37 results about "Restriction Enzyme Cut Site" patented technology

The invention provides a tryptamine hydroxylase mutant and application thereof. The tryptamine hydroxylase mutant is selected from any one of the following amino acid sequences: (a) the tryptamine hydroxylase mutant is obtained by deleting 30-45 amino acids from the N terminal of tryptamine hydroxylase as shown in SEQ ID NO: 1, and (b) the tryptamine hydroxylase mutant is obtained by deleting 30-45 amino acids from the N terminal of tryptamine hydroxylase as shown in SEQ ID NO: 2; (b) is formed by mutation of the amino acid sequence shown in (a), mutation sites are selected from one or more of the following amino acid residue sites: the 114 site, the 133 site, the 247 site, the 309 site, the 401 site and the 464 site, and the positions of the mutation sites correspond to the wild tryptamine hydroxylase with the sequence of SEQ ID NO: 1; (c) an amino acid sequence obtained by adding a tag sequence, a restriction enzyme cutting site sequence or a signal sequence to one end or two ends of (a) or (b). The catalytic activity of the tryptamine hydroxylase mutant is greatly improved compared with that of a wild enzyme, and the yield of 5-hydroxytryptamine is remarkably improved.

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

The invention belongs to the field of molecular biological detection, and particularly relates to a PLS3 gene SNP marker for rapidly detecting economic traits of Tibetan chicken, a detection method and application. It is found for the first time that G / A mutation at the 28469th site of the Tibetan chicken PLS3 gene is closely related to whether the gene has a BstZ17 I restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention relates to the technical field of aquaculture fish vaccines, in particular to a fusion gene of grass carp NPM1a and II type reovirus VP38, recombinant protein, a composition and application. The nucleotide sequence of the fusion gene is as shown in SEQ ID NO: 1. The fusion gene is obtained by connecting grass carp NPM1a and II type reovirus VP38 through a linker sequence, a BamH restriction enzyme cutting site is added to the 5'end of the fusion sequence, and an XhoI restriction enzyme cutting site is added to the 3 'end of the fusion sequence; the sequence of the linker is as shown in SEQ ID NO: 2. The subunit vaccine of the fusion protein of the grass carp NPM1a and the II type reovirus VP38 is prepared, and indole-3-lactic acid is used as an immunopotentiator in a combined manner, so that the technical bottlenecks of low protection rate, poor intestinal mucosal barrier repairability and the like of the existing vaccine are overcome.

The invention discloses a dipeptide synthetase and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the dipeptide synthetase is as shown in SEQ ID NO: 1; the method comprises the following steps: cloning a dipeptide synthetase gene from streptomyces albogriseolus 1-36 by virtue of a PCR (Polymerase Chain Reaction) technology; the recombinant plasmid is connected to NcoI and XhoI restriction enzyme cutting sites of a high-efficiency expression vector pET-28a (+), and is transformed into competent cells of escherichia coli DH5alpha, and plasmids are extracted; and transforming to an expression host strain BL21 (DE3) to obtain a genetic engineering strain for efficiently expressing the dipeptide synthetase. The dipeptide synthetase is applied to synthesis of a dipeptide compound or synthesis of a compound taking the dipeptide compound as a precursor. The dipeptide synthetase obtained by the invention can identify carboxyl donors and amino donors from non-natural amino acids to generate dipeptide compounds, and has the advantages of mild enzyme catalysis conditions and high catalysis efficiency.

The invention relates to a framework based on a miR-30 framework, a construction method of the framework and application of the framework in RNAi (Ribonucleic Acid Interference). The skeleton construction method comprises the following steps: taking an existing shRNAmiR30 vector as a PCR (Polymerase Chain Reaction) template; the method comprises the following steps: designing two pairs of complementary overlapping primers of siRNA containing a target gene, namely primers F1 and R1 and primers F2 and R2, respectively performing amplification, and performing OE-PCR by taking the obtained P1 and P2 as templates and the primers F1 and R2 as upstream and downstream primers to obtain miR-OE. Furthermore, according to the construction method disclosed by the invention, reverse mutation of a stem-loop structure in a miR-OE skeleton and an EcoRI restriction enzyme cutting site is also carried out. The primers required to be synthesized in the miR-OE skeleton and miR-OE-EL construction method are short chains with the length of less than 60nt, the synthesis difficulty and cost are effectively reduced, and the primer design is more flexible; moreover, the interference efficiency of the miR-OE-EL skeleton on the target gene is obviously improved, and the miR-OE-EL skeleton can be well applied to preparation of shRNA drugs.

