159 results about "EcoRI" patented technology

Disclosed are DNA segments encoding <DEL-S DATE="20010821" ID="DEL-S-00001">hyaluronic acid synthase which are employed to construct recombinant cells useful in the production of hyaluronate synthase or hyaluronic acid (HA)<DEL-E ID="DEL-S-00001"> <INS-S DATE="20010821" ID="INS-S-00001">the recombinant DNA segment identified in FIG. 5. <INS-E ID="INS-S-00001">In preferred aspects, chromosomal DNA from Streptococcus equisimilis is partially digested with EcoRI and the resultant fragments are ligated to form recombinant vectors. These vectors are useful in the transformation of host cells such as E. coli and or Streptococcal hosts. <DEL-S DATE="20010821" ID="DEL-S-00002">Resultant transformants are screened by the novel screening assays to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies may be selected and employed in the production of the enzyme itself or its product, HA.<DEL-E ID="DEL-S-00002"> <INS-S DATE="20010821" ID="INS-S-00002">The recombinant DNA segment identified in FIG. 5 is then inserted into a recombinant Streptococcal host for the production of hyaluronic acid (HA).<INS-E ID="INS-S-00002">

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.

Methods for determining methylation profiles of single polynucleotide molecules, especially genomic DNA molecules, by using sequence specificity and methylation sensitivity of restriction endonucleases and other proteins that selectively bind to methylated or unmethylated residues.

The invention belongs to the technical field of genetic engineering, and provides a method for acquiring chicken whole genome high-density SNP marker sites. The method comprises the following steps: (1) predicting distribution of restriction fragments acquired from double-restriction chicken genomes of EcoRI and MseI; (2) designing a universal joint, a barcode joint and a PCR amplification primer according to the distribution characteristics of the restriction fragments of EcoRI and MseI; (3) constructing a simplified genome sequencing library; (4) sequencing by virtue of the library constructed in the step (3) through a computer; and (5) according to a sequencing result, acquiring SNP marker sites. The method disclosed by the invention provides a universal strategy for the construction of a whole genome high-density SNP map by virtue of double-restriction GBS for different varieties of chickens, so that the cost on acquiring every SNP marker site is reduced by an order of magnitude compared with a conventional chip technology; and the method is stable in technology and high in repeatability.

The invention relates to a method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of restriction endonuclease digest of DNA, adaptor-ligation, optional pre-amplification, selective amplification, pooling of the amplified products, sequencing the libraries with sufficient redundancy, clustering followed by identification of the genetic markers within the library and/or between libraries and determination of (co-) dominant genotypes of the genetic markers.

The invention relates to a preparation method of HRPII protein monoclonal antibody of plasmodium falciparum. The preparation method comprises the following steps of: adopting HRPII protein of plasmodium falciparum as target antigen and respectively analyzing and selecting two dominant antigen epitopes of A and B; respectively repeating the two dominant antigen epitopes of A and B, then continuously connecting four glycine and forming recombinant protein C; adopting most securest code of escherichia coli and converting the amino acid sequence of the recombinant protein C into corresponding nucleotide sequence; carrying out chemical synthesis to the former step to obtain the nucleotide sequence, and respectively adding enzyme cutting sites BamHI and EcoRI at the upstream and downstream thereof; inserting nucleotide fragment obtained by the former step into expression carrier PET-28a(+), constructing recombinant protein C expression carrier and inducing to express the recombinant proteinC in the escherichia coli BL21 (DE3); carrying out ultrasonic bacteria breaking and low-temperature centrifugation, then taking supernatant of the solution, affining a chromatographic column by nickel-agarose, eluting and obtaining purified recombinant protein C; after immunizing Balb/c mouse with the recombinant protein C for a plurality of times, taking and fusing spleen cells with sp2/0 myelomacells, and obtaining six hybridoma cell lines by multiple rounds of screening; and purifying monoclonal antibody, respectively marking horse radish peroxidase and prorating matching and combination of optimum monoclonal antibody by ELISA orthogonal experiment.

