17 results about "Restriction Enzyme Site" patented technology

This invention belongs to the field of human Ly6d protein secretion expression technology, and particularly relates to a recombinant expression plasmid, recombinant cells, and construction method for extracellular expression of human Ly6d protein. The recombinant expression plasmid includes a basic plasmid pcDNA3.1 and a target sequence for expressing recombinant human Ly6d protein inserted at the restriction enzyme sites of the basic plasmid. When this recombinant expression plasmid is transformed into EXPI293F cells to construct recombinant cells, it can highly express human Ly6d extracellularly, reaching an expression level of 255.67 mg / 1000 mLCells, which is highly beneficial for the purification and collection of this recombinant protein, providing assistance for subsequent research and production.

This invention belongs to the field of molecular biology detection, specifically relating to a rapid detection method for economic traits of Tibetan chickens. PLS3 Gene SNP markers, detection methods, and applications. This invention is the first to discover: Tibetan chickens... PLS3 The G / A mutation at position 28469 of the gene and its presence BstZ17 The presence or absence of the I restriction enzyme site is closely related to the main economic traits of Tibetan chickens. Based on this new discovery and the SNP site, this invention provides a method for rapidly detecting this SNP site and screening for economic traits in Tibetan chickens. The method is simple, convenient, fast, low-cost, and provides reliable results.

This invention discloses the chitin synthase 1 (LmCHS1) gene of the leafminer moth, its dsRNA, its synthesis method, and its insect-resistant applications. Through transcriptome analysis, specific primer pairs for the open reading frame of the LmCHS1 gene were designed and synthesized. The LmCHS1 gene sequence was obtained through gene cloning (nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2). The gene fragment with sequence SEQ ID NO: 3 was selected as the RNAi target sequence. Primer pairs SEQ ID NO: 6 and SEQ ID NO: 7 with restriction enzyme sites were designed and synthesized. PCR amplification was performed, and a prokaryotic expression vector was constructed to induce dsRNA expression. Feeding leafminer moth larvae with dsRNA interfered with LmCHS1 gene expression. After 24 hours, abnormalities were observed in the larvae's body surface, with some larvae failing to complete molting or dying due to an inability to form a normal body surface after molting. After 96 hours, the larval mortality rate was 72.22%, compared to 40% in the GFP control group, demonstrating a significant effect. This invention has broad application prospects in the control of the leaf borer pest in forestry due to its high efficiency, specificity, and safety.

The invention belongs to the technical field of biological detection, and particularly relates to a molecular beacon probe composition, a reagent and a kit for detecting miR-122. The composition comprises a molecular beacon and a specific hairpin probe, the nucleotide sequence of the molecular beacon is shown as SEQ ID NO.1, and the molecular beacon is modified with a fluorescence reporter group and a quenching group; the hairpin probe comprises two restriction enzyme cutting sites, two segments of repetitive sequences and a recognition site capable of being specifically combined with miR-122. According to the application, miR-122 is specifically combined with a hairpin probe to start a reaction, a dual-cycle cascade mechanism of target triggering, initiation chain generation and secondary chain displacement is constructed, and rapid quantitative analysis of miR-122 is realized in a single tube by a one-step method through high-fidelity recognition and exponential signal amplification of an initiation chain and a molecular beacon. The system has ultrahigh sensitivity, single-base resolution specificity and high accuracy, provides key technical support for noninvasive early screening of diseases such as liver cancer and the like, and has important clinical application value.

The invention discloses a plant insect-resistant related protein as well as a coding gene and application thereof. According to the invention, the full length of the SlJAIB14 gene is amplified from tomato, and two restriction enzyme cutting sites of BamHI and Sac I are introduced to the 5'and 3 'ends of the gene through primer sequences. The newly synthesized gene is connected with a pYPX158 plant expression vector containing double 35S promoters through a T4 DNA (Deoxyribose Nucleic Acid) ligase, so that a recombinant plasmid pYPX-S1JAIB14 containing a target gene S1JAIB14 is obtained. According to the invention, plasmids are introduced into agrobacterium tumefaciens (GV3101) by using an electric shock method. The SlJAIB14 gene is transformed into tomato by using an agrobacterium-mediated method, and the resistance of the transgenic plant to cotton bollworm is verified through a feeding experiment.

