331 results about "Restriction Enzyme Cut Site" 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 discloses a method for connecting a DNA molecule fragment with a carrier. The carrier comprises at least three different restriction enzyme cutting sites E1, E2 and E3, wherein the E2 is located between the E1 and E3; the DNA molecule fragment to be connected at least comprises the restriction enzyme cutting sites E1 and E3. The method comprises the following steps: firstly carrying out enzyme cutting and dephosphorylation on the restriction enzyme cutting sites E1 and E2 of the carrier; and then carrying out a first step of the enzyme cutting and connection on the restriction enzyme cutting site E1 of the DNA molecule fragment to obtain a linear connection product of the DNA molecule fragment and the carrier, and then recycling the connection product with a correct size; finally carrying out a second step of the enzyme cutting and connection on the restriction enzyme cutting site E3 of the connection product to obtain a self-connected annular connection product. The invention can realize the effective connection between the DNA molecule fragment and the carrier, so that the generation of incorrect connected polymers with different forms is reduced, the proportion of the correct connected product accounts for over 30% in the connection system so as to realize an efficient electro-transformation.

The invention discloses a method for enhancing black streaked dwarf resistance of paddy rice by using an artificial microRNA (micro Ribonucleic Acid) and a special double chain RNA thereof. The double chain RNA consists of a sequence A and a sequence B. The method comprises the following steps of: replacing a mature body sequence of an entogenous microRNA of paddy rice and an inverse complementary sequence of a mature body thereof respectively by the sequence A and the sequence B; simultaneously, designing a restriction enzyme cutting site and a protecting basic group on 5' and 3' of the microRNA, and obtaining an artificial microRNA vector through artificial synthesis; and transferring the vector into a plant expression vector with a strong promoter through restriction enzyme ligation, and introducing the plant expression vector into paddy rice to obtain transgenic paddy rice which is resistant to black streaked dwarf. The disease resistance of paddy rice is enhanced through an artificial microRNA technology, target missing is prevented, high safety is achieved, and the disease-resistant property can be inherited stably.

The invention relates to a gene assembly method of phage genetically engineered antibody library, belonging to the field of biomedical study and clinical applications. The method aims to overcome the problems of the prior art, including poor stability, low efficiency, high mutation probability and low fidelity, and indirect application to humanized single chain antibody study. The technical proposal adopted by the invention comprises the following steps of: obtaining heavy chain variable region gene and light chain variable region gene of a humanized antibody by RT-PCR, and ligating the antibody heavy chain variable region, a linker and the antibody light chain variable region with the pre-designed restriction enzyme cutting sites on two ends of primers using PCR and restriction enzyme method at the same time.

The invention relates to a method for screening non-essential regions for replication of a goat pox virus and universal transfer vectors for same. The method comprises the steps of amplifying two-end gene segments of any two regions of a goat pox virus gene by using a PCR (Polymerase Chain Reaction) method; then, inserting an enhanced green fluorescent protein (EGFP) gene and a xanthine-guanine phosphoribosyl transferase (gpt) gene expression cassette into the segments; establishing two universal transfer vectors of the goat pox virus; and acquiring a recombinant virus expressing an exogenous gene stably from the transfer vectors, thereby determining the selected regions to be non-essential regions for replication of the goat pox virus, wherein each universal transfer vector contains one unique restriction enzyme cutting site Sal I and allows gene expression cassettes of other items to insert in. The recombinant virus obtained by means of the two universal transfer vectors provided by the invention not only has a growth performance similar to a parent virus, but also has better safety because a plurality of toxicity related genes in a genome are knocked out in an orientation way, and has the potential to be developed into an attenuated vaccine strain for gene engineering.

The invention discloses a construction method of genetically engineered bacterium for producing astaxanthin, which is characterized by comprising the following steps: A)constructing a gene expression module, wherein the gene expression module comprises relative gene for producing astaxanthin, a promoter at the upstream of relative gene for producing astaxanthin and a terminator at the downstream of the relative gene for producing astaxanthin; and B)performing cotransformation of the gene expression module to saccharomyces cerevisiae. The method has the advantages of simple, rapid and high efficiency performance, multi-grade clone is avoided, restriction enzyme cutting site is not required for depending on, multiple fragments enable cotransformation, the homologous recombination efficiency is high, and the engineering bacteria construction time can be obviously shortened.

