116 results about "HindIII" patented technology

The invention provides a method for preparing a chicken genetic engineering immunopotentiator by an akirin2 gene sequence, relating to a method for preparing a chicken genetic engineering immunopotentiator. The invention solves the problem that the existing genetic engineering immunoenhancement nucleic acid vaccines can not extensively improve the immunity effects of vaccines and bacterins. The method comprises the following steps of: acquiring a chicken akirin2 gene coded sequence; acquiring a recombinant pMD18-T vector containing the chicken akirin2 gene; designing a primer and enabling the 5'- terminals to have HindIII and EcoRI restriction sites, subcloning the chicken akirin2 gene in the recombinant pMD18-T vector into an expression vector to construct a recombinant expression vector; and diluting the recombinant expression vector by a sterile PBS (phosphate buffer solution). The chicken genetic engineering immunopotentiator provided by the invention can improve the expression levels of multiple immunity related factors such as IL-1, IL-6, toll-like receptors and the like, and has the characteristics of wide applicable range and the like for various vaccines and bacterins.

The invention belongs to the technical field of the livestock genetic engineering, and particularly relates to the molecular mark used as auxiliary choice of pig marks and relating to immunity and reproductive trait and application thereof. The molecular mark is obtained from ISG15 gene clone, and the cDNA sequence thereof is described in a sequence list SEQ ID NO: 1. A base mutation of A390-G390 exists in a 390bp place of the sequence list SEQ ID NO: 1, thereby resulting in the HindIII-RFLP polymorphism. The invention also discloses the primers used by the HindIII-RFLP polymorphism and a polymorphism detection method, and provides a new molecular mark for the auxiliary choice of pig marks.

The invention discloses a building method of a silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system, comprising: taking silkworm wild type BmNPV genome DNA as the template; respectively taking P10-upF/P10-upB and P10-downF/P10-down B as a primer PCR for amplification to obtain p10-up and p10-down; after processed, inserting into BamHI-PstI-HindIII locus in pUC 19 to obtain recombinant plasmid pUC19-p10-up-down; using pUC-19-p10-up-polh-down, liposome lipofectin and MilliQ H2O to prepare DNA-Lipofectin mixed liquor; adding the mixed liquor into BmN cells for cultivating; collecting transfection cell supernatant; inoculating BmN cells, and recovering a polyhedral body; extracting virus DNA from the polyhedral body and electrically converting DH10 beta competent cells; screening locus ceruleus and cultivating; after cultivating PCR positive bacterial plaque, extracting macro-molecular DNA to transfect to the BmN cells; separating to obtain helper plasmids from DH10Bac culture bacteria of AcMNPV Bac-to-Bac; converting the helper plasmids into E.coli DH10 beta containg Ploh+BmBacmid; and screening the DH10 beta bacterial strain of the helper plasmid containg Ploh+BmBacmid. The invention can produce recombinant virus capable of infecting by eating with mouth, and recombinant virus does not need to infect by intracutaneous inoculation, thus improving the production efficiency of the silkworm rhabdovirus expression system.

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 present invention provides an obesity gene susceptivity and pre-warning detection kit, which is characterized in that a plurality of nucleic acid (DNA and RNA) molecule markers are combined, wherein the nucleic acid (DNA and RNA) molecule markers comprise APMI (+45), FTO (rs9939609), FTO (rs8050136), GNB3 (825C/T), LPL (HindIII), MC4R (rs17782313), PPARgamma2 (Pro12Ala), UCP2 (Ala55Val), and beta3-AR (Trp/Arg). The kit comprises: specific amplification primer pairs and DNA sequencing primer pairs of the gene polymorphism loci, PCR reaction regent components, PCR product enzymolysis regent components and DNA sequencing reaction regent components. According to the present invention, detection is performed by using the kit, and data processing and statistical analysis are performed on the related gene information, such that obesity susceptivity of a subject can be evaluated.

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 belongs to a molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim. The method can be used for monitoring drug resistance of Fusarium graminearum to carbendazim and prevalence warning, wherein Fusarium graminearum can cause wheat scab. The detection method includes the following three main steps: (1) nuclear genome DNA of bacterial strain to be detected is respectively extracted: (2) internal and external primer pairs are utilized to carry out nested PCR, thus obtaining a target segment; (3) the target segment is respectively restricted by restriction enzymes HindIII and TaaI (Tsp4CI), and genotype of medium resistance level bacterial strain can be accurately identified by virtue of a restriction product electrophoresis spectrum. By adopting PIRA-PCR (primer-introduced restriction analysis PCR) technology, quantity of Fusarium graminearum with medium drug resistance in field and ratio thereof in group can be rapidly and accurately detected. Detection accuracy reaches more than 95%.

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.

