21 results about "Molecular cloning" patented technology

The invention discloses a bone targeting forsythiaside A loaded exosome and a preparation method and application thereof, and relates to the technical field of biomedicine.The preparation method comprises the following steps that bone marrow mesenchymal stem cells are separated and cultured, and GLG1 is over-expressed through a molecular cloning technology to obtain GLG1-BMSCs; the exosome GLG1-Exos of the GLG1-BMSCs is collected through an ultracentrifugation method; and loading forsythiaside A into the exosome through an ultrasonic method and an extrusion method to obtain the GLG1-Exos-FTA. The traditional Chinese medicine active ingredient forsythiaside A is loaded, and forsythiaside A is delivered to bone marrow in a targeted manner, so that the effect of preventing and improving diabetic osteoporosis is achieved; due to the targeting effect, the bioavailability of forsythiaside A can be enhanced, and meanwhile, the curative effect of resisting diabetic osteoporosis is improved. Experiments show that the loading rate of forsythiaside A of the GLG1-Exos-FTA reaches 36.9%, the particle size distribution peak value is 122.4 nm, and the GLG1-Exos-FTA has typical exosome characteristics and bone targeting, and can significantly improve the bone microstructure of diabetic osteoporosis mice.

The invention belongs to the technical field of animal reproductive biology and cell engineering, and particularly relates to a fluorescence labeling expression system for visualizing an oocyte nucleus maturation process and application of the fluorescence labeling expression system. The fluorescence labeling expression system comprises an eukaryotic expression vector pVenus-LMNB1; the eukaryotic expression vector pVenus-LMNB1 comprises a nucleotide sequence for coding the fusion protein Venus-LMNB1. According to the invention, a nuclear fiber layer protein stably expressed on a nuclear membrane is selected as a research object, a eukaryotic expression vector pVenus-LMNB1 is constructed by using a molecular cloning technology, an LMNB1 gene containing a green fluorescent label Venus is overexpressed in a porcine oocyte by using a microinjection technology, fluorescent protein labeling of a structural protein on the nuclear membrane is utilized, and the expression vector pVenus-LMNB1 is constructed. The expression positioning condition of the LMNB1 in the maturation process of the porcine oocytes is observed, and a powerful tool is provided for development of in-vitro culture related researches of the porcine oocytes.

This invention relates to the fields of molecular cloning and protein expression, and particularly to a Pichia pastoris engineered strain expressing full-length human type III collagen and its applications. The engineered Pichia pastoris strain is obtained by cloning the enzymatically digested human type III collagen gene sequence into an expression vector to obtain a recombinant expression vector, which is then transformed into Pichia pastoris. This engineered Pichia pastoris strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 6, 2024, with accession number CGMCC NO.32510. The Pichia pastoris engineered strain obtained by this invention can efficiently produce the desired human type III collagen through fermentation, exhibiting excellent characteristics such as high yield and absence of endotoxins.

The invention belongs to the technical field of bioengineering and analytical chemistry, and provides a probe eZinCh-2 modified plasmid based on FRET (Fluorescence Resonance Energy Transfer) and application of the probe eZinCh-2 modified plasmid in zinc ion detection. According to the invention, the plasmid is constructed through molecular cloning and homologous recombination technologies, and is microinjected into a zebrafish embryo, so that a transgenic strain capable of stably inheriting and expressing the probe protein is successfully obtained; as a living body biological tool, the zebrafish of the strain is combined with a confocal microscope spectrum FRET technology, and real-time and quantitative monitoring and visual imaging of dynamic changes of zinc ions at living body, cell and tissue levels are achieved. The method solves the problem that real-time dynamic visual monitoring of the zinc ions cannot be realized in a living body model in the prior art, and is suitable for real-time microscopic quantitative analysis of the content of the zinc ions in model organisms such as zebra fish and the like.

The invention provides an UTR element Hh and a construction method and application thereof, and relates to the technical field of mRNA. Ribosome load prediction and secondary structure optimization are respectively carried out on 5 'UTR of a gene of which the NCBI number is AF339412.1 and 5' UTR of a gene of which the NCBI number is MK475748.1 through a PaddleHelix platform, and an inhibitory hairpin structure is avoided by the obtained optimized sequences, so that the luciferase expression quantity is obviously improved compared with that of natural UTR. On the basis of sequence optimization, HBB and hbb are directionally assembled through a molecular cloning technology, an HBB-hbb chimera (Hh) is constructed, the DNA sequence of the Hh is as shown in SEQ NO.1, the RNA sequence of the Hh is as shown in SEQ NO.2, and the protein expression quantity is further improved.

