31 results about "Restriction Enzyme Cleavage Site" patented technology

The present invention relates, e.g., to a method for isolating a DNA molecule of interest in a form suitable for sequencing at least a portion of the DNA by a high throughput sequencing method, comprising (a) digesting a double-stranded (ds) DNA molecule with two different restriction enzymes, A and B, to generate a ds form of the DNA molecule of interest, which is bounded by the two restriction enzyme cleavage products, and (b) attaching to each end of the DNA molecule of interest an adaptor molecule which comprises at one end a restriction enzyme cleavage site that is compatible with the restriction enzyme A or the restriction enzyme B cleavage product, and which also comprises a sequence and/or element that allows the DNA of interest to be sequenced with a high throughput sequencing apparatus. The method can be adapted for sequencing DNA with a variety of high throughput sequencing apparatuses, including machines manufactured by the 454, Illumina (Solexa Sequencing technology) and ABI (SOLiD™ Sequencing technology) companies. A method is also described for sequencing regulatory elements within a cell, comprising subjecting a collection of ds DNA molecules that are enriched for regulatory elements and that are generated by digestion with two restriction enzymes, A and B, which generate sticky ends, to an isolation method of the invention, and sequencing the collection of ds DNA molecules with a high throughput sequencing apparatus.

The invention provides a method for detecting kanamycin. The kanamycin is detected by using double restriction enzyme cleavage sites and color change caused by G-tetrad. The preparation method specifically comprises the following steps: carrying out specific binding on a hair pin and kanamycin; adding a color developing agent into a homogeneous phase reaction mixed liquor for display detection. According to the method, high specific detection for the target object kanamycin is realized by using the oxidability of the G-tetrad; the effect of signal amplification is reached by using nucleic acid tool enzymes.

The type III restriction enzyme EcoP15I is used to isolate from cDNA of an expressed gene a tag comprising more than 25 nucleotides and capable of identifying the expressed gene, wherein the 3′ end of the tag is defined by a cleavage site of the type III restriction enzyme and the 5′ end of the tag is defined by the cleavage site of another restriction enzyme that is closest to the 3′ end of the cDNA of the expressed gene. The tag of the invention allows accurate quantitative gene expression analysis and rapid gene expression profiling in any organism for which no expressed sequence tag (EST) database is available.

The invention provides tumor acidity responsive autophagy-induced polypeptide as well as a preparation method and application thereof. The preparation method comprises the following steps: chemicallysynthesizing gene sequences of pH low insertion peptide (pHLIP) and Beclin 1 active fragments and connecting the sequences to a pET-32a expression vector through a restriction enzyme cutting site to obtain a recombinant plasmid; converting the recombinant plasmid to escherichia coli BL-21(DE3) and obtaining engineering bacteria pET-32a-pHLIP-Beclin 1/BL21(DE3) through screening; performing IPTG induced expression on the engineering bacteria, collecting thalli and obtaining target proteins through separation and purification. The tumor acidity responsive autophagy-induced polypeptide provided by the invention can be positioned to a tumor acidic microenvironment through pHLIP; the Beclin 1 active fragment is conveyed to tumor cells in a targeted manner, so that the tumor cells are induced togenerate autophagic death; the tumor acidity responsive autophagy-induced polypeptide can be applied to the preparation of a targeted anti-cancer drug.

The invention relates to an ultra-binary vector as well as a construction method and an application thereof. The ultra-binary vector is characterized in that a framework vector of the ultra-binary vector is pCMBIA1301-hpt<->, and Xba I-RB-LB-Sal I is inserted between restriction enzyme cutting sites Xba I and Sal I of the framework vector, thereby forming two T-DNA (transferred-deoxyribose Nucleic Acid) regions together with original left and right border sequences; and hpt II is connected into a T-DNA2 region through the restriction enzyme cutting sites, so as to acquire the ultra-binary vector pDT1301 containing the hpt II. The pDT1301 disclosed by the invention contains the hpt II and is convenient for screening callus-resistant and transgenic regenerated plants; and exogenous target genes of the hpt II are not in the same T-DNA region, so that the self-fertilization and separation of transgenic descendants are facilitated, the safe transgenic plants which do not contain selective marker genes are acquired, and obstacles for the safe release of the transgenic plants are cleared.

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 provides a preparation method and a use of a medicine over-expressing a PCNP gene. The medicine includes a plasmid over-expressing the PCNP gene and a PCNP protein obtained through the over-expression of the PCNP gene. The construction method of the plasmid over-expressing the PCNP gene comprises the following steps: designing a primer according to the sequence of the PCNP gene, andcarrying out PCR amplification on a target gene; carrying out enzyme digestion on restriction enzyme digestion sites Xho I and Kpn I in a GV230 vector to form a linearized GV230 vector; and connectingthe target gene with the linearized GV230 vector to construct a GV230-PCNP vector which is the plasmid over-expressing the PCNP gene. When the medicine over-expressing the PCNP gene is used in humanneuroblastoma cells, the overexpression of the PCNP gene in the human neuroblastoma cells can promote apoptosis, inhibit cell growth, inhibit cell migration and inhibit the growth of human neuroblastomas.

The invention discloses a CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors and further provides a molecular cloning method suitable for modifying large plasmid vectors. The CRICPR-Cas9-based method includes: designing proper sgRNA according to a to-be-modified site of a starting plasmid; adopting Cas9 protein and the sgRNA to in-vitro cut the starting plasmid; connecting the starting plasmid which is cut with a target gene on the basis of a homologous recombination method to complete modifying. By using the CRICPR-Cas9-based method, the problem that proper restriction enzyme cutting sites are difficult to find for modifying the large plasmid vectors through conventional molecular biologic means is solved effectively. The method is an improvement of a site mutation kit which generally includes site mutation of target protein and building of truncation with deleted structural domain. By the method, the technical problem that secondary mutation (random mutation caused by PCR enzyme fidelity) is easy to be caused when the site mutation kit is used for mutation of large vectors exceeding 10kb is solved effectively.