The invention discloses a colony, expression and appliance of lactic acid bacteria aminoglutaric acid decarboxylase gene in food industry biotechnics domain, which is characterized by the following: coming from spittle streptococcus thermophilic subspecies (Streptococcus thermophilus) with length at 1380bp; augmenting from gene group DNA through PCR; adding NcoI and EcoRI on the two ends of the gene; limiting enzyme identification sequence; connecting to pET-DsbA of same limited enzyme alimentary; transforming expressing host bacteria BL21(DE3)pLysS of bacillus coli; realizing retooling expression in bacillus coli; getting glutamic acid decarboxylase molecule with molecular weight at 52. 4kDa; transforming L- glutamic acid decarboxylase to gamma-aminobutyric acid with retooling enzyme; providing a great amount of and high active rough enzyme for enzymatical synthesis of gamma-aminobutyric acid; decreasing cost of enzymatical synthesis of gamma-aminobutyric acid. This method possesses warm condition, which belongs to biological synthesis method.

The invention discloses a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for distinguishing a goose parvovirus from a muscovy duck parvovirus. According to the PCR-RFLP method, NS gene sequence digestion site difference is used for distinguishing the goose parvovirus from the muscovy duck parvovirus. The method comprises the following steps of: extracting DNA (deoxyribonucleic acid), carrying out PCR amplification to obtain an NS gene segment, and carrying out RFLP analysis after EcoRI digestion is carried out. The PCR-RFLP method for distinguishing the goose parvovirus from the muscovy duck parvovirus is simple and high in efficiency and accuracy.

The invention provides recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase as well as a construction method and application thereof and belongs to the technical field of genetic engineering. The invention provides a recombinant plasmid pMD-GDH containing FAD-dependent glucose dehydrogenase genes. After the FAD-dependent glucose dehydrogenase genes are subjected to codon preference adjustment, EcoRI restriction sites and NotI restriction sites are added at two ends of the sequence, and six histidine tags are added at the 3' end. The recombinant pichiapastoris takes Pichia pastoris as an expression host and comprises the recombinant plasmid. The invention further provides application of the recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase in fermentation production of the FAD-dependent glucose dehydrogenase. The embodiments show that when the recombinant pichiapastoris is subjected to induction culture for 136 hours during culture in a 10L of fermentation tank, the enzyme activity reaches 257600U/L and is 5.3 times that of the enzyme activity reported in the prior art.

The present invention relates to a streptococcus antigen C5a and a preparation method thereof. The streptococcus protective antigen C5a is an SEZC5a recombinant protein, which consists of 571 amino acids and has a molecular weight of 60.3kDa; a forward primer has one BamHI restriction enzyme cutting site, and a reverse primer has one EcoRI restriction enzyme cutting site. According to the preparation method of the streptococcus protective antigen C5a, the SEZ C5a recombinant protein is treated with cloning, expression and purification; and a series of biological engineering technologies and experiments on mice are applied to conduct system analysis on an rSCPZ. After vaccination, the rSCPZ can provide high protective efficacy; an anti-rSCPZ mice double-immunized serum has significant passive immune protection on mice; and the mice immunized by the rSCPZ show high level of antibody titer in serum. The anti-rSCPZ antibody can induce high level of bactericidal capability; an scpZ gene has a transcription level in SEZ (Streptococcus zooepidemicus) infected mice higher than that of culture in vitro; and the rSCPZ can adhere to hep-2 cells and inhibit cell infection ability of SEZ.