The invention discloses a construction approach for bacillus coli of transpolyphosphokinase gene, in which the said method includes gene-clone and carrier-construction; the clone includes extracting master DNA of bacillus coli; designing primers; augmenting ppk gene; restructuring the augmented object gene into clonic carrier pMD18-T and converting bacillus coli DH5 alpha; the construction includes adopting restriction enzymes of BamHI and HindIII bisenzyme to cut pMD18-T plasmid with ppk object gene and idle expression carrier pET-28a(+); directionally connecting the object gene by T4 to the expression carrier pET-28a(+), then converting the DH5 alpha; by the PCR and bisenzyme pressure methods screening and verifying the positive recon; extracting the recombination plasmid Pet28a-PPK and converting the acceptor strain BL21(DE3). The invention is of simple process and the obtained gene has efficient phosphorous removal ability.

The invention relates to a method for expressing hABCG2 in an insect cell sf9. The method comprises the following steps: cloning a human ABCG2 gene with BamHI and HindIII enzyme digestions at two ends into BamHI and HindIII sites of a pFastBac1 carrier respectively; converting an obtained recombinant plasmid pFastbac1-hABCG2 into a DH10Bac escherichia coli competent cell to obtain a recombinant baculovirus shuttle vector Bacmid-hABCG2 which is inserted into the hABCG2 gene; mediating the recombinant baculovirus shuttle vector Bacmid-hABCG2 with lipidosome to transfect an sf9 insect cell to obtain hABCG2 recombinant virus, and further culturing to obtain the expressed hABCG2 protein. According to the method disclosed by the invention, a novel way is provided for obtaining hABCG2 proteins on a large scale, and a working foundation is laid for the application and development of the hABCG2 proteins.

The invention discloses a method for using silkworm cultured cell to express antibacterial peptide Cecropin B. The method comprises the following steps: (1) using the total RNA extracted from the fat body cells of wild silkworm chrysalis as template, adopting RT-PCR amplification to obtain wild silkworm antibacterial peptide Cecropin B gene; using 1% agarose gel electrophoresis to perform PCR product analysis to the antibacterial peptide Cecropin B gene, using a PCR purification kit of Qiagene for purification, then cloning the purified PCR product to TA vector pCR2.1 to obtain pCR2.1-Cecropin B, utilizing the dideoxynucleotide chain termination to confirm the correctness of cloned gene order; using restriction endonuclease BamHI and HindIII to digest pCR2.1-Cecropin B and obtain Cecropin B genetic fragments, then cloning rhabdovirus transfer vector pBlueBacHisa in the genetic fragments to obtain reconstituted transfer vector; performing cotransfection of the reconstituted transfer vector and silkworm wild-type nuclear polyhedrosis virus BmNPV DNA in silkworm cultured cell, performing homologous recombination to generate recombinant virus; and (3) inoculating the recombinant virus containing wild silkworm antibacterial peptide Cecropin B gene in the silkworm cultured cell, infecting at 27 DEG C for three days to express the infection, and centrifuging to collect silkworm cultured cell.

The present invention discloses method of preparing transgene calmodulin conjugated protein kinase gene and its application. The preparation process includes the following steps: A. restricting cloned calmodulin conjugated protein kinase gene with BamHI; B. connecting the gene in the step A to the intermediate vector pAHC17 to form pAHC17-R19(+); C. double restricting pAHC17-R19(+) with HindIII and EcoRI, and connecting to similarly restricted expression vector pCAMB1A1300 or pCAMB1A1301, to set calmodulin conjugated protein kinase gene between the ubiquitin promoter and NOS tail to form convertible or expressible vector; D. transferring the prepared vector into agrobacterium EHA105 and introducing mature embryo introduced rice callus; and E. screening positive plant. The calmodulin conjugated protein kinase gene is used in rice to produce transgenic plant with later florescence, longer growth period, and increased yield.

The invention provides a recombinant vector, a recombinant baculovirus prepared by from the vector and application of the virus in preparation of malaria vaccines. The recombinant vector is constructed by inserting a section of recombinant sequence into the pFastBacDual vector. The recombinant sequence is formed by: according to CMV, Ph, SP and TM sequences, adding EcoR I enzyme site and Xho I enzyme site between SP and TM, adding KpnI enzyme site in front of CMV-F, and adding HindIII enzyme site after TM-R. The recombinant baculovirus is obtained by: inserting a Plasmodium yoelii Pys48 antigen gene into the recombinant vector, conducting homologous recombination with the genome of a shuttle vector Bacmid through transposition, then transfecting a bombyx mori cell, and performing packaging in the bombyx mori cell. Through simple separation and purification of the recombinant baculovirus, an antibody with good titer can be prepared, thus providing a reference for study of P48 malaria vaccines.

