137 results about "XhoI" patented technology

The invention discloses a genetically engineered bacteria used for producing stevia glycosyltransferase UGT76G1; a UGT76G1 coding gene is inserted between the restriction enzyme cutting sites EcoRI and XhoI of a PYes2 carrier, so as to construct a recombinant plasmid, and then the recombinant plasmid is introduced to expression host Saccharomyces cerevisiae YPH499 to obtain the engineered bacteria; and the coding gene of the UGT76G1 is GenBank, No. GenBank: AY345974.1, and the gene sequence of the coding gene is named as UGT (Udp Glucuronyl Transferase). The invention also discloses a construction method of the genetically engineered bacteria and the application of the bacteria to the production of rebaudioside A. According to the invention, under the condition that expensive UDPG (Uridine Diphosphate Glucose) is not added, cheap carbon source glucose is used as a substrate, the metabolic pathway of UDPG in the yeast is regulated, and then the rebaudioside A is produced from St glycosides through whole cell catalysis.

The invention relates to a recombinant human III-type collagen and a prokaryotic expression method thereof. The amino acid sequence of the humanized III-type collagen is shown as SEQ ID NO.1, and thenucleotide sequence of an encoding gene is shown as SEQ ID NO.2. The method comprises the following steps: carrying out codon optimization and design to obtain the gene sequences; inserting the gene sequences into a position between enzyme cutting sites NdeI and XhoI of an expression vector PET30a(+) to construct a recombinant expression vector pET30-1230; transforming Escherichia coli BL211(DE3)competent cells; selecting positive clones; and performing culture induction for efficient expression.

The invention discloses a method for constructing a tandem expression small interfering RNA recombinant lentiviral vector. The method comprises the following steps of: (1) constructing the siRNA recombinant lentiviral vector with single target spot: a, chemically synthesizing an oligonucleotide chain; b, annealing a sense chain and an anti-sense chain; and c, connecting an annealing fragment to the vector; (2) constructing the siRNA recombinant lentiviral vector with double target spots: a, amplifying a U6/H1-siRNA expression frame from the siRNA recombinant lentiviral vector with single target spot by utilizing the PCR; b, connecting the expression frame to the siRNA recombinant lentiviral vector with single target spot undergoing enzyme digestion by the XhoI; and c, performing the enzymedigestion, identification and screening of the clone forwardly inserted in the expression frame; and (3) constructing the siRNA recombinant lentiviral vector with multiple target points in tandem connection: inserting the U6/H1-siRNA expression frame in the siRNA recombinant lentiviral vector with double target spots to construct the siRNA recombinant lentiviral vector with triple target spots, and obtaining the siRNA recombinant lentiviral vector with multiple target points in tandem connection by repeating the step. The construction method is suitably used for the expression of the siRNA with multiple target genes as well as the research on the multiple target genes which are silent for a long time.

The invention discloses a PCR-RFLP primer for distinguishing DHV-1 and a new serotype and a method for distinguishing the DHV-1 and the new serotype. The primer is an upstream primer P1:5'-CAATTGAGGACATGGCTAAGAAA-3' and a downstream primer P2:5'-TCAGAGASCCRTCRAAWCCAGAAA-3. According to the method, distinguishing is conducted according to the DHV-1 and the restriction enzyme cutting site difference contained in a new serotype coding area and includes the steps that RNA extraction is conducted, and after corresponding target fragments are obtained through RT-PCR amplification, RFLP analysis is conducted after XhoI restriction enzyme cutting. The distinguishing method is simple and fast in operation and high in accuracy.

The invention relates to the fields of molecular biology and immunology, in particular to a vibrio harveyi secretory type vaccine and a structure and application thereof. Particularly, the secretory type vaccine is a base sequence in a sequence table SEQ ID No.1, and the construction process thereof is as follows: utilizing vibrio harveyi T4 as a template and F11 and R8 as primers for PCR amplification, connecting the product with pBS-T at room temperature, utilizing NdeI/XhoI double-enzyme cleavage to recover 1.2kb segment from plasmids pBSVhP1, simultaneously utilizing NdeI/XhoI double-enzyme cleavage to recover 4.3kb segment of plasmids pBT3, connecting the two segments at room temperature by ligase T4DNA for 2-4h, transforming connection liquid into colon bacillus DH5 alpha, culturing on an LB solid medium containing Ap for 24h to screen out white transformant, i.e. the Vibrio harveyi secretory type vaccine of base sequence in the expression sequence table SEQ ID No.1. The obtained vaccine has immune and protective functions on Vibrio harveyi. The obtained vaccine has the immune and protective effects on the Vibrio harbeyi, and the immune and protective efficiency of the vaccine of the invention on the Vibrio harveyi can reach to 90%. The preparation process is simple, devitalization and other steps do not needed, and no adjuvant is needed.