The invention relates to an electrochemiluminescent biosensor for detection of endonuclease, preparation and application thereof. The preparation steps include: S01. pretreating a glassy carbon electrode; S02. preparing a gold nano-graphene modified glassy carbon electrode by means of cyclic voltammetry; S03. respectively preparing an Ru(bpy)3<2+> labeled B-DNA beacon with a concentration of 1-10microM and a ferrocene labeled C-DNA beacon with a concentration of 1-10microM; S04. immersing the gold nano-graphene modified glassy carbon electrode obtained in S02 into mercapto modified A-DNA to conduct cultivation for 3-6h, and then conducting immersion in the Ru(bpy)3<2+> labeled B-DNA beacon and the ferrocene labeled C-DNA beacon obtained in S03 in order for 20-40min. The electrochemiluminescent biosensor provided by the invention can be used for detecting endonucleases EcoRI and ApaI. The sensor provided by the invention has good stability and reproducibility and high specificity to endonucleases EcoRI and ApaI, is hardly interfered by other substances in a detection sample, has very strong sensitivity, and is simple, convenient and quick to operate during detection, thus having certain help to research and development of antibacterial and antiviral drugs.

The invention discloses a method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2. The invention provides a DNA molecule having a nucleotide sequence 1 shown in the sequence table. The invention also provides a recombinant vector, a transgenic cell line or recombinant bacteria containing the DNA molecule. The recombinant vector is prepared by inserting the DNA molecule between recognition sites HindIII and EcoRI of a shuttle plasmid. An experiment proves that according to a plasmid incompatibility principle, an incompatiple plasmid is constructed and a strain A16QT which does not contain virulence megaplasmids is obtained. The method has the characteristics of simpleness, speediness, good singularity and high safety. The method has a very important meaning for construction of a plasmid-less bacillus anthracis strain and a research on the interaction between chromosome and megaplasmids pXO1 and pXO2, provides a novel experimental means for construction of a novel vaccine, and provides a novel idea for preventing and controlling bacillus anthracis.

The invention discloses a method for high efficient preparation of novel Mytilus coruscus antimicrobial peptide Mytichitin-A in pichia pastoris. A gene engineering method is adopted and the full-length gene of the antimicrobial peptide Mytichitin-A is inserted into an exosome type expression vector pPICZalphaA through EcoRI and KpnI restriction enzyme cutting sites, so as to obtain a pPICZalphaA-Mytichitin-A recombinant expression vector. Through an electrotransformation method, a SacI enzyme linearized recombinant plasmid pPICZalphaA-Mytichitin-A is transferred into the pichia pastoris GS115. Through optimizing, the optimum expression conditions are determined as follows: methanol concentration is 1.0%, fermentation time is 96h, and the yield is 45.5 microgram/mL. The result of bacteriostasis experiments shows that the recombinant antibacterial peptide has an obvious inhibition effect on both staphylococcus aureus and bacillus subtilis 151-1.

The invention relates to a vitreoscilla hemoglobin vgbS nucleotide sequence and a plasmid and a preparation method thereof, and belongs to the technical field of biological engineering. The vitreoscilla hemoglobin vgbS nucleotide sequence is prepared by introducing restriction enzyme cutting sites of NdeI and EcoRI at both ends of a vgb nucleotide sequence of streptomyces serving as a host cell respectively. In addition, the plasmid pJTU4406 is generated by cutting off the vgbS nucleotide sequence on pJTU4405 through the NdeI and the EcoRI, and inserting the vgbS nucleotide sequence into corresponding sites of a carrier pIB139. An amino acid sequence of protein expressed and generated by the vgbS nucleotide sequence is totally identical to that of the natural vitreoscilla hemoglobin, and the vitreoscilla hemoglobin vgbS nucleotide sequence can improve the utilization efficiency of the streptomyces to oxygen, promote the growth velocity of thalli and improve the yield of antibiotics.