HCMV glycoproteins B and H have been identified. The gB protein is encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary. The gH protein is encoded by DNA in the HindIII L fragment lying between 228 and 2456 bases from the L/D boundary. The genes have been incorporated in recombinant vaccinia vectors and expressed in host animals to raise HCMV-neutralizing antibody, thereby indicating vaccine potential. The glycoproteins can also be used in a variety of different ways, as vaccines or in the production, purification or detection of HCMV antibody.

The invention discloses a method of simultaneously performing enzyme-cut and link up co-system based on a CcdB lethal gene and a SmaI restriction enzyme cutting site. The method comprises the following steps of: inserting the ccdB gene between EcoRI and HindIII sites of a carrier pUC18 to obtain a pU18-ccdB plasmid; and then mixing the pU18-ccdB plasmid, a DNA fragment, a restriction enzyme SmaI, T4 ligase, T4 ligase buffer and water to form a reaction system for reaction so as to obtain a link product. By taking a common pUC18 carrier as a framework carrier, a quick, efficient and low background cloning method is constructed by combining a screening mechanism of the ccdB lethal gene and the SmaI restriction enzyme cutting site contained in the CcdB lethal gene without steps of performing double digestion on a ccdB recombinant carrier and a PCR product and recovering glue and the like, and the PCR product can be directly linked to a cloning carrier, so that the method is high in efficiency; and the whole time is controlled within 20 minutes, so that the experimental cost is greatly lowered, and the experimental progress is accelerated. The cloning system has very wide application prospects.

The invention discloses an application method of a regulation gene GmRSD in the middle and later periods of soybean nodulation. The method comprises the following steps: firstly, constructing an overexpression vector containing a nucleotide sequence as shown in SEQ IDNO:1, then constructing a transformant by utilizing the overexpression vector, infecting a receptor soybean root system by utilizingthe obtained transformant, screening positive plants, and acquiring transgenic soybeans with high nodulation and nitrogen fixation capabilities compared with normal soybeans. The overexpression vector takes pEGAD as a skeleton vector, after EcorR I and HindIII enzyme digestion, a target gene segment is connected to the skeleton vector, and the construction of the overexpression vector is completed. Agrobacterium tumefaciens is transformed by the overexpression vector to obtain the transformant. Then, the transgenic plants are obtained by an agrobacterium tumefaciens K599-mediated hairy root transformation method, and the positive plants are screened so as to obtain the transgenic plants with high nodulation and nitrogen fixation capacities compared with normal plants. The overexpression vector constructed by the method transforms receptor soybean plants to obtain the transgenic plants. The method remarkably improves nodulation and nitrogen fixation capacity of soybeans, and has greatpractical significance in increasing soybean yield.

The invention discloses a method for preparing a P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen. The P2X7R protein extracellular section amino acid sequence is optimized; the whole gene synthesis is used; the P2X7R extracellular section gene coding sequence is inserted into an expression carrier pET30a through restrictive enzyme cutting sites NdeI and HindIII; by using a enzyme digestion method and sequencing, the accuracy of the final expression carrier is ascertained; the built protein expression recombinant plasmid containing P2X7R extracellular section gene coding sequence is converted into a BL21 expression strain for IPTG induced expression; P2X7R protein is subjected to affinity chromatography purification in liquid supernatant; detection is performed. The method has the beneficial effects that the immunogenic P2X7R extracellular section can be obtained; the method can be used for P2X7R antibody preparation.

The invention provides recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof and belongs to the technical field of gene engineering. The recombinant plasmid pET28a-Ag43 is inserted into EcoRI and HindIII multiple cloning sites of plasmid pET28a from an auto-transport protein Ag43 encoding gene, and the auto-transport protein Ag43 encoding gene has a nucleotide sequence shown by SEQ ID No.1 in a sequence table. A heterologous protein gene segment can be quickly connected to the multiple cloning sites of the recombinant plasmid in a double enzyme digestion mode to construct the recombinant engineering bacteria, so that heterologous protein can be displayed and expressed on the surface of a cell, and wall breaking for releasing endoproteinase is avoided; meanwhile, zymoprotein is fixed to the surface of the cell, and convenience is brought to recycling the zymoprotein.

The invention provides a cell fusion expression pre-T vector, which is prepared by the following method: firstly, a DNA1 fragment of BspTI-GSGSG-HHHHHH-XcmI-Amp-XcmI-HindIII is established and encoded; secondly, the DNA sequence of three XcmI sites in an LacI gene in a site-directed mutation pET39 plasmid is changed under the condition that the encoding amino acid sequence is unchanged, so as to lose XcmI recognition sites, and site-directed mutation pET39-1 which eliminates the XcmI sites is obtained; thirdly, DNA1 is directionally recombined into pET39-2 by utilization of BspTI and HindIII; and fourthly, a DsbA signal peptide gene in the pET39-2 is removed, and the cell fusion expression pre-T vector is obtained. Besides the XcmI for preparing a linear T vector, the invention does not need to use other restriction enzymes, can further establish fusion expression genetic engineering bacteria in one step, and provides a feasible technical method for preparing a plurality of bioactive peptide engineering bacterium libraries.

