21 results about "EcoRI" patented technology

The invention discloses an AAV2 recombinant gene system for double expression of sFLT-1 and shVEGF as well as a preparation method and application of the AAV2 recombinant gene system, and co-expression of the sFLT-1 and the shVEGF is realized. A target gene (sFLT-1: NM001159920.2, shVEGF: NM001025366.3) is cloned to a pAAV-CMV vector through BamHI, HindIII, EcoRI and MluI sites, a pAAV-CMV-sFLT-1-shVEGF plasmid is constructed, and the plasmid is subjected to PCR (Polymerase Chain Reaction), enzyme digestion and sequencing verification, so that the recombinant plasmid is obtained. And then, co-transfecting HEK293 cells with pAAV-RC2 and pHelper plasmids to produce rAAV particles, carrying out gradient ultracentrifugation purification on the rAAV particles by virtue of iodo-pentose, and storing the rAAV particles at the temperature of 80 DEG C below zero. The AAV2 recombinant gene system has the following advantages that the sFLT-1 and the shVEGF are stably expressed, the immunoreactivity is low, and the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF plays a role for a long time, so that the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF can be applied to various neovascularization diseases.

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 relates to the field of gene vector construction, in particular to construction and application of a gene vector integrating fluorescent screening and self-deletion functions, the gene vector comprises a conditional knock-out (cKO) vector and a gene knock-in (KI) vector which are both integrated with a Dre-Rox mediated self-deletion module and a fluorescent screening module, the construction method comprises the following steps: carrying out HindIII / EcoRI double enzyme digestion on a pUC19 vector, carrying out homologous recombination amplification on a target fragment, carrying out connection transformation and screening verification to obtain a qualified vector; the vector is applied to preparation of cKO / KI gene modified mice, and efficient screening of positive individuals is realized through fluorescence preliminary screening, genotype identification and fluorescence quenching detection. The positive screening workload and cost can be reduced by 70% or above, non-target elements are accurately cut off, interference is avoided, time-space accurate regulation and control are achieved, carrier construction is easy and convenient, repeatability is high, and the method is suitable for gene function research, disease model construction and drug target verification.

The invention discloses pichia pastoris for highly expressing porcine epidermal growth factor fusion protein and application of the pichia pastoris, and belongs to the technical field of fermentation engineering. A porcine epidermal growth factor gene sequence is cloned to a pichia pastoris expression vector pPIC9K-fu through an EcoRI / NotI site to obtain a recombinant vector pPIC-fuEGF, then the recombinant vector pPIC-fuEGF is converted to pichia pastoris GS115, an RT01 strain capable of highly expressing the porcine epidermal growth factor fusion protein is obtained through screening, the preservation number is CCTCC NO: M 2025014, and the recombinant vector pPIC-fuEGF can be used for efficiently expressing the porcine epidermal growth factor fusion protein. The pig epidermal growth factor expression level of the strain in a fermentation tank experiment can reach 600mg / L. According to the porcine epidermal growth factor fusion protein expressed by pichia pastoris, the expression quantity of soluble epidermal growth factors can be increased, and the porcine epidermal growth factor fusion protein has a good application prospect in modern intensive breeding.

The invention relates to the technical field of viruses, in particular to a construction method and application of a porcine parvovirus full-length infectious clone carrying a stable genetic marker. The method comprises the following steps: cloning a complete genome of a porcine parvovirus SXND18 strain to a plasmid pBluescript II SK (+) through an In-fusion seamless cloning technology, so as to obtain a recombinant plasmid pSXND18; meanwhile, a new EcoRI restriction enzyme cutting site is introduced into a genome by utilizing an overlapping PCR (Polymerase Chain Reaction) technology and is used as a genetic marker for identifying a wild virus and a rescue virus; and mixing the recombinant plasmid with a transfection reagent, and transfecting ST cells to obtain the rescue virus rSXND18. The porcine parvovirus reverse genetic operating system established by the invention can be used for research in the directions of porcine parvovirus virulence analysis, cross-species transmission mechanism and the like, and also lays a foundation for research on novel vaccines of the porcine parvovirus.

This invention relates to the field of molecular marker technology, and in particular to a method for detecting maize... MADS1 Primer pairs for gene mutation and their application in screening high-folate maize. The nucleotide sequences of the primer pairs described in this invention are shown in SEQ ID NO.1 and SEQ ID NO.2. This invention, through comparison and analysis of the gene sequences of maize B73 and MADS1 gene mutant materials, discovered a large deletion in the MADS1 gene in the mutant materials. Based on the MADS1 gene deletion, this invention designed a molecular marker primer pair. Using this primer pair to identify the folate content of maize materials, maize with a 234bp band amplified, and which remained 234bp after EcoRI digestion, were identified as high-folate maize. The primer pairs provided by this invention can be used for assisted molecular breeding, providing a rapid detection method for crop folate fortification.

