprises the following steps of carrying out plasmid recombination, induced protein expression and purification on the natural main protease to generate fusion protein, and carrying out purification, enzyme digestion and separation on the fusion protein to obtain the natural main protease without redundant residues at the N-terminal and the C-terminal. By adopting the method, the problems of tedious experimental steps and low yield in the existing traditional method can be solved.

The invention discloses a mannase gene VbMan26A, a recombinant plasmid, a recombinant strain, mannase and application thereof. The mannase gene VbMan26A is used for encoding the mannase, the nucleotide sequence of the mannase gene VbMan26A is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded mannase is shown as SEQ ID NO: 2. The mannase gene VbMan26A provided by the invention has the advantages that the mannase encoded by the mannase gene VbMan26A has high activity under low-temperature and alkaline conditions, mannan can be effectively hydrolyzed into mannan oligosaccharide with 2-3 residues, and the mannase gene VbMan26A can be applicable to preparation of probiotic mannan oligosaccharide and can also be used for preparing washing products.

The invention provides a recombinant vector pET32a-fhlA assisting hydrogen production, a recombinant strain and its construction method and application, belonging to the technical field of genetic engineering. The recombinant vector assisting hydrogen production includes an initial vector and an fhlA gene, and the initial The vector is pET32a vector; the recombinant strain includes the recombinant vector assisting hydrogen production. The recombinant strain can use sodium formate as a substrate to produce hydrogen, and when the final concentration of sodium formate is 60mmol / L, the amount of hydrogen produced is the highest, reaching 9.8mmol H 2 / L / h, the hydrogen production capacity is significantly higher than that of wild bacteria and empty plasmid recombinant bacteria, which can improve the hydrogen production efficiency.

The invention discloses a method for synthesizing p-hydroxymandelic acid, which belongs to the technical field of bioengineering. The present invention constructs a dual-plasmid recombinant bacterium by means of molecular biology, co-expresses pyruvate decarboxylase, phenylacetaldehyde dehydrogenase and NADH oxidase genes, and introduces the constructed expression plasmid into E. coli BL21 (DE3). Convert p-hydroxybenzaldehyde and glyoxylate to p-hydroxymandelic acid, and through the NADH oxidase coenzyme regeneration system, by converting NADH to NAD + , making NAD + Recycling, conversion can be carried out efficiently. And by optimizing the substrate concentration and ratio, optimizing the conversion temperature and pH, the efficient production of p-hydroxymandelic acid was realized. Under the conditions of 30° C. and pH 7 for 24 hours, 30 mM of the substrate p-hydroxybenzaldehyde can be converted into 28.5 mM of p-hydroxymandelic acid, and the conversion rate can reach 95%.

The invention discloses a recombinant plasmid for constitutive expression of an aspergillus niger surface display lipase and application of the recombinant plasmid. An aspergillus niger surface display carrier pCALB-C serves as a departure plasmid, a constitutive promoter PgpdA substitutes for an inducible glucoamylase promoter Pgla, and accordingly, the recombinant plasmid pGpdA-CALB-CwpA is obtained. The recombinant plasmid transforms escherichia coli to obtain escherichia coli DH5alpha with the plasmid pGpdA-CALB-CwpA; the recombinant plasmid transforms an aspergillus niger protoplast to obtain an engineering bacterium AN-GpdA-CALB-CwpA displaying the lipase CALB on the surface of aspergillus niger; after fermented cultivation of the engineering bacterium, mycelia are collected centrifugally, refrigerated and dried to obtain an aspergillus niger surface display CALB whole-cell catalyst which can be used for efficient compounding of glyceryl phosphatidylcholine.

The invention discloses an alginate lyase SHA-6 gene with a nucleotide sequence as shown in SEQ ID NO:1; the alginate lyase SHA-6 gene is recombined with pET-32a(+) plasmids for being converted into escherichia coli BL21 to obtain engineering bacteria of alginate lyase SHA-6; the bacterial strain is induced to express to generate a great number of inclusion body proteins SHA-6; the alginate lyaseSHA-6 inclusion body is subjected to protein purifying and renaturation, so that the inclusion body recovers activity; the re-natured alginate lyase has wide substrate specificity, polymannose aldehyde acid PolyM and polygulose aldehyde acid PolyG can be utilized as a substrate, and enzyme activity reaches 13.5U / mg, so that the alginate lyase SHA-6 gene is alginate lyase with a wide application prospect.

Provided herein are improved copy number plasmids, particularly those plasmids capable of replication in a bacterial cell. The improved copy number plasmid contain a deletion, insertion, or substitution in the replication control region, particularly a Pseudomonas-specific replication control region, that results in an increase in plasmid copy number in comparison to a control plasmid. Also provided are host cells containing the improved copy number plasmids, as well as methods of using the improved copy number plasmids for the recombinant production of a protein of interest. Further provided are methods for generating plasmids with improved copy number. The methods disclosed herein involve the reiterative selection of improved copy number plasmids by the growth and selection of plasmids capable of growth under increasing selective pressure, wherein the selective pressure is applied utilizing a selection agent to which the control plasmid confers resistance.

The invention relates to a Seneca Valley virus recombinant plasmid, a recombinant virus and a construction method, belonging to the technical field of virus construction. The Seneca Valley recombinant virus provided by the present invention is: the 177th amino acid of the GH loop of capsid VP2 is mutated from S to A. The Seneca Valley recombinant virus provided by the invention has high virus titer and virus proliferation ability.

The invention discloses a plasmid recombinant listeria monocytogenes. A green fluorescent protein (GFP) gene is cloned on a plasmid carried by the plasmid recombinant listeria monocytogenes. The invention also discloses a construction method and application of the plasmid recombinant listeria monocytogenes. The plasmid recombinant listeria monocytogenes disclosed by the invention can be used as an internal standard bacterium for extracting bacterial plasmid group DNA (Deoxyribonucleic Acid) from animal waste. And the method for detecting the DNA extraction rate of the bacterial plasmid group in the excrement as an internal standard bacterium has specificity.

The present invention relates to a product expressed by a housefly embryonic cell construction antibacterial peptide attacin eukaryotic expression system and a construction method, housefly antibacterial peptide attacin is used as a target gene, constructed housefly embryonic cell line MDEC-07114 is transfected by recombinant baculovirus Bacmid-pFastBacTM-attacin, and an antibacterial peptide attacin housefly embryonic cell eukaryotic expression system is constructed. The housefly antimicrobial peptide attacin is obtained by complete gene synthesis, the target gene attacin is cloned into PUC57plasmid, after double enzyme digestion, recombinant plasmid is connected with pFastBacTMHTA donor plasmid for recombination of baculovirus Bacmid DNA; the recombinant baculovirus Bacmid DNA is transfected into housefly embryonic cell MDEC-07114, culture supernatant and cell lysates crude protein are extracted, and purified by a nickel column, SDS-PAGE and Western-Blot are used for detection and identification of protein expression, and the antimicrobial activity of the expressed product can be detected.