The invention provides high-stability recombinant I-type collagen and application thereof, and belongs to the technical field of collagen. The invention aims to avoid potential restriction enzyme cutting sites and unstable sequence fragments during sequence design through directional selection of the existing amino acid sequence, so that the amino acid sequence can be efficiently expressed in pichia pastoris, and the recombinant I-type collagen obtained after purification has very high stability; the recombinant I-type collagen gives consideration to factors such as related biological active sites, hydrophilicity and the like, has related biological activity while being relatively high in stability, and has very good practicability.

The invention discloses a micro-ring plasmid synthesis method, and belongs to the technical field of gene editing. Two pairs of primers are designed, eukaryotic expression plasmids containing selection markers and target fragments are used as templates for PCR amplification to obtain two target fragments, vector plasmids loxp-R6K with specific sequences and the target fragments form recombinant plasmids, the recombinant plasmids contain sequences such as the target fragments, restriction enzyme cutting sites and R6K replication start sites, and the recombinant plasmids are used for preparing the recombinant plasmids. The recombinant plasmid is transformed in Escherichia coli GT115 competent cells, the recombinant plasmid is verified to have the recombination efficiency of 81% after bacterial colony PCR amplification, a correctly cloned seed solution is screened and subjected to induced expression to form a bacterial solution containing a screening-label-free micro-plasmid, the micro-ring plasmid is separated and purified, and the screening-label-free micro-ring plasmid is obtained. The sequence of the microring plasmid only contains a target fragment and an R6K replication start site, the accuracy of the size sequence of the microring plasmid in a bacterial colony cultured and cloned by the microring plasmid is as high as 90%, and the microring plasmid can be efficiently and stably obtained.

The invention discloses a matrix metalloproteinase-responsive multifunctional bionic ferritin nano material as well as a preparation method and application thereof. The method comprises the following steps: modifying melittin on the surface of a ferritin nanoparticle through a genetic engineering method, connecting by using a linker containing a matrix metalloproteinase 9 (MMP-2 / 9) restriction enzyme cutting site, expressing the obtained recombinant ferritin (SEQ ID NO: 6) in escherichia coli, and self-assembling to form the enzyme response functionalized bionic nanoparticle. According to the method, melittin can be connected to ferritin nanoparticles in series, the toxicity of melittin is remarkably reduced, and the melittin responds to matrix metalloproteinase in a tumor microenvironment so as to kill tumor cells; meanwhile, intravenous administration of melittin can be realized, and the melittin injection has a good treatment effect on in-situ and metastatic tumors.

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention relates to the technical field of biological pharmacy, and discloses a messenger ribonucleic acid sequence design method and device, computing equipment and a storage medium. According to the method, on the basis that a given 5 'UTR sequence serves as initial input, new codons are added one by one through a growth method, the overall-length minimum free energy and codon use efficiency of the whole sequence are calculated, scored and sorted, an elimination method is used for excluding sequences containing restriction enzyme cutting sites / poor properties / poorer scores, and finally, the sequences containing the restriction enzyme cutting sites / poor properties / poorer scores are obtained. And selecting representative sequences by using a cluster clustering search method to increase diversity, and finally obtaining the sequences for recommendation synthesis. The invention provides an mRNA sequence optimization design method in an environment with 5 'UTR and 3' UTR, the full-length minimum free energy of an mRNA sequence and the codon use efficiency of a CDS region are considered, the two targets can be optimized at the same time, the stability can be optimized from the perspective of overall molecules, the expressibility of the CDS region is optimized at the same time, and the method has the advantages of being simple in structure, convenient to operate and the like. And the stability and expressibility of the mRNA sequence are ensured.

The invention provides a preparation method and application of MBP-GL fusion recombinant protein. A C3 region of SPG and a B structural domain of protein L are connected in series, codon optimization is performed on a gene sequence, the front end of MBP is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site consistent with an expression vector are added to the tail end of the B region of the protein L, and the MBP-GL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a carrier subjected to the same double enzyme digestion, a recombinant protein MBP-GL gene expression carrier of a structural domain of strongest binding IgG of protein G and protein L connected with MBP is constructed, the carrier is subjected to transformation, induced expression and purification, and the recombinant protein MBP-GL gene expression carrier which not only has glucan ligand specific binding capacity, but also can be used for preparing the recombinant protein MBP-GL gene expression carrier. And the recombinant protein can be combined with all antibodies, subtypes and antibody fragments with high load.