The invention discloses a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method for differentiating duck circovirus from goose circovirus. The PCR-RFLP method is used for differentiating by utilizing Rep protein gene sequence enzyme cutting site difference and comprises the following steps of: extracting DNA (Deoxyribonucleic Acid), carrying out PCR amplification to obtain a Rep protein gene segment, and carrying out RFLP analysis after carrying out XhoI digestion. The PCR-RFLP method disclosed by the invention is simple and higher in efficiency and accuracy.

The invention discloses a bat SARS-like coronavirus spike protein immunity determining area and a preparation method and application thereof. The method comprises the following steps of: A, amplifying by taking the bat SARS-like coronavirus S gene as a template design primer; B, performing enzyme digestion of the fragment obtained by the amplification by use of BamHI and XhoI, connecting to an expression vector pET32a, and sequencing to assure correctness; and C, purifying the recombinant plasmid, transforming the BL21 competent cell, culturing the monoclonal antibody, and performing 30-degree induction in a culture medium with the final concentration of 0.3mMIPTG; collecting the thalli, performing ultrasonic breaking, and then purifying with a HisTag purification kit; and after the purification, detecting the concentration of protein in SDS-PAGE to obtain the target protein. The method is simple and easy to implement, convenient to operate and easy in production; the peptide has the best immunogenicity; and a method of applying the protein to the identification of mice S monoclonal antibody epitope has high specificity, and is simple to operate and easy to repeat.

The invention provides a lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes. The lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes comprises fundamental sequences, resistance gene sequences, multiple-cloning-site sequences, promoter subsequences, and CYP3A4 gene cDNA (complementary DNA) sequences in a pLVX-AcGFP-C1 expression vector. Multiple cloning sites include an XhoI restriction enzyme cutting site and an XmaI restriction enzyme cutting site. The CYP3A4 gene cDNA sequences include XhoI restriction enzyme cutting sites, CYP3A4 gene coded sequences and XmaI restriction enzyme cutting sites. The CYP3A4 gene cDNA sequences are forwardly inserted into the multiple-cloning-site sequences. The lentiviral expression vector is capable of expressing the CYP3A4 gene specifically, continuously, efficiently and stably with high transfection efficiency and low consumption, and can be used as a powerful tool applied to pharmaceutical research and development related to CYP3A4.

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 discloses an efficient method for producing Trx-hCTRP2. The method mainly comprises the following steps of: (1) cloning a human hCTRP2 gene; (2) constructing a prokaryotic expression vector by carrying out a double digestion on an expression vector pET32-a (+) and a plasmid hCTRP2/ pMD20-T with XhoI and BamH I, purifying for recovery, and utilizing a T4DNA ligase for connection to obtain a recombinant vector pET32/ hCTRP2; (3) conversing the recombinant vector into a escherichia coli host; (4) carrying out soluble induction expression; (5) purifying. According to the method of the invention, pET32 is employed as the vector and inducible expression conditions are optimized, so as to highly express a soluble hCTRP2 fusion protein. The method not only guarantees biological activities of the hCTRP2 fusion protein, but also enables efficient obtainment of a large amount of stabilized proteins.

The invention provides a lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression. The lentiviral expression vector comprises a fundamental sequence, a resistant gene sequence, a multiple cloning site sequence, a promoter sequence and a CYP2E1 gene cDNA sequence of a pLVX-AcGFP-C1 expression vector, multiple cloning sites include XhoI enzyme cutting sites and EcoRI enzyme cutting sites, and the CYP2E1 gene cDNA sequence comprises XhoI enzyme cutting sites, a CYP2E1 gene coding sequence and EcoRI enzyme cutting sites and is forwardly inserted into the multiple cloning site sequence. The lentiviral expression vector has the advantages of high transfection efficiency, low consumption and capability of specifically, continuously, efficiently and stably expressing CYP2E1 genes, and can serve as a powerful tool applied to drug research and development related to CYP2E1.