The invention relates to an H5N1 nucleoprotein prokaryotic expression vector and a nucleoprotein induced expression method thereof, and belongs to the field of bioengineering.The H5N1 nucleoprotein prokaryotic expression vector is obtained by synthesizing an optimized H5N1 nucleoprotein base sequence in vitro and constructing the optimized H5N1 nucleoprotein base sequence into a PGEX-6P-1 plasmid through enzyme cutting sites BamHI and XhoI; the method comprises the following steps: transforming an H5N1 nucleoprotein prokaryotic expression vector into BL21 competent bacteria, inoculating the BL21 competent bacteria into an ampicillin-resistant LB solid culture medium for culture, selecting single bacteria, cloning and inoculating the single bacteria into an ampicillin-resistant LB liquid culture medium for culture, and finally adding IPTG (isopropyl-beta-d-thiogalactoside) for culture and induction to obtain induced expression nucleoprotein fusion protein. The acquisition of the fusion protein provides a basis for researching H5N1 vaccines with more remarkable and more lasting immunity and effectiveness.

The invention discloses a method for improving plant pilot editing efficiency, which comprises the following steps: replacing AtU6 with an NtU26 promoter through a restriction enzyme site directional cloning technology, and respectively connecting the NtU26 promoter with two serially connected PegRNA sites. The method comprises the following steps: constructing a '35S promoter-Cmylc enhancer-NtU6-26' expression unit to replace a '35S promoter-Cmylc enhancer-AtU6', and ensuring that the transcription initiation of pegRNA is regulated and controlled by a 35S promoter-Cmylc enhancer-NtU6-26 promoter; the unique transcriptional regulation ability of the NtU6-26 promoter in tobacco cells is utilized, sufficient PegRNA substrates are provided for assembly of an editing compound (nCas9-RT / pegRNA) by improving the pegRNA transcriptional level and increasing the accumulation amount of the PegRNA, the order of magnitude improvement of tobacco pilot editing efficiency is achieved, precise replacement of AtU6 by the NtU26 promoter is achieved by utilizing restriction enzyme sites, uncertainty caused by random integration is avoided, and the method has the advantages that the method is simple and convenient to operate and high in practicability. The stability and repeatability of the gene expression unit are ensured; the NtU6-26 is designed for tobacco cells, and the unique transcriptional regulation ability of the NtU6-26 can significantly improve the transcriptional level of pegRNA and increase the accumulation amount of pegRNA in the cells.

The invention belongs to the technical field of biology, and discloses a method for determining a multivalent antigen sequence of an anti-periodontitis red complex, which comprises the following steps: firstly, screening out key virulence factor antigens (FimA-II, RgpA, Msp and BspA) from three core pathogenic bacteria of the red complex: porphyromonas gingivalis, treponema denticola and fusisterone; then, independently cloning each antigen gene to a pUC57 cloning vector, constructing a single antigen recombinant plasmid, and avoiding intergene interference by designing a specific enzyme cutting site; and finally, through plasmid transformation, positive clone screening and double enzyme digestion verification, the plasmid quality is ensured. The obtained single antigen plasmid can be flexibly combined and used, is beneficial to subsequent multivalent vaccine development, and has the advantages of wide antigen coverage, high expression reliability, flexible construction strategy, easiness in industrialization and the like.

The invention provides an exponential rolling circle amplification method based on Argonaute protein. The exponential rolling circle amplification method comprises the following steps: providing a 5'end phosphorylated single-chain gDNA; the method comprises the following steps: carrying out hybridization annealing on target RNA (Ribonucleic Acid) and a Padlock probe to form an RNA-Padlock compound; under the action of ligase, cyclizing the RNA-Padlock compound to form a circular DNA (Deoxyribose Nucleic Acid) template; the gDNA and TtAgo are subjected to pre-incubation, and a TtAgo / gDNA compound is formed; the method comprises the following steps: mixing a circular DNA template, a TtAgo / gDNA compound, DNA polymerase, dNTPs and a fluorescent dye in a same reaction system, and carrying out an exponential rolling circle amplification reaction. According to the exponential rolling circle amplification method based on the Argonaute protein, accurate shearing can be achieved at any preset position of an RCA product, a specific enzyme cutting site does not need to be introduced, system construction is greatly simplified, and exponential amplification is achieved.