The invention provides a method and a device for simulating a pulsed field gel electrophoresis band based on a sequencing technology. The method for simulating the pulsed field gel electrophoresis band based on the sequencing technology comprises the following steps: acquiring nanopore sequencing data of a biological sample to be analyzed, and assembling based on the nanopore sequencing data to obtain a genome sequence; according to the sequence characteristics of the restriction endonuclease, identifying a restriction enzyme cutting site corresponding to the restriction endonuclease from the genome sequence; when the restriction enzyme cutting site is the restriction enzyme cutting site subjected to methylation modification, removing the restriction enzyme cutting site to obtain a residual restriction enzyme cutting site; calculating the distance difference between two adjacent restriction enzyme cutting sites according to all residual restriction enzyme cutting sites, and taking the distance difference as the molecular weight of a simulated band; aiming at the molecular weight of each simulated strip, determining a strip position corresponding to the molecular weight; and drawing a simulated pulse field gel electrophoresis band result according to the position of each band and the molecular weight corresponding to the band position so as to accurately simulate the band result.

The invention discloses a high-efficiency expression and purification method of 5 '-3' exonuclease, which comprises the following steps: based on a Bac-to-Bac system, adding a tag and a TEV restriction enzyme cutting site to optimized 5 '-3' exonuclease Xrn1, then connecting the 5 '-3' exonuclease Xrn1 to a multiple cloning site behind a Polyhedrin promoter of a pFastBac expression vector to prepare expression baculovirus plasmids, and carrying out purification on the expression baculovirus plasmids to obtain the 5 '-3' exonuclease. Then transfecting the recombinant baculovirus particles into insect cells to generate recombinant baculovirus particles; finally, the 5 '-3' exonuclease Xrn1 is obtained through nickel column affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the 5 '-3' exonuclease Xrn1, and a technical foundation is laid for large-scale production of the 5 '-3' exonuclease.

The invention belongs to the technical field of biology, and relates to a cloning primer pair of an okra AeC4H gene, a cloning method and application of the okra AeC4H gene, and the primer pair can efficiently amplify a full-length CDS sequence of the AeC4H gene; the cloning method comprises the following steps: extracting total RNA (Ribonucleic Acid) of okra tissues, carrying out reverse transcription, carrying out PCR (Polymerase Chain Reaction) amplification, recovering a product, connecting a vector, screening positive clones and the like. The recombinant expression vector contains an AeC4H gene coding sequence which is inserted into the vector through a specific restriction enzyme cutting site. The invention also provides a method for transforming a plant by using the vector and a method for increasing the flavonoid content of the plant, which are realized by overexpressing the AeC4H gene. The invention also provides an application of the AeC4H gene or the encoded protein thereof in regulation and control of plant flavonoid biosynthesis. According to the invention, overexpression of the AeC4H gene in the plant is realized, so that the flavonoid content of the plant is increased, and the application of the AeC4H gene in regulation and control of biosynthesis of the flavonoid of the plant is disclosed.

The invention provides a site-specific mutagenesis vector and a use method thereof, and relates to the field of gene engineering in the biotechnological pharmaceutical industry, the vector contains BbsI restriction enzyme cutting sites except for a polyclone region, and site-specific mutagenesis comprises the steps of carrying out base mutation on the BbsI restriction enzyme cutting sites, removing the BbsI sites and keeping amino acid unchanged. According to the invention, a Golden gate method is adopted to construct the multi-copy vector, and a plurality of copies are directly assembled on a system in one step, so that the time is saved, and the tedious steps of multiple times of enzyme digestion and connection are omitted. According to the invention, a multi-copy vector is constructed in vitro, the copy number of the antibacterial peptide is increased, simultaneous expression of each unit is realized, and the expression quantity is improved. Meanwhile, the antibacterial peptide genes are connected in series by adopting a serial connection technology of the antibacterial peptide genes, so that the expression quantity of the antibacterial peptide is improved on the DNA level.