The invention discloses a construction method of genetically engineered bacterium for producing beta-carotene, which is characterized by comprising the following steps: A)constructing a gene expression module, wherein the gene expression module comprises relative gene for producing beta-carotene, a promoter at the upstream of relative gene for producing beta-carotene and a terminator at the downstream of the relative gene for producing beta-carotene; and B)performing cotransformation of the gene expression module to saccharomyces cerevisiae. The method has the advantages of simple, rapid and high efficiency performance, multi-grade clone is avoided, restriction enzyme cutting site is not required for depending on, multiple fragments enable cotransformation, the homologous recombination efficiency is high, and the engineering bacteria construction time can be obviously shortened.

The invention discloses fusion protein containing a leucine-rich repetitive sequence, and a preparation method and application thereof. Concretely, the invention relates to fusion protein, and the fusion protein possesses the following structure from N terminal to C terminal: A-X-E-Y1-Y2 (formula Ia) or A-Y2-Y1-E-X (formula Ib), wherein A is an optional secretion signal peptide, X is a member rich in leucine repetitive sequence 1-2 (LRRs1-2) of Slit2 protein (neuronal guidance factor 2), E is a restriction enzyme cutting site, Y1 is a first tag peptide member, Y2 is a second tag short peptide member and the second tag short peptide member is His tag member, and '-' represents a peptide bond or a peptide joint for connecting the above members. The fusion protein is beneficial for correct folding of LRR (leucine-rich repeat) and forming active functional polypeptide, and is capable of simply removing two tag members through once resection enzyme cutting, thereby preparing high-purity high-activity LRR sequence.

The invention provides a plasmid for preparing a DNA Marker, and a construction method and application thereof. The plasmid comprises different DNA fragments forming the target DNA marker; the copy number of the different DNA Marker fragments in the plasmid is more than 1, and the same single restriction enzyme cutting site is positioned between the DNA Marker fragments. The invention also provides a method for quickly preparing the DNA marker at low cost. The DNA marker prepared by the method has the characteristics of sharp band, uniform brightness or random controllability, high repeatability between batches and the like.

The invention discloses a preparation method of a rice genetic engineering sterile line. The preparation method comprises the following steps: by using the Afl2 restriction Enzyme cutting sites introduced to the upstream and downstream, connecting the corn lethal gene ZMAA1, through a single restriction Enzyme cutting site, to a pCAMBIA1300 binary vector already connected with a DsRED gene expression element and the rice EAT1 gene. The restriction enzyme cutting sites Kpn I and Sma I introduced into the upstream and downstream primers and the restriction enzyme cutting sites Kpn I and Sma I onthe pCAMBIA1300 binary vector are utilized. According to the method disclosed by the invention, the F1-generation hybrid conforms to the Mendel's law of segregation in the self-seed process, and theoffsprings include the hybrids capable of maintaining the triple-linked genes, as well as the sterile line without fertility.

The invention relates to a phage antibody library which assembles an ScFv gene fragment between Restriction Enzyme cutting sites of SfiI and NotI of a pCANTAB5E carrier. The phage antibody library is characterized in that the ScFv gene fragment can be affinitive and enriched with antigens of 16-membered macrolide agricultural chemicals to form a soluble single-chain antibody and the phage antibody library is applied to immunoassay of pesticide residue. The phage antibody library has the advantages that the antibody with high affinity can be obtained without animal immunization, the test period is short, the antibody library is the phage antibody library which takes small molecular milbemycin oxime of a 16-membered macrolide generic structure as immunogen to construct, the antibody library theoretically can directly obtain a specific antibody library of 16-membered macrolide compound through screening, the screening flux is high, the efficiency is high, the specificity is strong, and the affinity selecting range is wide, so that the phage antibody library has wide application prospect in the aspects such as agricultural chemical antibody preparation, testing technique development and the like.