The invention discloses an HSV1-TK (Herpes Simplex Virus 1-Thymidine Kinase) molecular imaging probe for detecting cell apoptosis as well as a construction method and application thereof. A preparation method of the HSV1-TK probe comprises the following steps: (1) performing enzyme digestion on a carrier pcDNA3.1 by using restriction enzymes HindIII and XhoI to linearize the carrier; (2) amplifying the terminal C of DnaE, the terminal C of a TK gene, a DEVD sequence, the terminal N of the TKgene and the terminal N of protein intron DnaE by virtue of PCR (Polymerase Chain Reaction); (3) recombining each segment with the linearized pcDNA3.1 carrier in a certain sequence by virtue of a gene recombination technology; (4) carrying out transformation, plasmid extraction and enzyme digestion identification on the recombinant carrier, thereby obtaining the HSV1-TK probe. The molecular imaging probe is capable of monitoring the occurrence and development of cell apoptosis in vivo, and thus overcoming the defect that the cell apoptosis only can be observed at an in-vitro level by virtue of technologies such as an electron microscope and flow cytometry and provides a favorable tool for monitoring in vivo.

The invention discloses a bidirectional promoter bi-visual fluorescent protein report gene plant expression vector. The promoter bi-visual fluorescent protein report gene plant expression vector is a recombinant expression vector of which the multiple cloning sites of an initial vector are inserted with the following DNA molecules: an EcoRI enzyme recognition sequence, an NoS terminator, an SacI enzyme recognition sequence, a kpnI enzyme recognition sequence, a red fluorescent protein gene, an SmalI enzyme recognition sequence, a BamHI enzyme recognition sequence, an XbalI enzyme recognition sequence, a bidirectional promoter, an XholI enzyme recognition sequence, an SpeI enzyme recognition sequence, a green fluorescent protein, an HindIII enzyme recognition sequence, an HpaI enzyme recognition sequence, an SalI enzyme recognition sequence, an NoS terminator and a Clal contained in turn from 5' end; and the nucleotide sequence of the bidirectional promoter is from the 5917th site to the 6834th site from the 5' end in a sequence 1 of a sequence list. The bidirectional promoter bi-visual fluorescent protein report gene plant expression vector can be applied to fusing functional genes by simple operation of direct enzyme cutting and connection.

The invention belongs to the technical field of tuberculosis diagnosis, and particularly discloses a recombinant protein for serodiagnosis of multimedicament-resistant tubercle bacilli. The recombinant protein is Rv1793 recombinant protein, and is obtained by the following steps of: inserting Rv1793 into the position between Pet28a EcoRI and HindIII endonuclease sites to form a recombinant plasmid, transforming the recombinant plasmid into E. coli BL21 to form recombinant E. coli, and expressing the protein. The sensitivity of the novel recombinant protein on the serodiagnosis of medicament resistance can reach 75 percent, and is far more than 53.3 percent of diagnosis rate of medicament resistance of a control CFP-10 recombinant protein. The Rv1793 recombinant protein becomes an effective biomarker applied to the serodiagnosis of medicament-resistant tuberculosis.

The invention discloses a preparation method of a human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and the application thereof; the method comprises the steps that: A, the obtainment of hTRIM 25 gene: a pair of primers is designed by referring to a human-infected TRIM25 gene sequence; B, the construction of an eukaryotic expression vector pCA-hTRIM25eGFP: hTRIM25 is connected into the pCA vector through EcoRI and XhoI to obtain pCA-hTRIM25, then Sall and XhoI are respectively used for digesting pEGFP-C1 and pCA-hTRIM25, the vector pEGFP-C1 and a fragment containinga promoter and hTRIM25 are recovered, and are connected with T4ligase, recombinant plasmid is extracted, and the recombinant plasmid is respectively digested and identified by EcoRI and HindIII; andC, the construction of a BHK21-hTRIM25eGFP cell line which stably expresses the hTRIM25 gene: a liposome method Lipofectamine2000 is adopted to transfect the pCA-eGFP and pCA-hTRIM25eGFPG to BHK-21 cells, the screening is carried out after the transfection, and the expression of hTRIM25 protein is identified through indirect immunofluorescence and Westernblotting to obtain BHK21-hTRIM25eGFP. The application of the line in preparing FMDV treating or preventing medicine is that: the constructed BHK21-hTRIM25eGFP cell line can stably express the hTRIM25 protein; the cell line BHK21-hTRIM25eGFP which stably expresses the hTRIM25 gene can inhibit the replication of the foot and mouth disease virus; and the hTRIM25 gene is used for constructing transgenic mouses, transgenic pigs and other transgenic animals.