The invention discloses a preparation of method of a plasmodium vivax aldolase protein monoclonal antibody. According to the antibody, aldolase protein is specifically used as targeting antigen; three dominant antigen epitopes A, B and C are selected and connected by flexible segments so as to form a recombinant D; enzyme cutting sites of BamHI and XhoI restriction enzymes are added on the upper course and the lower course of the recombinant, respectively; then the product after double enzyme cutting processes is inserted into PET-28a (+) carrier so as to structure a recombined protein D expression carrier; the recombined protein D is expressed by escherichia coli BL21; a Balb/c mouse is immunized; spleen cells of the mouse are taken and fused with sp2/0 myeloma cells; and finally ten hybridoma cell lines capable of stably secreting aldolase protein monoclonal antibody are obtained after filtering. The monoclonal antibody obtained by the method provided by the invention is capable of specifically identifying tertian plasmodium aldolase protein so that the monoclonal antibody is beneficial for the specific detection of tertian plasmodium infection; in addition, the specificity is high, the response is sensitive, and the experiment cost is low, so that the antibody is suitable for the high-pass rapid detection.

The invention relates to a preparation method of a monoclonal antibody of human ARMET protein and a testing reagent kit. Restriction enzyme NcoI/XhoI is used for cloning the cDNA coding sequence of the human ARMET protein to the carrier pET28a (+), which expresses that the C-terminal fuses together with the recombination ARMET protein of 6 histidines; the plasmid of pET28a(+)-ARMET is converted into a competent cell BL21, which induces and expresses the ARMET protein, collects the thalli and purifies the human ARMET protein; meanwhile, the monoclone of anti-ARMET is prepared and the kit for testing ARMET is established. The method can be applied to the study on the functions of the ARMET, the basic studies on the physiological mechanism and pathomechanism of a human body and can also be used in the auxiliary diagnosis to the clinical diseases.

The invention discloses a recombinant plasmid for degrading nitrobenzene, gene engineering bacteria and a preparation method thereof. The recombinant plasmid has gene sequences which are represented by SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 between a locus BglII and a locus XhoI. The nitrobenzene degrading engineering bacteria strain PET-NB disclosed by the invention can effectively degrade nitrobenzene in solution to form a ring-opening product; and thus, the biochemical quality of water is improved.

The invention discloses a recombined rhabdovirus for expressing a porcine bocavirus VP2 protein and application thereof. A porcine bocavirus VP2 gene is amplified by PCR (polymerase chain reaction), and the nucleotide sequence of the porcine bocavirus VP2 gene is represented by SEQ ID NO.1. The amplified porcine bocavirus VP2 gene is inserted into BamHI and XhoI locuses of a rhabdovirus transmissible plasmid pFastBacTMHTB to construct a recombined transmissible plasmid pFastBac HTB-VP2; the recombined transmissible plasmid is converted into a DH10Bac competent cell comprising a rhabdovirus framework plasmid; the VP2 gene is integrated into the rhabdovirus framework plasmid Bacmid through homologous recombination; an sf9 cell is transfected by extracting the DNA of the recombined Bacmid to obtain the recombined rhabdovirus rBV-VP2 of which the preservation number is CCTCC V201401. A large quantity of VP2 protein and virus sample particles are expressed and produced in insect cells, and an effective PBoV VLPs subunit vaccine is developed.

The invention pertains to the research field of animal pharmaceutical and biological engineering, which is a DNA vaccine pIRES-N/IL2 co-expressed by N gene of an bird infectious bronchitis virus and bird IL-2 gene. pIBVN, pIRES-EGFP/DsRed plasmid is double digested with NheI and XhoI, N gene and carrier segment are gel recovered and connected together, and eucaryon expression plasmid pIRES-N/DsRed is constructed. Bird original IL-2 gene is PCR obtained using pDNAIL-2 plasmid as template. After that plasmid of IL-2, pIRES-N/DsRed are double digested by BamHI and NotI, IL-2 gene and carrier segment is gel recovered and connected, and DNA vaccine pIRES-N/IL2 co-expressed by N gene of Chinese isolate SAIBk of bird infectious bronchitis virus and bird IL-2 gene is constructed. The vaccine can be used in preventing bird infectious bronchitis.