The invention provides a mercuric ion detection kit based on constant-temperature cascading nucleic acid amplification and a detection method thereof. Thymine-containing DNA identification elements are specifically bound with mercuric ions and then are folded to form an intra-molecular stem-loop structure; The 3' terminal of the DNA can be identified by polymerase, the DNA itself serves as a template to start strand displacement amplification reaction so as to produce a large amount of single-stranded DNA products; the single-stranded DNA products can open a restriction enzyme cutting site containing molecular beacon stem-loop structure to produce fluorescence signals; at the same time, the single-stranded DNA products can be also used as primers, and hybridization combined molecular beacons are used as templates to trigger secondary strand displacement amplification reaction, and released SDA products can form heteroduplexes with new molecular beacons so as to produce cascaded-amplified fluorescent signals. The detection method is high in sensitivity, ingeniously achieves cascading amplification of strand displacement amplification reaction without system complexity increase, is high in amplification efficiency and response speed and can achieve mercuric ion quantification within 30 minutes, and the detection limit is as low as 2 nM.

The invention discloses a small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification. Through nucleic acid complementary hybridization, target small RNA specifically identifies a 3-WJ primer and a 3-WJ template, a stable three-way cross structure is formed, and the 3-WJ primer starts an SDA (strand displacement amplification) reaction along with the phosphorothioate-modified 3-WJ template and produces a large number of single-chain SDA products with restriction enzyme cutting sites. The single-chain DNA opens stem-loop structures of molecular beacons to enable fluorescence to recover. A double-chain complementary structure formed by the molecular beacons and the single-chain SDA products contains nucleic acid nickase recognition sites, the sites are recognized by nickase after the double-chain structure is formed, the cut molecular beacons fall off from the double-chain structure to produce fluorescence signals, and the released SDA products and new molecular beacons can form hybrid double chains and produce more fluorescence signals. The quantitative method and the kit are high in sensitivity, and cascade amplification of the SDA reaction is realized skillfully by the aid of the phosphorothioate-modified template.

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.

Aspects of the present invention are drawn to methods and compositions for sorting nucleic acid molecules into physically separate compartments according to the identity of a nucleotide base or sequence of bases at a specific location, resulting in the production of reduced complexity samples that find use in any number of downstream genetic analyses. Aspects of the methods of the invention include fragmenting a nucleic acid sample, e.g., with a restriction enzyme, ligating an adaptor (or adaptors), and sorting the fragments based on the identity of the nucleotide base(s) positioned adjacent to the fragmentation site (e.g., the restriction enzyme cut site/or recognition site). Each round of sorting produces binned samples having reduced complexity over the parent sample.

The invention discloses an mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector. In the vector, a plasmid vector is pEGFP-C1, an mbr sequence with the length of 279bp is contained on the upstream of a cytomegalovirus (CMV) promoter of the pEGFP-C1, and a protein coding region of FPGS is contained between two restriction enzyme cutting sites, namely Hind III and EcoR I of the pEGFP-C1; and the mbr sequence is shown in a sequence table 2. An IC50 result of the constructed mbr-FPGS efficient expression plasmid which is measured by a Western Blot experiment and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) experiment proves that: the FPGS expression level of the mbr-FPGS efficient expression plasmid is improved by 30 percent (p is less than 0.05) compared with that of an FPGS comparison plasmid, the mbr-FPGS efficient expression plasmid can obviously improve the sensitivity of cancer cells to methotrexate (MTX) and has a certain dose-time dependence,and the IC50 value (72h) of an mbr-FPGS efficient expression group cell is 2.63ng/ml which is one seventh (p is less than 0.05) of the IC50 value (19.01ng/ml) of a comparison group cell approximately.

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 starch induction type recombinant bacillus subtilis as well as a preparation method and application thereof. The starch induction type recombinant bacillus subtilis contains a recombinant vector, wherein the recombinant vector is characterized in that in front of a BamHI restriction enzyme cutting site of a PHT43 plasmid, a Pgrac promoter is replaced with an alpha-amylase promoter PamyQ by performing for three times a mode of overlapping PCR continuously, and then an MTSase-MTHase fusion enzyme gene is inserted behind the BamHI restriction enzyme cutting site. When the used alpha-amylase promoter and an amylase signal peptide are combined with the expression gene, i.e., the MTSase-MTHase fusion enzyme gene, the expression effect of the starch induction type is better than that of other induction types.