The invention discloses an antibiotic-free screening gene expression system, plasmid, engineering bacteria and application thereof, and relates to the technical field of bioengineering. The expression system provided by the invention comprises a first strain, a second strain, a third strain and plasmids of antibiotic-free screening genes, the first strain, the second strain and the third strain are escherichia coli strains with a thyA gene knocked out, the first strain is used for molecular cloning, the second strain is used for expression of target protein, and the third strain is used for biosynthesis of natural products such as polyketone and non-cellulose peptide; a thyA complementary gene expression cassette is inserted into the plasmid of the antibiotic-free screening gene, and a resistance gene is knocked out; the thyA complementary gene expression box contains a thyA gene.

The invention belongs to the field of molecular biology, and particularly provides a PCR (Polymerase Chain Reaction) amplification method capable of directly generating sticky end products in a PCR process and application. The novel double-stranded sticky end primer is matched with the high-fidelity polymerase without 5 '-3' end excision enzyme activity, and a DNA product with a sticky end structure can be generated in one step in the PCR process, so that the efficiency of connecting DNA in molecular cloning or sequencing library building is remarkably improved.

The present application relates to the recombinant microorganism for producing 3-amino propanol by fermentation method and its construction method and application. Specifically, the present application provides a construction method of the recombinant microorganism for producing 3-amino propanol, which is introducing the coding gene of omega-transaminase into the microorganism producing 3-hydroxy propionaldehyde, wherein the omega-transaminase can catalyze the conversion of 3-hydroxy propionaldehyde into 3-amino propanol in the presence of amino donor. The present application introduces the omega-transaminase capable of catalyzing the synthesis of 3-amino propanol with 3-hydroxy propionaldehyde as the substrate into the microorganism producing 3-hydroxy propionaldehyde based on gene editing, molecular cloning and other technologies, which first creates a brand new full-path biosynthesis process route of 3-amino propanol.

The application discloses application of insect adult cytochrome b5 in pest control and belongs to the field of plant protection. Through a molecular cloning technique, the application successfully obtains full-length sequences of two Cyt b5 genes Cyt b5v1 and Cyt b5v2, and confirms that the two genes are in a high expression state in a whitefly resistant population. Double-stranded RNAs, namely dsCyt b5v1 and dsCyt b5v2, are synthesized by using an RNA interference (RNAi) technique, and after feeding treatment, the expression of target genes is significantly inhibited, and the sensitivity of the whitefly to different neonicotinoid insecticides is effectively improved. The application has the core advantages of low toxicity, high efficiency and high safety while realizing high insecticidal activity, and provides important technical support for a green pest control system in a field.

The invention discloses a serine protease inhibitor SPINK8 as well as a preparation method and application thereof in acute lung injury caused by sepsis, and belongs to the fields of molecular biology, functional protein technology and biological medicine. The amino acid sequence of the SPINK8 protein is as shown in SEQ ID No. 1. According to the invention, the SPINK8 recombinant vector is constructed through molecular cloning for the first time, and the reSPINK8 recombinant protein is obtained through prokaryotic expression and purification. When the acute lung injury caused by sepsis occurs, the reSPINK8 protein can remarkably inhibit lung inflammatory response and improve the pathological state of the lung, and has immunoregulation and protection effects on the acute lung injury caused by sepsis. In view of the advantages that SPINK8 is small in molecular weight, easy to express and purify and easy to prepare medicines, the SPINK8 has important application value in prevention and treatment of sepsis acute lung injury and targeted medicine screening.

This invention belongs to the field of genetic engineering and relates to a method for preparing amylase derived from halophilic archaea and its application; based on halophilic archaea... Haloarcula sp. ZJK10-12、 Haladaptatus The genes for α-amylase, maltose amylase, and glucoamylase were obtained from the whole genome sequence of sp. ZJK29-3. Recombinant plasmids were constructed using primer design and molecular cloning, and transformed into prokaryotic host bacteria to obtain the corresponding recombinant strains. The bacteria were enriched and cultured in Hv-YPC liquid medium, and the cells were collected and lysed by sonication. α-amylase, maltose amylase, and glucoamylase were obtained using nickel column affinity chromatography. The amylases exhibit excellent enzymatic properties and outstanding salt tolerance. Their application in the fermentation of high-salt, dilute soy sauce can improve raw material utilization and product quality, demonstrating good industrial applicability and promotional value.