The invention relates to a quantitative fluorescent multiplex PCR test kit for Epstein-Barr virus, and the kit comprises the following reagents: a, specific primer pairs for amplifying mutations of key target genes in the Epstein-Barr virus; and b, specific probes for the mutations of key target genes in the Epstein-Barr virus, wherein the mutations are P-thr, V-val and V-leu in the coding region of EBNA-1 gene, promoters Cp, Fp and Zp of the EBNA-1 gene, and XhoI-loss, 30bp Deletion and Ser 366 Thr of LMP1 gene. The test kit is advantageous in that: the aim of precise prediction of the risk of nasopharyngeal carcinoma in high risk population can be achieved; the main carcinogenic mutations can be well known for the combination of population screening and population intervention for nasopharyngeal carcinoma; and the kit has the advantages of high in sensitivity, fast in test, and low in cost, having broad market prospect.

The invention relates to an antitumor and thrombolytic double-effect chimeric protein, a preparation method thereof and the use thereof. The amino acid sequence of the chimeric protein is represented by an amino acid sequence SEQ ID No.4 or an amino acid sequence SEQ ID No.5. The preparation method of the chimeric protein comprises: constructing a chimeric gene; inserting the chimeric gene subjected to enzyme digestion into the EcoRI/XhoI locus of a carrier plasma Pet28a(+) to construct a recombinant plasma; transferring the recombinant plasma into the BL 21 competent cells of Escherichia coli to obtain recombinant Escherichia coli; culturing the recombinant Escherichia coli in a shake flask, disrupting the recombinant Escherichia coli by ultrasonic waves, passing collected supernate through a Ni-NTA affinity chromatographic column, performing gradient elution, condensation and desalination to obtain the chimeric protein. The chimeric protein combines antitumor and antithrombotic effects.

The invention discloses a Latcripin-9 gene segment of a shiitake mushroom C91-3 strain, coding protein, a preparation method and application. A nucleotide sequence of the Latcripin-9 gene segment is shown as SEQ ID NO:1, and a nucleotide sequence of the coding protein is shown as SEQ ID NO:2. The preparation method of the Latcripin-9 gene segment includes following steps: extracting mycelium total RNA of the shiitake mushroom C91-3 strain; performing reverse transcription on the mycelium total RNA to synthesize cDNA; performing PCR amplification on a target gene; taking obtained cDNA as a template, and using a PCR primer with two restriction enzyme cutting sites EcoRI/XhoI for PCR; recycling and purifying a DNA segment.

The invention discloses preparation and application methods of a genetically engineered bacterium expressing thymosin beta4. The preparation method comprises the following steps: S1, synthesizing a gene sequence of the thymosin beta4 which can be efficiently expressed in escherichia coli; S2, recombining the obtained gene into a SapI digested pTWIN1 plasmid vector to obtain a pTWIN-Tbeta4 fusion vector; S3, by taking the pTWIN-Tbeta4 as a template, carrying out PCR (Polymerase Chain Reaction) amplification on a contained peptide sequence and a Tbeta4 sequence on the vector; S4, recombining the obtained gene sequence to NcoI and XhoI digested plasmid pET-28a to obtain a pET-Tbeta4 expression vector; S5, converting the obtained vector to a competent escherichia coli cell BL21 to obtain a pET-Tbeta4 engineering bacterium; and S6, adding IPTG (Isopropyl-beta-D-Thiogalactoside) to induce Tbeta4 to express when the OD600 value of the obtained bacterial liquid is 0.4-0.6. The invention further discloses an application method of the genetically engineered bacterium expressing the thymosin beta4. The preparation and application methods of the genetically engineered bacterium expressing the thymosin beta4 provided by the invention lay a foundation for environment-friendly production of the thymosin beta4 with a low cost.

The invention relates to genetic engineering and protein expression technology, in particular to wild esterase B3 genetically engineered bacteria and a building method and application thereof. The wild esterase B3 genetically engineered bacteria have a base sequence shown as SEQ ID No.1. The building method comprises the following steps of: performing polymerase chain reaction (PCR) amplification by taking a complete sequence in a culex tarsalis cDNA library as a template and a DNA fragment with NcoI and XhoI restriction enzyme cutting sites and 5'end protection basic groups respectively as a primer; performing digestion on the PCR amplification product by using a restriction enzyme; cloning the digested product to a prokaryotic expression vector; and transforming the product to an Escherichia coli expression host, wherein the expression product is the wild esterase B3 genetically engineered bacteria. The wild esterase B3 genetically engineered bacteria after being fermented can degrade organic phosphorus, organic chlorine, carbamate and pyrethroid compounds. In the method, simple Escherichia coli is used as the genetically engineered bacteria, so that a large number of esterase B3 proteins with biological activity are produced and microbiological culture capable of degrading organic phosphorus compounds is obtained; and the pathogenicity of the microbiological culture is low and can be used for bioremediation of an area which is polluted by organic phosphorus.