The invention discloses a preparation method of mixed standard plasmids, and belongs to the field of preparation of mixed standard plasmids, and the preparation method comprises the following steps: synthesizing target gene segments with sequence numbers as shown in SEQ ID NO: 1-120 and a pESI-T vector to obtain 120 synthetic plasmids, and respectively inserting the target gene segments into EcoRI loci of the pESI-T vector; dissolving the synthetic plasmids with a TE buffer solution until the final concentration is 25ng / mu L, transforming escherichia coli competent cells, and introducing the synthetic plasmids into the cells to obtain a transformed product; uniformly coating the converted product on an LB flat plate containing corresponding antibiotics; screening positive clones to obtain 120 positive clones; carrying out multiplication culture on 120 positive clones, and extracting plasmids from a culture; verifying the plasmid; and measuring the plasmid concentration, and preparing diluted standard plasmids.

The invention discloses a dsRNA fragment capable of improving wheat amylopectin content and a synthesis method and application thereof, and relates to the field of wheat starch content regulation, pET28a is used as an initial skeleton, a sequence containing a reverse T7 promoter, multiple cloning sites and ISA1 or ISA2 gene fragment is inserted, a recombinant vector is constructed through restriction enzyme digestion connection of restriction enzymes SalI and EcoRI, and the recombinant vector is used as a recombinant vector for improving wheat amylopectin content. And transforming the recombinant expression vector into an escherichia coli HT115 competent cell, and synthesizing the dsRNA fragment after induced expression. According to the invention, a non-transgenic transient silencing technology is adopted, and target gene expression is inhibited in a targeted manner, so that the problems of long period, low efficiency, dependence on complex gene cloning and vector construction processes and potential off-target effect and ecological risk existing in the traditional gene technology are avoided.

The invention discloses a method for preparing dsRNA through large-scale in-vitro transcription based on a magnesium ion feeding strategy. The method comprises the following steps: S1, carrying out enzyme digestion on a plasmid by using EcoRI incision enzyme to obtain a target gene segment with T7 promoters at two ends; s2, performing in-vitro transcription in a transcription system by taking a target gene segment with T7 promoters at two ends as a template; s3, digesting the template to obtain a final product dsRNA; wherein linkage control of pH and Mg < 2 + > is carried out in the in-vitro transcription process in the step S2, the pH value of a transcription system is controlled to be 7.5-7.8 through alkali liquor, and the concentration of Mg < 2 + > in the transcription system is controlled to be 60-120 mM through fed-batch magnesium salt. The invention develops a novel large-scale in-vitro transcription method, magnesium acetate is supplemented in a material supplementing manner to improve the reaction yield, at least 20g of dsRNA can be obtained through one-time reaction, and the problem of low yield of an in-vitro transcription method is solved.

The invention relates to the technical field of non-coding RNA (Ribonucleic Acid) of parasites, and discloses a method for researching regulation and control of variant antigen genes of plasmodium non-coding RNA, which comprises the following steps: S1, constructing a modified plasmid pLN-ENR-GFP by overexpression plasmid: replacing a promoter region with a plasmodium falciparum U6 promoter; after PCR amplification, carrying out HindIII / EcoRI double enzyme digestion on a product, and inserting the product into a pLN-ENR-GFP carrier; adding a 9 * T terminator at the 3'end, introducing EcoRI / ApaI enzyme cutting sites at the two ends, and carrying out enzyme cutting connection: inserting an RUF6-15 fragment into the modified pLN-ENR-GFP vector; s2, synchronization of plasmodium and plasmid transfection and synchronization: taking a plasmodium falciparum in-vitro culture with a plasmodium falciparum body proportion of more than 60% in a cyclic body stage, and adding 5% of D-sorbitol (1 ml of cell volume: 14 ml of sorbitol); according to the method for researching the regulation and control of the variant antigen gene of the plasmodium non-coding RNA, a plasmodium falciparum RUF6-15 transgenic overexpression strain (138nt) is constructed, efficient and stable expression of target ncRNA is realized through optimization of a U6 promoter (SEQ ID NO: 1) and a PolyT terminator, and the technical bottleneck of functional verification of medium-length ncRNA of the plasmodium is broken through.