The invention discloses an engineering bacterium for high yield of N-acetylglucosamine as well as a construction method and application of the engineering bacterium, and the construction method comprises the following steps: sequentially knocking out nagA, nagB, nagP, gamA and gamP genes in a bacillus subtilis 168 strain to obtain an MT101 strain; the method comprises the following steps: carrying out series connection on a GNA1 gene in Saccharomyces cerevisiae W303 yeast and a glmS gene in a Bacillus subtilis 168 strain by adopting a chemical synthesis method, so as to obtain a GNA1 and glmS series connection gene; the method comprises the following steps: introducing a GNA1 and glmS tandem gene into a pP43NMK plasmid, and enabling the GNA1 and glmS tandem gene to be positioned between a KpnI restriction enzyme cutting site and a HindIII restriction enzyme cutting site, so as to obtain a pP43NMK-GNA1-glmS plasmid; and transferring the pP43NMK-GNA1-glmS plasmid into an MT101 strain, so as to obtain the engineering bacterium capable of producing the N-acetylglucosamine at high yield. According to the engineering bacterium for producing the N-acetylglucosamine at high yield, high expression of the N-acetylglucosamine can be realized, and the yield of the N-acetylglucosamine is remarkably improved.

The invention discloses a multi-copy expression construction method of an insecticidal fusion peptide U10-MYRTX-Mri1a-GNA with bacteriostatic activity in pichia pastoris, which comprises the following steps: 1, carrying out reverse PCR (Polymerase Chain Reaction) on a plasmid pGAPZalpha A, introducing a Hind III restriction enzyme cutting site to obtain a plasmid pGAPZalpha A-Hind III, 2, constructing a fragment containing a U10-MYRTX-Mri1a gene, and 3, carrying out multi-copy expression on the fragment containing the U10-MYRTX-Mri1a gene to obtain the insecticidal fusion peptide U10-MYRTX-Mri1a gene. The method comprises the following steps: 1, constructing a U10-MYRTX-Mri1a gene fragment, 2, constructing a U10-MYRTX-Mri1a gene fragment, 3, introducing a GGGSAAA linker and a snowflake agglutinin (GNA)-6 * His label to the C end of the U10-MYRTX-Mri1a gene fragment, 4, constructing a multi-copy expression vector pGAPZalpha A-HindIII-Mira1-GNA * 3, 5, preparing a pichia pastoris competent cell X33, and 6, carrying out electrochemical conversion to obtain a genetically engineered bacterium X33-pGAPZalpha A-HindIII-Mira1-GNA * 3. According to the invention, a linearized plasmid is transferred to pichia pastoris X33, so that a genetically engineered bacterium for expressing the fusion peptide U10-MYRTX-Mri1a-GNA is obtained.

The invention provides a nucleic acid amplification method, a single-stranded DNA multi-copy catenary and application thereof. The method comprises the following steps: connecting nucleic acid with a known sequence to obtain nucleic acid to be amplified; designing a primer pair according to a known sequence contained in nucleic acid to be amplified, and carrying out amplification ligation reaction to obtain a first double-stranded DNA multi-copy catenary; two primers of the primer pair can respectively contain different restriction enzyme cutting sites 1 and 2. An enzyme 1 can be used for cutting an enzyme cutting site 1 contained in the double-stranded DNA multi-copy catenary, a single-stranded gap is formed in a double-stranded DNA ring, and a digestive enzyme is used for digesting the single-stranded gap so as to obtain a first single-stranded DNA multi-copy catenary. Taking the first single-stranded DNA multi-copy catenary as a template, and carrying out amplification ligation reaction by utilizing the primer to obtain a second double-stranded DNA multi-copy catenary; and cutting an enzyme cutting site 2 contained in the second double-stranded DNA multi-copy catenary by using an enzyme 2, forming a notched single strand in the second double-stranded DNA multi-copy catenary, and digesting the notched single strand by using a digestive enzyme so as to obtain the second single-stranded DNA multi-copy catenary. The single-stranded DNA multi-copy catenary is used for nucleic acid sequencing.

The invention belongs to the field of molecular genetic breeding, and discloses a CAPS molecular marker of a rice blast-resistant gene Pi2, a primer group and application, and the nucleotide sequence of the CAPS molecular marker is as shown in SEQ ID NO.1; the SEQ ID NO. 1 contains a Pst I restriction enzyme cutting site. The invention designs a CAPS marker primer Pi2-CF / Pi2-CR and restriction enzyme Pst I for distinguishing a disease-resistant gene Pi2 from other disease-resistant genes and susceptible genes, so that the gene Pi2 is accurately identified, and a favorable tool is provided for subsequently cultivating a broad-spectrum durable rice blast-resistant new rice variety. The invention discloses an intragene molecular marker of a rice blast resistant gene Pi2, which is a co-dominant functional marker, and a disease-resistant homozygous genotype, a susceptible homozygous genotype and a heterozygous genotype can be accurately distinguished by only using one marker.