The invention provides a replicating type recombinant adenovirus HAdV-5 carrier system. The system comprises a framework plasmid pKAd5, an intermediate plasmid pMDXE3YA and a shuttle plasmid pKNXE3M;a target gene coding region is cloned to multiple cloning sites of the shuttle plasmid, EcoRI digests the shuttle plasmid carrying a target gene, and an obtained fragment is used for replacing the corresponding part of the intermediate plasmid; then NdeI is used for digesting the newly-constructed intermediate plasmid, and an obtained fragment is used for replacing the corresponding part of the framework plasmid so as to obtain a recombinant adenovirus plasmid; and the PacI linearized recombinant adenovirus plasmid is used to transfect a 293 cell so as to prepare a recombinant HAdV-5 adenovirus with target gene expression controlled by an HAdV-5 major later promoter (MLP). according to the system, the recombinant adenovirus can be replicated and proliferated in various human cells, is a replicating type virus and is expected to have a wide application prospect in the research and development of animal oral carrier vaccines.

The invention provides a recombinant pichia pastoris for producing (+) gamma-lactamase as well as a construction method thereof, and belongs to the field of genetic engineering technology. The invention provides a recombinant plasmid containing (+) gamma-lactamase gene, EcoRI restriction site and NotI restriction site are added at two ends of the sequence of (+) gamma-lactamase gene at a 591 siteafter codon preference adjustment, and six histidine labels are added at the 3' end. The recombinant pichia pastoris takes louis Pasteur pichia pastoris as an expression host, and comprises the recombinant plasmid. The recombinant pichia pastoris for producing (+) gamma-lactamase is applied in fermentation production of (+) gamma-lactamase. Compared with a prokaryotic expression vector, the productivity of lactase of (+) gamma-lactamase adopting yeast as the expression vector is improved by 58%.

The invention relates to the field of genetic engineering and the field of protein secretion expression and provides a method for constructing a pichia pastoris engineered strain used for preparing dextranase. The method comprises the following steps: (1) designing an oligonucleotides primer and taking lipomyces 1390 genome DNA as a template; cloning to obtain dextranase genes; (2) utilizing a secretion expression carrier pPIC9K to insert the dextranase genes into multi-cloning sites (EcoRI and NotI) of the pPIC9K so as to construct an integral secretion-type expression carrier pPIC9K-139; and(3) linearizing the reconstructed expression carrier pPIC9K-139 so as to realize the high-efficiency secretion expression of the dextranase, and obtaining the reconstructed pichia pastoris engineeredstrain GS115-139 by means of selection. The method successfully realizing the effective expression of the dextranase genes in pichia pastoris, constructs the engineered strain which can efficiently secrete and express the dextranase, and realizes the expansion of a fermentation process of the dextranase.

The invention discloses a lentiviral expression vector pLOX-CMV-E/P and construction and application thereof. Multiple cloning site which is inserted into the downstream part of a CMV promoter of the vector can be recognized by 7 restriction endonucleases, including SpeI, BclI, EcoRI, Paci, XhoI, SalI and BamHI. The application of the vector comprises the following steps: inserting a target gene to the multiple cloning site to realize the stable expression of the target gene; using Puromycin resistance gene and EGFP which is driven by EF1alpha promoter to perform fluorescence tracing and drug resistance screening; based on LoxP site inserted in 3'LTR region and the transcription and reverse transcription characteristic of lentivirus, realizing effective deleting of an exogenous gene and a reporter gene under the condition of Cre recombinase expression. The vector disclosed by the invention can realize the package of the lentiviral particles by commonly transfecting 293T cell together with pCMVR8.74 and PMD2.G vector.

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD protein gene, a recombinant baculovirus and a preparation method and application thereof, and belongs to the technical field of veterinary vaccines. The recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene is prepared by inserting HBM signal peptide-gD-His tagor HBM signal peptide-gD-IgGFc-His tag as an exogenous gene into an enzyme cutting site between BamHI and EcoRI and an enzyme cutting site between SpeI and HindIII of the baculovirus transfervector respectively; and the gD is a gD protein gene deleted from a encoding transmembrane region fragment. The vector promotes the secretory expression of the porcine pseudorabies virus gD protein and the gD-IgGFc fusion protein. The subunit vaccine prepared by using the gD protein and the gD-IgGFc fusion protein has the advantages of good safety, strong specificity and high virus attack protection rate.