The invention relates to the technical field of plant genetic transformation, and provides a plant genetic transformation system based on direct delivery, a genetic transformation method and application. An original vector pMKV057 is directionally modified to obtain a modified vector, and the modification method comprises the following steps: replacing an LUC expression cassette of the vector with a modular expression cassette integrated with an epitope tag; the method comprises the following steps: designing multiple cloning sites on a vector, introducing restriction enzyme cutting sites HindIII and EcoRI or HindIII and XbaI, and inserting a Kozak sequence into a 5'end upstream adjacent region of a nucleotide sequence of the multiple cloning sites to obtain a modified vector. And transforming the modified vector into agrobacterium tumefaciens to carry out plant genetic transformation. Through the technical scheme, the problems of low plant genetic transformation efficiency and long transformation time in related technologies are solved.

This invention discloses a recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO: 3. The recombinant humanized type III collagen is an engineered humanized collagen derived from natural type III collagen (NP_000081.2), exhibiting good water solubility and avoiding immune rejection when applied to the human body. Furthermore, this invention provides a method for preparing the recombinant human type III collagen. Using a recombinant strain with a recombinant vector containing a codon-optimized sequence (as shown in SEQ ID NO: 2) inserted between the BamHI and EcoRI sites, high-density fermentation and purification are used to obtain the recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID NO: 3.

The invention discloses a standardized environment-tolerant microbial element library and application thereof, and belongs to the technical field of bioengineering. According to the element library, site modification is carried out on an environment-tolerant gene or a regulatory element thereof, and a prefix and a suffix containing specific enzyme cutting sites are added for sequence standardization and cloning construction, so that a plurality of standardized environment-tolerant microbial elements are obtained and form the element library. Wherein the 5'end of the standardized sequence contains EcoRI and XbaI sites, and the 3 'end of the standardized sequence contains SpeI and PstI sites, so that modular assembly among elements can be realized, and no redundant enzyme cutting sites exist after connection. The element library covers various functional elements such as freezing resistance, high salt resistance, high temperature resistance, acid resistance, heavy metal resistance and the like, microbial hosts can be modified in a plug-and-play mode, the microbial hosts are endowed with physical and chemical environmental stress tolerance or specific functions, and the element library has important application value in the fields of industrial fermentation, biomining industry, environmental governance, agriculture, space biology and the like.

The invention discloses a baculovirus expression vector as well as a construction method and application thereof. The vector takes pFastBacDual as a skeleton, 2-9 repetitive BS sequence modification promoters are connected in series to the downstream of a polh promoter, an extremely advanced transcription factor VLF-1 gene is introduced, and the two components synergistically enhance the transcriptional activity. According to the invention, three types of recombinant vectors pBSX-eGFP, pBSX-eV and pBSX-GV are constructed, and the optimal combination is determined as overexpression of two repeated BS sequences and VLF-1, so that the expression quantity of the target protein can be increased. The vector construction process is standardized, enzyme cutting sites such as BamHI and EcoRI are reserved, and high-expression protein can be obtained after Sf9 cells are transfected and cultured for 4-5 days. The recombinant protein expression efficiency of a baculovirus expression system is improved, the advantages of correct folding and post-translational modification of the recombinant protein are reserved, and the method is suitable for large-scale industrial recombinant protein production scenes such as biopharmacy and vaccine research and development and has important application value.

The invention discloses a kit for rapidly constructing an endothelin converting enzyme ECE1 knock-down plasmid and application of the kit, and relates to the technical field of molecular biology and genetic engineering. Comprising the following components: a high fidelity PCR reagent which comprises # imgabs0 # High-Feliability DNA (deoxyribonucleic acid) Polymerase, a buffer solution and dNTPs (deoxyribonucleoside triphosphates) and is used for amplifying an ECE1 gene; the double enzyme digestion reagent comprises AgeI, EcoRI and a buffer solution thereof, and is used for linearizing the carrier; and the T4 DNA ligase and the buffer solution are used for connecting the ECE1 gene and the vector. By adopting high-fidelity PCR and seamless cloning technologies, the plasmid construction time is remarkably shortened, and the experiment efficiency is improved; according to the method, PCR (Polymerase Chain Reaction) amplification is carried out by using # imgabs1 # High-Felidity DNA (Deoxyribose Nucleic Acid) Polymerase, so that the high fidelity and the specificity of an amplified product are ensured.

The application discloses a kind of recombinant human fibronectin and its coding gene and application.The amino acid sequence of the recombinant human fibronectin is codon-optimized according to the codon preference of Pichia pastoris first, and the nucleotide sequence of the coding gene is obtained as SEQ ID NO.2, then it is cloned into the EcoRI and NotI enzyme cutting sites between pPIC9K of Pichia pastoris expression vector, and the recombinant expression vector pPIC9K-FNc is obtained, and then it is transferred into P.pastoris GS115 expression host cell, and recombinant P.pastoris GS115 / pPIC9K-FNc is obtained by geneticin G418 screening, then the recombinant bacteria are fermented and expressed by methanol induction, and the recombinant human fibronectin is obtained after purification.The fibronectin produced by the application has large molecular weight and better biological activities such as wrinkle resistance and barrier repair.