The invention discloses a linker peptide mediated enzyme immobilized BaPAD catalyst, and a preparation method and application thereof, and belongs to the field of preparation and application of catalysts. The method comprises the following steps of: firstly, inserting linker peptide, which specifically adsorbs zeolite, into a plasmid pET28a-bapad vector containing an encoded phenolic acid decarboxylase BaPAD gene through an Nco I/Xho I restriction enzyme cutting site, and then, immobilizing to obtain the enzyme immobilized BaPAD catalyst on a pre-treated Na-Y zeolite carrier through fusion enzyme, wherein the phenolic acid decarboxylase BaPAD is from bacillus atrophaeus. The biocatalyst prepared by the invention is used for catalyzing FA to produce 4-VG; compared with a method for catalyzing FA to produce 4-VG in a whole-cell manner, the concentration and productivity of 4-VG are improved to 1.97 M and 22.8 g/L/h under the same condition; and the catalyst has good reusability, and canmeet industrial production requirements of 4-VG.

The invention discloses a recombinant plasmid of expression L-lysine transportprotein and engineering bacteria and application. The gene is from corynebacterium glutamicum ATCC13032, and amplified from a glutamate corynebacterium genome by polymerase chain reaction (PCR); EcoRI and HindIII restriction enzyme cutting sites are respectively added to two ends of the L-lysine transportprotein gene; pKk223-3 connection and transformation after digestion are carried out by using the same restriction enzyme; plasmids are screened and extracted, and electrically converted into the corynebacterium glutamicum ATCC13032; multiplication of the transportprotein in the corynebacterium glutamicum is realized; the molecular weight of the expression product L-lysine transportprotein is 25081.9Da. By adopting the recombinant L-lysine transportprotein or engineering bacteria of expressing the recombinant transportprotein, the L-lysine can be transferred to the outside from inside of the cell, and the recombinant plasmid has the advantages of high transport efficiency and high transfer speed, and the yield of the L-lysine is improved.

The invention discloses a method for remaking multiple cloning sites of a known vector, namely an adapter primer-PCR (Polymerase Chain Reaction) method. The method comprises the following steps of: amplifying the known DNA (Deoxyribonucleic Acid) fragment of any species by applying an adapter primer which respectively adds multiple restriction enzyme cutting sites to a 5' tail end; carrying out double enzyme cutting by using restriction enzymes positioned on both ends of the amplified fragment, and connecting to the multiple cloning sites of a vector to be remade by using a T4DNA joining enzyme; transforming a connected product into competent escherichia coli; and selecting positive clone, extracting plasmids, verifying whether the remade vector is carried with new enzyme cutting sites or not through enzyme cutting, and retaining a correct plasmid vector. The method disclosed by the invention can meet the requirements of a specific experiment by fast and conveniently adding the new enzyme cutting sites to the multiple cloning sites of the known vector, is low in cost and high in flexibility and can be applied to remaking the multiple cloning sites of the known vector.

The invention provides a monkey type 1 adenovirus SAdV-1 vector system. The monkey type 1 adenovirus SAdV-1 vector system comprises an adenovirus plasmid pKSAV1-EG and a packaging cell line 293SE13. A target gene coding sequence can be inserted into a SpeI restriction enzyme cleavage site of the pKSAV1-EG plasmid through DNA (Deoxyribonucleic Acid) assembly or restriction enzyme cleavage-ligation cloning to generate an adenovirus plasmid carrying the target gene; and an exogenous gene expression cassette containing an expression regulatory sequence can be similarly inserted into an FseI site of pKSAV1-EG, and an adenovirus plasmid carrying the exogenous gene expression cassette is generated. By transfecting a 293SE13 cell with the adenovirus plasmid subjected to SwaI linearization, and a recombinant virus is rescued; and the 293SE13 is also used for amplifying the recombinant virus. The generated SADV-1 virus genome is deleted in an E1/E3 part, and the SADV-1 virus is a replication-defective virus. The human lacks the pre-stored immunity to the SADV-1, and the target cells of the SADV-1 are different from those of a common HAdV-5 vector, so that the vector system is predicted to have a wide application prospect in the fields of gene therapy and vector vaccines.

The invention belongs to the technical field of biology, and particularly relates to a prokaryotic expression and purification method and application of a PK34 antibacterial peptide. The method comprises the following steps: 1, optimizing a PK34 polypeptide amino acid sequence, inserting a PK34 gene into an expression vector through a restriction enzyme cutting site by adopting whole-genome synthesis, and constructing a recombinant expression plasmid; 2, the constructed recombinant expression plasmid containing the protein of the PK34 gene coding sequence is transformed into a prokaryotic cell, and PK34 polypeptide is expressed through induction; 3, splitting the expressed prokaryotic cells, and detecting expression positions in a supernatant and a precipitate; and 4, purifying the PK34 protein. The simple and convenient method for rapidly preparing the PK34 polypeptide in a large scale is achieved, the target polypeptide which is high in purity and still has antibacterial activity without treatment is obtained through the steps of large-scale expression of the PK34 polypeptide in prokaryotic cells and simple and convenient purification, the production period is short, the production cost is low, the protein yield is high, the product quality is high, and the method is suitable for industrial production. And the method can be used for preparing the anti-PK34 monoclonal antibody.