The application provides a biotin-labeled exogenous circular DNA and a construction method and application in protein interaction thereof, and belongs to the technical field of gene interaction. The application provides a construction method of the biotin-labeled exogenous circular DNA, wherein a target sequence is amplified by using a biotin-labeled primer to obtain linear DNA containing a preset restriction enzyme cutting site; the obtained linear DNA is subjected to single enzyme cutting treatment by using a corresponding restriction endonuclease; and the DNA after enzyme cutting is subjected to a self-ligation reaction to generate closed circular DNA. The preparation process of the biotin-labeled circular DNA is stable and controllable, and has high repeatability; the biotin labeling of the circular DNA can be combined with a streptavidin system to realize high-affinity and specific enrichment of a DNA-protein complex; and the circular DNA can be stably delivered in cells and maintain the structural integrity of the circular DNA, thereby providing an effective means for truly reflecting the interaction between the circular DNA and the protein in a physiological environment.

The invention discloses a method for detecting the loss degree of a polyA sequence of a plasmid template and application of the method in quality control of an mRNA (messenger Ribonucleic Acid) product prepared by in-vitro transcription, and the method comprises the following steps: inserting a restriction enzyme cutting site E1 into a spacer sequence contained in the polyA sequence, enzyme digestion is carried out on E1 and another enzyme digestion site E which is different from the E1 and is contained in the plasmid template, and the integrity of the E1 is detected on the basis of electrophoresis of an enzyme digestion product or an HPLC (High Performance Liquid Chromatography) result, so that the integrity of the polyA sequence is detected. Compared with traditional sanger sequencing for detecting the integrity of the polyA, the method does not depend on special sequencing equipment, the result can be obtained within 1-2 hours, and quantitative or semi-quantitative analysis can be carried out on the integrity of the polyA. The method has important practical value for ensuring good quality of a DNA plasmid template and complete biological functions of mRNA obtained by in-vitro transcription.

The invention discloses a preparation method of a deer whole blood small molecule peptide, which comprises the following steps: constructing a special compound enzyme system for layering and distributed enzymolysis through specific recognition of a functional domain sequence, and solving the technical problem of poor accessibility of a restriction enzyme cutting site caused by a quadrupole structure of hemoglobin and a heme binding characteristic; the obtained deer whole blood small molecule peptide is good in color and luster, the yield of a 500-3000 Da peptide fragment is high, and meanwhile characteristic microelements such as iron and zinc are reserved.

The invention relates to a construction method of a database for discriminating proteolytic enzyme restriction enzyme cutting sites. Comprising the following steps: S1, acquiring family classification information and restriction enzyme cutting site data of proteolytic enzyme from an MEROPS database, acquiring sequence and function annotation data of substrate protein from a UniProt database, and acquiring three-dimensional structure data of the substrate protein from an AlphaFold database; s2, migrating and integrating the data in the S1 to the local; and S3, dividing the processed data into four correlative core data tables, and storing the four core data tables in a local database. According to the method disclosed by the invention, by combining MEROPS, UniProt and AlphaFold databases, the restriction enzyme cutting site of the proteolytic enzyme is accurately predicted by adopting an artificial intelligence algorithm. Compared with a traditional method, the method has the advantages that the restriction enzyme cutting sites can be recognized more efficiently and more accurately, and the protein function analysis speed and accuracy are remarkably improved.

The invention belongs to the field of biological nano sensors, and particularly discloses a method for ultrasensitively detecting the activity of beta-glucosyltransferase (beta-GT) based on a CRISPR (clustered regularly interspaced short palindromic repeats) nano sensor. The invention designs a 5-hmC modified dumbbell probe integrating multiple functions of target recognition, palindromic isothermal amplification and restriction enzyme cutting sites, and realizes ultra-sensitive detection of beta-GT activity at a single molecule level by utilizing palindromic primer hyperbranched rolling circle amplification (PP-HRCA) based on a CRISPR (clustered regularly interspaced short palindromic repeats) nano sensor. The nano sensor can accurately quantify the activity of beta-GT in a complex biological matrix, and the method is convenient and fast, low in price, high in sensitivity and suitable for being popularized and applied to inhibitor screening and enzyme kinetic analysis.