The invention relates to a method for constructing highly expressed trehalose synthase engineering bacteria by using a Pcry3Aa promoter. For a recombinant carrier, a Pcry3Aa-PhoD fragment by which a Pcry3Aa promoter fragment and a PhoD signal peptide fragment are connected by an overlap PCR (Polymerase Chain Reaction) is inserted at the upstream of a restriction enzyme cutting site BamHI of a shuttle plasmid PHT01, and a target protein trehalose synthase TreS fragment is inserted between two restriction enzyme cutting site, i.e., BamHI and AatII. The invention further relates to a method for constructing the highly expressed trehalose synthase engineering bacteria by using the recombinant carrier. According to the method disclosed by the invention, the Pcry3Aa promoter is adopted to naturally induce the synthesis of trehalose synthase; because the Pcry3Aa promoter contains a special STAB-SD structure, the stability of the Pcry3Aa promoter to transcribe mRNA is improved, the half-life period of mRNA is prolonged, the mRNA translation level of a downstream target gene is improved, and therefore the trehalose synthase is highly expressed.

The invention discloses a double-LAMP (loop-mediated isothermal amplification) detecting method, and belongs to the technical field of molecular biology. By the aid of the method, vibrio parahaemolyticus and vibrio vulnificus can be simultaneously detected. An LAMP detection system comprises primer groups for detecting vibrio parahaemolyticus genes OmpA and vibrio vulnificus metalloprotease genes, and each primer group contains a pair of outer primers F3 and B3 for the OmpA genes and the metalloprotease genes and a pair of inner primers FIP and BIP; PstI and BamHI restriction enzyme cutting sites are respectively arranged on the inner primers BIP. The double-LAMP method has the advantages of high sensitivity and detection speeds, good specificity, simplicity in operation and visual and clear result observation.

The invention discloses a group-I type-4 aviadenovirus DNA (Deoxyribonucleic Acid) vaccine. The antigen of the group-I type-4 aviadenovirus DNA vaccine contains a group-I type-4 aviadenovirus penton gene. The invention also discloses a preparation method and application of the group-I type-4 aviadenovirus DNA vaccine. The preparation method comprises the following steps of (1) designing a group-I type-4 aviadenovirus penton gene primer with EcoR I and Sal I restriction enzyme cutting sites, and amplifying the target gene fragment by using the group-I type-4 aviadenovirus DNA as a template; (2) connecting the penton gene fragment onto a pMD-18T vector; (3) connecting the penton gene to an eukaryotic expression vector pCI; (4) transfecting pCI-penton into an LMH cell; and (5) performing amplification and extracting and purifying pCI-penton. The group-I type-4 aviadenovirus DNA vaccine has the advantages that the preparation is simple; the use is convenient; the cost is low; the humoral immunity and the cellular immunity can be excited at the same time; the comprehensive protection is effectively provided for SPF chickens; and the toxicity enhancing risk does not exist.

The invention provides a method for preparing recombinant human amyloid protein A Beta42 and application of the recombinant human amyloid protein, which belongs to the technical field of fusion protein. In the method, PCR sense primer P1 of the human amyloid A Beta42 is designed; a BamHI restriction enzyme cutting site, an anti-sense primers P2, a Sall restriction enzyme cutting site and a terminator codon TAG are introduced according to the sequence of the precusor protein APP of the human amyloid and multiple cloning sites of the cloning vector pGEX-4T-1. cDNA of human SH-SY5Y cells is taken as a templet for amplifing PCR; the length of the fragment of the product is 147bp. The Beta42 fragment of human APP from 672 to 713 is encoded. The A Beta42 gene fragment sequence is combined into a prokaryotic expression vector pGEX-4T-1 to form the prokaryotic expression plasmids pGEX-4T-1/A Beta42 of human A Beta42. PGEX-4T-1/A Beta42 is transformed into colibacillus BL21 (DE3), and purified activated recombinant human amyloid A Beta42 is got through induced expression, separation and enzyme cutting.