The application discloses a sequencing data calibration system and method based on microbial amplicon sequencing, and belongs to the field of microbial detection and bioinformatics analysis. The application takes ddPCR technology as the core, first screens common human intestinal microorganisms, designs and obtains 14 reconstructed sequences containing bacterial 16S and fungal 18S / ITS, prepares high-purity DNA standard substances through chemical synthesis and molecular cloning, completes standardization and uniformity verification, then designs high-specificity primers and probes, optimizes ddPCR reaction conditions, establishes a calibration method, completes uncertainty evaluation through joint calibration of 9 laboratories, and prepares multi-target amplicon abundance standard substances. The application realizes the transformation of amplicon sequencing data from relative quantification to absolute quantification, corrects the system error of the whole sequencing process, improves the comparability and reliability of cross-platform and cross-batch data, and provides technical support for the standardization of microbiome research.

The application belongs to the technical field of biotechnology, and particularly discloses a DNA molecular cloning method based on GgeAgo and application thereof. The method comprises the following steps: a vector plasmid is cut by a cutting system; the cutting product of the vector plasmid is mixed with an exogenous fragment with a homologous region, and then a recombination-based cloning method is used for assembly, followed by transformation of Escherichia coli to generate a recombination plasmid. The cutting system comprises a guide nucleic acid molecule and a programmable endonuclease GgeAgo or / and a mutant thereof. The GgeAgo cuts the plasmid under the guidance of the guide nucleic acid molecule to linearize the plasmid, an exogenous fragment with a homologous region is added, and then Escherichia coli is transformed to realize efficient and accurate insertion of the exogenous fragment into the target plasmid. The molecular cloning method provided by the application has high efficiency, short time and simple operation, and effectively reduces the mutation caused by PCR. The molecular cloning method provided by the application provides a new idea for the Ago-based molecular cloning technology and the synthesis and assembly technology of DNA.

The application discloses a beta-1, 6-glycosidic bond hydrolytic enzyme and application thereof in traditional Chinese medicine resources. The method screens a key gene sequence with a function of hydrolyzing beta-1, 6-glycosidic bond from a Candida parapsilosis L7 genome, and names the key gene sequence as CoExg1. A gene engineering bacterium capable of efficiently heterologously expressing CoExg1 is constructed by using a molecular cloning technology. After fermentation and enzyme production, an enzyme catalytic reaction system is established, and a traditional Chinese medicine active molecule in mogroside V is directionally converted, beta-1, 6-glycosidic bonds in different regions of a substrate structure are selectively hydrolyzed, and therefore, efficient and phased synthesis of different products such as sianoside I and mogroside IIIE is realized. In addition, CoExg1 can also hydrolyze beta-glycosidic bonds in the structures of icariin and lupeol, and directionally synthesize icariin II and lupeol, thereby providing important technical support for high-value utilization of traditional Chinese medicine resources and quality improvement and efficiency enhancement of traditional Chinese medicine glycosides.

The invention relates to the technical field of plant genetic engineering, and particularly provides molecular cloning of a rice endosperm specific promoter pROLM24 and application of the rice endosperm specific promoter pROLM24. Three tissue-specific promoters with nucleotide sequences as shown in SEQ ID NO.1-3 are separated from a rice alcohol-soluble protein gene, the three tissue-specific promoters are fused with an RUBY visual marker, and a result shows that the content of betacyanin in an expression product is measured to be up to 2.9% of the dry weight of rice seeds. The method has very high application value in the field of plant synthetic biology. The invention further discloses application of the promoter in the aspects of improving crop seed quality, improving crop traits, cultivating new varieties of transgenic plants by using key elements of the promoter or expressing high value-added proteins by using a seed bioreactor and the like.

The application discloses a construction method and application of a modular gene editing vector. Specifically, a modular vector assembly system is developed based on a type II restriction enzyme mediated Golden Gate molecular cloning technology. Since the type II restriction enzyme has the characteristics of different recognition sites and cutting sites, different cohesive ends can be generated by using the same type II restriction enzyme, and seamless cloning can be realized by using a T4 ligase. The whole operation of the system only needs two enzymes AarI and BsmBI , simplifies the experimental process, reduces the cost, improves the stability of the whole experimental process, and retains an assembly site of BsmBI on the final vector bone, so that multiple elements can be expanded without reengineering assembly, and more assembly requirements can be flexibly realized. Therefore, the modular gene editing vector has a good application prospect in plant gene editing or breeding.