The invention relates to the technical field of DNA cloning, and discloses a universal short-fragment DNA cloning method based on reverse PCR and template-free PCR and application of the universal short-fragment DNA cloning method, and the universal short-fragment DNA cloning method comprises the following cloning steps: taking an annular plasmid as a template, and adopting reverse PCR amplification to obtain a linearized vector; an upstream primer and a downstream primer are designed and synthesized, the 3'ends of the two primers have complementary overlapping regions, the 5 'ends of the two primers contain plasmid homologous sequences, and the two primers are subjected to thermal cycle self-assembly and extension in a PCR reaction system without exogenous template DNA to form a blunt-end double-stranded DNA fragment; and purifying the blunt-end double-stranded DNA fragment and a linearized vector, connecting through homologous recombination, and transforming a host cell to obtain a recombinant plasmid containing a target gene. The universal cloning method for the short-fragment DNA does not depend on any restriction enzyme cutting site, and the vector is universal; meanwhile, an exogenous template DNA is not needed; time and cost are greatly reduced, and the method is suitable for construction of high-throughput sgRNA, epitope tags, promoter elements and the like.

The invention belongs to the technical field of biology, and particularly relates to a phage vector, a construction method and application of the phage vector in construction of a high-titer and high-insertion-rate phage antibody library. The phagocytosis vector comprises a functional region and a non-functional region, the functional region comprises multiple cloning sites, the multiple cloning sites comprise single-type restriction enzyme cutting sites, and the restriction enzyme cutting sites are used for connecting a target gene sequence. The construction of the single-enzyme digestion phage plasmid vector and the phage library constructed by the single-enzyme digestion phage plasmid vector have the advantages that the insertion rate of gene fragments is higher, the storage capacity is higher, the titer reaches 1010 cfu / mL, and the optimized single-enzyme digestion system is more suitable for construction of the phage library than a double-enzyme digestion system.

The invention discloses PTP (protein tyrosine phosphatase) sigma Ig1-3 fusion protein as well as a preparation method and application thereof, and belongs to the field of genetic engineering. The recombinant fusion protein is obtained by adding an induction peptide to the N end of a PTP sigma Ig1-3 structural domain and sequentially adding a TEV restriction enzyme cutting site, a GFP fluorescent protein and a His tag to the C end of the PTP sigma Ig1-3 structural domain, the amino acid sequence of the PTP sigma Ig1-3 structural domain is as shown in SEQ ID NO.1, the amino acid sequence of the induction peptide is as shown in SEQ ID NO.2, and the amino acid sequences of the TEV restriction enzyme cutting site, the GFP fluorescent protein and the His tag are as shown in SEQ ID NO.3. Experiments prove that the recombinant fusion protein has obvious binding activity with glycosaminoglycans such as Haitong heparin sodium, chondroitin sulfate and the like, and has a good application prospect in mechanism research of axonal injury and regeneration and drug delivery therapy.

The invention relates to the technical field of gene engineering, and particularly discloses a double-display phage as well as a construction method and application thereof, and the construction method comprises the following steps: synthesizing a pM8A plasmid as shown in SEQ ID NO.1 and a pM8C plasmid as shown in SEQ ID NO.2; the preparation method comprises the following steps: inserting a gene segment for coding RGD peptide into NcoI and HindIII restriction enzyme cutting sites of a pM8A plasmid to obtain a pM8A-RGD plasmid, inserting a gene segment for coding BMP2 peptide into NcoI and HindIII restriction enzyme cutting sites of a pM8C plasmid to obtain a pM8C-BMP2 plasmid, co-transfecting the pM8A-RGD plasmid and the pM8C-BMP2 plasmid into a TG1 competent cell, adding a helper phage, incubating, and purifying to obtain the double-display phage pM8-RGD-BMP2. The double-display phage provided by the invention has the effects of promoting proliferation of the mesenchymal stem cells and promoting osteogenic differentiation of the mesenchymal stem cells.

The invention discloses a visual plant polygene editing vector and application thereof. The invention finds that the anthocyanin synthesized by regulation and control of the RH1 gene is taken as a reporter molecule, is visible to naked eyes under natural light, and can simply and quickly indicate the generation of plant transgenosis and improve the probability of obtaining a gene editing material, so that the application of the RH1 gene as the pigment reporter gene for plant genetic transformation is determined. The method comprises the following steps: further inserting an expression cassette taking an RH1 gene or an RUBY gene as a pigment reporter gene into a plant polygene editing vector to construct a visual plant polygene editing vector; a plurality of restriction enzyme cutting sites are reserved in the reporter gene expression box, one or more exogenous genes can be inserted or replaced, multi-gene overexpression is achieved while multi-gene editing is achieved, a promoter can be replaced with promoters with different expression intensities or tissue specific expression, and expression of inserted genes is accurately regulated and controlled.