The invention discloses a bacterial strain of producing pyruvic acid and a construction method of the bacterial strain. The construction method comprises the following steps: cloning a D-amino acid oxidase gene DAAO (D Amino Acid Oxidase) into the pUC57 plasmids through EcoRI or BamHI to obtain a plasmid pUC57-DAAO; carrying out gene amplification by taking plasmids pUC57-DAAO as a template; cloning a product obtained by gene DAAO amplification to a plasmid pET28A or pET21a; selecting and culturing positive clone plasmids connected to a DNA segment containing DAAO from the plasmids; converting the constructed positive clone plasmids to an expression host bacterium BL21(DE3); and selecting and culturing to obtain a first bacterial strain which can convert D-alanine to pyruvic acid, wherein the fragment size of the DAAO gene is 1124bp, and the gene contains cleavage sites of endonuclease NdeI and EcoRI. The bacterial strain constructed by the construction method can directly convert D-alanine into pyruvic acid through a biotransfer approach.

The invention provides construction and application of ganoderma laccase pichia pastoris genetic engineering strain. The construction comprises the following steps: cloning a laccase gene GlLac from ganoderma; carrying out EcoRI and XbaI double-enzyme cleavage; cloning the double-enzyme cleavage product into a pichia pastoris expression plasmid to construct an expression vector; finally, electrically converting the expression vector into a pichia pastoris expression host strain; carrying out cultivation, amplification and screening to obtain a pichia pastoris engineering strain. According to the construction, the yeast secreting type co-expression vector pPICZ<Alpha>A-GlLac of ganoderma laccase (Lac) is constructed, and converted into pichia pastoris X33; recombinant of high Zeocin resistance is selected out to serve as a pichia pastoris engineering strain. The extracellular expression vector pPICZ<Alpha>A of pichia pastoris is a carrier for judging whether a multi-copy gene is inserted into a pichia pastoris genome or not, and can express high-copy genes.

The invention relates to a method for loose head cabbage molecular markers by utilizing the SAMPL technology comprising DNA extraction, DNA enzyme digestion-connection system optimization, SAMPL pre-amplification system optimization, SAMPL amplification system optimization, electrophoresis, silver staining color development and cluster analysis, wherein, a DNA sample is performed enzyme digestion through using EcoRI, MseI or PstI enzyme, and then corresponding joints of EcoRI,MseI or PstI are connected on the DNA sample through using T4-DAN ligase; the SAMPL pre-amplification is performed through using a 10mu L reaction system; a pre-amplification primer is corresponding to the chosen enzyme in the enzyme digestion; the SAMPL amplification is performed through using a 20mu L reaction system, one amplification primer is corresponding to the pre-amplification primer, and the other is an SAMPL primer. The method for loose head cabbage molecular markers by utilizing the SAMPL technology performs optimization and primer design for the complicated system of the SAMPL, constructs an SAMPL reaction system applicable to loose head cabbages and realizes loose head cabbage molecular markers.

The invention discloses an engineering bacterium for efficiently expressing recombinant ChIFN-alpha. The engineering bacterium is obtained by the following steps of S1, performing codon optimization synthesis on the ChIFN-alpha gene sequence published in Genebank according to the preference of the colibacillus codon; S2, designing a specific primer according to the optimized ChIFN-alpha gene; S3,performing PCR amplification on the ChIFN-alpha gene; cloning the PCR products onto prokaryotic expression vectors pET-41a through EcoRI and HindIII double enzyme digestion; S4, converting pET-41a-ChIFN-alpha recombinant plasmids with the correct sequencing into BL21(DE3) competent cells; S5, performing recombinant ChIFN-alphaprokaryotic expression and antiviral activity determination. The engineering bacterium disclosed by the invention can realize the efficient expression of the ChIFN-alpha; the antiviral activity of the obtained ChIFN-alpha can reach 1.71*10<7> UI/mg.