The invention discloses a brucella three-gene recombinant plasmid, a construction method thereof and expression and application thereof in escherichia coli, belonging to the technical field of geneticengineering. According to the technical scheme, a pET-28a(+) plasmid is taken as as a carrier, and a synthetic full-length gene Omp10-Omp28-L7/L12 is inserted between pET-28a(+) restriction enzyme cutting sites BamHI and XhoI so as to establish a pET-28a(+)recombinant plasmid containing an Omp10-Omp28-L7/L12 gene segment and a kanamycin screening label, namely pET-28a(+)-Omp10-Omp28-L7/L12 recombinant plasmid. The brucella three-gene recombinant plasmid has the beneficial effects that brucella Omp10-Omp28-L7/L12 is successfully cloned and is linked and converted to the recombinant plasmid, sothat the efficient soluble expression of the recombinant plasmid in an escherichia coli expression system is realized, an expressed protein is capable of inhibiting the infection, proliferation and transfer capabilities of brucella in the body fluid and cell levels, promoting the level of an antibody in a mouse and prolonging the immune time, and a novel treatment through is provided for the treatment of brucella diseases.

The invention discloses a Latcripin-16 gene segment, an encoding protein and a preparation method of a mushroom C91-3 strain. The Latcripin-16 gene segment has a nucleotide sequence shown in SEQ ID NO:1; the encoding protein has an amino acid sequence shown in SED ID NO:2. The preparation method of the Latcripin-16 gene segment comprises the following steps: extracting mycelium total ribonucleic acid (RNA) of the mushroom C91-3 strain; carrying out reverse transcription on the mycelium total RNA to synthesize cDNA; carrying out PCR amplification on a target gene; taking obtained cDNA as a template, and carrying out polymerase chain reaction (PCR) by taking a PCR reaction primer with EcoRI and XhoI restriction enzyme cutting sites; and recovering and purifying the DNA segment.

The invention discloses a bioconversion method for anti-AIDS-medicine-reyataz midbody, and belongs to the technical field of medical biology. Secondary structures of genes and codon usage biases are adjusted, the length of primers is controlled within 60 base, the primers are obtained, the obtained primers are dissolved with double distilled water, then the dissolved primers are added into a reaction system, a prepared PCR reaction system is amplified, obtained DNA fragments are subjected to gel cutting purification, and then are cloned into NdeI/XhoI loci of pET30a with a homologous recombination method, a successfully-sequenced DNA sequence is SEQ ID NO.3, and is named as Sst-2, and the corresponding amino acid sequence of the Sst-2 is SEQ ID NO.4. The bioconversion method is mild in reaction condition, and has the low requirement for equipment, the high temperature or cooling is not required in the production process, energy consumption is low, purification is convenient, single enzyme catalysis is used in the whole system, the cycle number of coenzyme is high, and the reaction condition is mild.

The present invention relates to a novel endogenous gene expression substituting lentivirus vector. According to the present invention, a U6 promoter is inserted between the 5' LTR and the 3' LTR of lentivirus so as to perform transcription of a segment of a shRNA sequence, and the shRNA insertion region contains double restriction enzyme cutting sites such as BamHI and XhoI so as to insert the shRNA sequence after annealing; an EFla promoter is used for transcriptional expression of a Flag tag fused exogenous gene, the Flag tag is fused on the N terminal of the exogenous gene, and the multiple cloning site contains three restriction enzyme cutting sites such as HpaI, EcoRI and NheI3 so as to insert the exogenous gene, wherein the HpaI is the blunt-ended restriction enzyme cutting site so as to ensure insertion of any genes; IRES is used for starting the EGFP reporter gene; and the production of two proteins through the one RNA transcripted from the EFla can be achieved. In addition, the disadvantages of the conventional vectors are overcome, and the fast and efficient intracellular genetically modified way is provided.