The invention discloses a plant pilot editing method which comprises the following steps: replacing AtU6 with an NtU26 promoter through a restriction enzyme site directional cloning technology, and respectively connecting the NtU26 promoter with two PegRNA sites which are connected in series. The method comprises the following steps: constructing a '35S promoter-Cmylc enhancer-NtU6-15 'expression unit to replace a '35S promoter-Cmylc enhancer-AtU6', and ensuring that the transcription initiation of pegRNA is regulated and controlled by a 35S promoter-Cmylc enhancer-NtU6-15 promoter; the unique transcriptional regulation ability of the NtU6-15 promoter in tobacco cells is utilized, sufficient PegRNA substrates are provided for assembly of an editing compound (nCas9-RT / pegRNA) by improving the pegRNA transcriptional level and increasing the accumulation amount of the PegRNA, the order of magnitude improvement of tobacco pilot editing efficiency is achieved, precise replacement of AtU6 by the NtU26 promoter is achieved by utilizing restriction enzyme sites, uncertainty caused by random integration is avoided, and the method has the advantages that the method is simple and convenient to operate and high in practicability. The stability and repeatability of the gene expression unit are ensured; the NtU6-15 is designed for tobacco cells, and the unique transcriptional regulation ability of the NtU6-15 can significantly improve the transcriptional level of pegRNA and increase the accumulation amount of pegRNA in the cells.

The invention discloses recombinant human hyaluronidase as well as a preparation method and application thereof, and relates to the technical field of enzyme engineering. The amino acid sequence of the recombinant human hyaluronidase is shown as SEQ ID NO.1, SEQ ID NO.2 is obtained after signal peptide is added, and coding nucleotide (SEQ ID NO.3) is obtained by optimizing according to CHO cell codon preference and adding enzyme cutting sites and a Kozak sequence; according to the present invention, the rHuPH20 cell strain is obtained by connecting the rHuPH20 to a PLK002-GS vector (SEQ ID NO: 4), carrying out electric shock transfection on the CHO-K1BN cell, and finally carrying out 25 [mu] M MSX pressurization culture to obtain the rHuPH20 cell strain, the culture process is optimized, the rHuPH20 yield in the 2L reactor supernatant exceeds 29000 IU / mL, the protein purity is more than 99% after three-step chromatography purification, the enzyme activity can achieve 49989 IU / mg, and the cell strain is stable after 75 PDL passage.

The invention belongs to the technical field of synthetic biology and genetic engineering, and particularly discloses an anti-leakage Cre recombinase expression cassette and application thereof in pichia pastoris gene editing. The expression cassette is composed of lox71-selection marker-sh blele-Cre coding sequence (SEQ ID NO: 1)-TetO manipulation sequence-lox66, wherein the Cre coding sequence (SEQ ID NO: 1) is optimized by pichia pastoris codon and is introduced into escherichia coli rare codon, and IIS type restriction enzyme sites such as PaqCI, BsmBI, BsaI, AarI and the like are completely eliminated; the gene is cloned to a pPICZ skeleton and introduced into an escherichia coli host of chromosome integrated tetR, Cre leakage expression can be thoroughly inhibited in a large-scale amplification stage, and recombination toxicity and plasmid mutation are avoided. An'upstream homologous arm-expression cassette-downstream homologous arm 'edit box can be obtained through one-step assembly by using Golden Gate, and construction is completed within 3 days; electrically transforming pichia pastoris after linearization, and integrating efficiency gt for the first time; according to the method disclosed by the invention, the content of the resistance marker is 70%, the resistance marker removal rate is 100% through methanol-induced Cre self-deletion, and only one lox71 / 66 heterozygous site is reserved. According to the invention, the problems that the traditional Cre / loxP system is easy to mutate in escherichia coli and low in efficiency in yeast are solved, and an efficient and safe universal gene editing tool is provided for industrial microbial metabolism engineering.