The present invention relates to a recombinant microorganism producing 3-aminopropanol by means of a fermentation method, a construction method therefor, and a use thereof. Specifically, the present invention provides a method for constructing a recombinant microorganism producing 3-aminopropanol. The method comprises: introducing an encoding gene for ω-transaminase into a microorganism producing 3-hydroxypropionaldehyde, wherein the ω-transaminase is capable of catalyzing the conversion of 3-hydroxypropionaldehyde into 3-aminopropanol in the presence of an amino donor. On the basis of techniques such as gene editing and molecular cloning, the present invention introduces ω-transaminase, which is capable of catalyzing the synthesis of 3-aminopropanol from 3-hydroxypropionaldehyde as a substrate, into a microorganism producing 3-hydroxypropionaldehyde, thereby creating a brand-new process route for full-path biosynthesis of 3-aminopropanol for the first time.

The invention discloses application of adult insect cytochrome b5 in pest control, and belongs to the field of plant protection. According to the invention, the full-length sequences of the Cyt b5v1 and Cyt b5v2 of the two genes Cyt b5 are successfully obtained through a molecular cloning technology, and the high expression state of the two genes Cyt b5v1 and Cyt b5v2 in a bemisia tabaci resistance population is proved. Double-stranded RNA (Ribonucleic Acid), namely dsCyt b5v1 and dsCyt b5v2, is synthesized by utilizing an RNA interference (RNAi) technology, and after feeding treatment, the expression of a target gene is remarkably inhibited, and the sensitivity of bemisia tabaci to different neonicotinoid insecticides is effectively improved. According to the present invention, the efficient insecticidal activity is achieved, the ecological environment safety is considered, the core advantages of low toxicity, high efficiency, high safety and the like are provided, and the important technical support is provided for the green prevention and control system of the field pests.

The invention relates to the technical field of biology, in particular to an insulating double-fluorescence reporting system for quantitatively measuring terminator efficiency and a construction method and application thereof. According to the method, RiboJ ribozyme is introduced, mRNA is automatically cut after transcription to eliminate errors caused by translation initial efficiency differences, a stable endogenous promoter is adopted to control fluorescent protein mCherry expression, internal initiation codon removal optimization is carried out on fluorescent protein, generation of truncated protein is avoided, and signal reliability is improved. The method is suitable for various prokaryotic hosts, the terminator efficiency from weak to strong can be accurately measured, the dynamic range is wide, the sensitivity is high, only conventional molecular cloning and flow cytometry are needed in operation, and an efficient and accurate evaluation method is provided for the terminator efficiency.

The molecular cloning device comprises a pipetting workstation, a peripheral equipment island platform used for storing and transferring consumables and a moving station capable of interacting samples with a central mechanical arm, the peripheral equipment island platform comprises an island platform mechanical arm, and a PCR instrument, a film sealing and tearing device and a centrifugal machine are arranged at the moving station; the plasmid extraction and cell transformation plate coating workstation is used for extracting plasmids and transforming competent cells for plate coating; the bacterial colony picking instrument is used for picking bacterial colonies in the culture dish into the liquid culture medium of the deep hole plate; the constant-temperature and constant-humidity automatic incubator is used for automatically culturing competent cells under a constant-temperature and constant-humidity condition; and the central mechanical arm is used for transferring samples and / or consumables among the mobile station, the plasmid extraction and cell transformation plate coating workstation, the bacterial colony picking instrument and the constant-temperature and constant-humidity automatic incubator. The automation degree can be improved, and pollution introduction is reduced.

The present application relates to a kind of methods for surface immobilization of bacteriophage lytic enzyme that infects gram-negative bacteria, the present application utilizes bacteriophage lytic enzyme LysSSE1 that can infect G-bacteria, by molecular cloning technology, amino acid type connector and / or affinity ligand SiAP1 / SiAP2 screened from database are introduced from the perspective of molecular biology, construct recombinant lytic enzyme containing peptide chain, then incubated with NaCl and Tween 20, 100 rpm oscillation 30-60 min.The method of the present application successfully realizes the stable and efficient sterilization of lytic enzyme in external environment, the immobilized enzyme containing amino acid chain obtained not only maintains the activity of original lytic enzyme, but also improves its resistance to environmental change and repeated sterilization ability, solves the problem of difficult addition of chemical type connector and environmental unfriendliness etc.It has important practical significance for effectively controlling the surface attachment pollution of G-bacteria in environment.