The invention discloses a recombinant humanized III-type collagen. The amino acid sequence of the recombinant humanized III-type collagen is shown as SEQ ID NO: 3. The recombinant humanized III-type collagen is an engineered humanized collagen, is derived from natural III-type collagen (NP000081.2), has better water solubility, and can avoid immunological rejection when being applied to a human body; the invention also provides a preparation method of the recombinant human III-type collagen, which comprises the following steps: carrying out high-density fermentation and purification on a recombinant strain recombined with a recombinant vector of which the nucleotide sequence is inserted into a codon optimization sequence as shown in SEQ ID NO: 2 between a BamHI site and an EcoRI site to obtain the recombinant humanized III-type collagen as shown in an amino acid sequence as shown in SEQ ID NO: 3.

The invention discloses a method for constructing a recombinant baculovirus for expressing L-amino acid oxidase of yellow-spotted bluefish. The method comprises the following steps: cloning a gene with a DNA sequence as shown in SEQ ID NO: 1 into a vector pMD18-T to construct a recombinant plasmid; using restriction endonucleases EcoRI and XhoI to digest the recombinant plasmid and a donor plasmid, respectively, and ligating the digestion products to obtain a recombinant donor plasmid; transforming the recombinant donor plasmid into Escherichia coli, allowing the target gene to specifically transpose with a baculovirus shuttle vector contained in the Escherichia coli, and screening and extracting the recombinant bacmid; and transfecting the recombinant bacmid into insect cells using a liposome transfection method. After the insect cells become pathological, the recombinant baculovirus is harvested.

The invention relates to the technical field of gene engineering, and provides a histone H1.4 NG end recombinant nucleic acid fragment which is formed by sequentially connecting an EcoRI restriction enzyme cutting site, a Myc tag coding sequence and a core nucleic acid sequence from the 5'end to the 3 'end, and the 3' end is connected with a BamHI restriction enzyme cutting site; the core nucleic acid sequence is as shown in SEQ ID NO. 1. The invention also provides a primer pair for amplifying the recombinant nucleic acid fragment. Meanwhile, the invention also provides a recombinant vector containing the recombinant nucleic acid fragment, a lentivirus, a cell model and application of the recombinant nucleic acid fragment in preparation of breast cancer cell proliferation and migration inhibition products. According to the invention, the NG end of the specific functional structural domain of H1.4 is locked for the first time, a biological material related to the NG end of histone H1.4 and application of the biological material in breast cancer cell inhibition are developed, and the application has important significance in enriching breast cancer targeted regulation strategies and researching and developing novel tumor inhibition products.

The present application relates to a kind of based on miR-30 skeleton and its construction method and its application in RNAi based on miR-30 skeleton.The skeleton construction method includes the following steps, with existing shRNAmiR30 vector as PCR template;Design two pairs of siRNA containing target gene complementary overlapping primers, respectively, primer F1 and R1, primer F2 and R2, respectively, to obtain P1, P2 as template, with primer F1 and R2 as upper and lower stream primers to carry out OE-PCR, obtain miR-OE.Further, the construction method of the present application also carries out the reply mutation of stem-loop structure and EcoRI enzyme cutting site in miR-OE skeleton.The miR-OE skeleton of the present application and the primer required to be synthesized in the construction method of miR-OE-EL are all short chain with length less than 60nt, and the difficulty and cost of synthesis are effectively reduced, and primer design is more flexible;And the interference efficiency of miR-OE-EL skeleton to target gene is significantly improved, and can be well applied to the preparation of shRNA drug.

The invention relates to a cucumber seed specific promoter pSEP5 and application thereof. The invention further discloses an expression vector pCAMBIA2300-enhance35S-pSEP5-DsRed2-d35S-eGFP containing a candidate promoter pSEP5, the expression vector is characterized in that restriction enzymes BamHI and EcoRI are used for carrying out linear treatment on a vector pCAMBIA2300-enhance35S-LTP2-DsRed2-d35S-eGFP, the vector pCAMBIA2300-enhance35S-LTP2-DsRed2-d35S-eGFP is used for constructing a vector pCAMBIA2300-enhance35S-pSEP5-DsRed2-d35S-eGFP with fragments pSEP5 and fragments DsRed2 through a homologous recombination method The invention also discloses application of the cucumber seed specific promoter pSEP5 in driving expression of a cucumber SPT system seed fluorescent module and in cucumber hybrid seed production.