The invention discloses a method convenient for plant pilot editing, which comprises the following steps: respectively carrying out cis-connection on a Cmylc promoter and two core editing elements through a restriction enzyme site directional cloning technology; a Cmylc promoter-nCas9-RT expression unit is constructed on the upstream of a gene (the sequence comprises a nuclear localization signal peptide coding region, an nCas9 nicking enzyme structural domain coding region and a reverse transcriptase functional domain coding region) for coding an nCas9-RT fusion protein, and it is ensured that transcription initiation of the fusion protein is regulated and controlled by a Cmylc promoter; the editing efficiency is enhanced from two dimensions by utilizing the unique transcriptional regulation ability of the Cmylc promoter in tobacco cells, on one hand, by improving the transcriptional level of nCas9-RT fusion protein and increasing the accumulation amount of functional fusion protein in cell kernels, a sufficient substrate is provided for assembly of an editing compound (nCas9-RT / pegRNA), and on the other hand, the editing efficiency is improved from two dimensions; finally, through cooperative high expression of'protein-nucleic acid 'double elements, the core limitation of'insufficient element expression quantity' in a traditional editing system is broken through, and the order of magnitude improvement of the tobacco leading editing efficiency is achieved.

The invention belongs to the technical field of synthetic biology, and particularly relates to an industrial preparation method of high molecular weight polynucleotide (PN). According to the method, a plasmid containing a PN base sequence is used as a template, long-chain DNA is generated by using a rolling circle amplification technology, and then efficient separation of PN fragments is realized through restriction enzyme digestion. The plasmid is subjected to optimization design, a restriction enzyme cutting site is introduced into a replication start site and a selection marker gene, after enzyme cutting treatment, a plasmid skeleton is degraded into small fragments smaller than 250 bp, and efficient separation of a target PN fragment is achieved by means of molecular weight difference. Through optimization design and large-scale preparation of a plasmid template, low-cost and easy-to-amplify production of PN prepared by rolling circle amplification is realized, and meanwhile, the length and sequence of a final PN product can be precisely designed, regulated and controlled. Particularly, a specific enzyme cutting site is introduced into a plasmid template, so that the subsequent precise removal of a plasmid skeleton sequence is facilitated, and the purity of a target product is remarkably improved. According to the process, the industrial preparation of the high molecular weight polynucleotide (PN) greater than 1500 bp is successfully realized, and a safe, efficient, convenient and large-scale brand new path is provided for the production of the high molecular weight PN.

Methods of detecting a target sequence of a nucleic acids molecule in a sample comprising amplifying an amplicon, contacting the amplicon with a restriction enzyme, incubating with a nucleic acid molecule adapter and a ligase, applying to a capturing moiety and detecting a tag in complex with the capturing moiety are provided. Methods of detecting single nucleotide polymorphisms (SNPs) by generating a de novo restriction enzyme cite or a non-canonical PAM are also provided, as are methods of designing a primer pair.

The application belongs to the technical field of biology and provides a molecular marker of microsatellite of Flourensia, specific primers, a preparation method and application. The application develops the molecular marker of microsatellite of Flourensia by using a restriction enzyme site related DNA genome scanning technology and obtains corresponding primers, establishes a method for preparing the molecular marker of microsatellite of Flourensia, and provides the molecular marker for researches on Flourensia germplasm resource identification, genetic diversity analysis, plant genetic map construction, population genetic structure analysis, gene positioning, quantitative trait gene analysis, evolution and genetic relationship, and lays a foundation for genetic researches on Flourensia germplasm resource genetic diversity, population structure and population historical dynamics, and is favorable for reasonable development of Flourensia resources.

The invention provides a vector combination, a multi-expression cassette vector construction composition, a method and application, and relates to the technical field of biology. The carrier combination comprises a donor carrier and an acceptor carrier, the donor vector sequentially comprises a multiple cloning site sequence 1, an expression cassette unit, a multiple cloning site sequence 3, a first resistance gene and a first resistance promoter from 5'end to 3 'end; the expression cassette unit contains a multiple cloning site sequence 2 for target gene insertion; the receptor vector sequentially comprises a second resistance gene, a second resistance gene promoter and a site-specific integration element from 5'end to 3 'end; the fixed-point integration element contains a multi-cloning-site sequence 4; the multiple cloning site sequences 1-4 respectively and independently contain at least two restriction enzyme sites; and the multiple cloning site sequences 1 and 3 respectively have the same restriction enzyme cutting sites as the multiple cloning site sequence 4. The vector combination for constructing the vector with the multiple expression cassettes has the advantages of simplicity in operation and lower experiment cost.