33results about How to "Increase positive rate" patented technology

The invention discloses a construction method of an agrobacterium tumefaciens mediated masson pine genetic transformation system, and belongs to the technical field of genetic engineering. According to the construction method of the agrobacterium tumefaciens-mediated masson pine genetic transformation system, immature masson pine cones are collected, embryogenic calluses are induced and cultured, the embryogenic calluses of the masson pine in a PEM III stage are used as a material, suspension pre-culture is performed to obtain an acceptor material, and the acceptor material is subjected to genetic transformation to obtain the agrobacterium tumefaciens-mediated masson pine genetic transformation system. The method comprises the following steps: firstly, carrying out tissue culture on a transgenic somatic embryo, infecting by adopting an agrobacterium infection solution with an OD600 value of 0.2-0.6, carrying out co-culture, recovery culture and screening culture, carrying out histochemical staining by adopting GUS, carrying out molecular identification to obtain a transgenic embryogenic callus, and carrying out maturation treatment to finally obtain the transgenic somatic embryo. The masson pine genetic transformation system established by the invention is stable in transformation performance and high in transformation positive rate, and when an acceptor material is masson pine embryonic callus with the genotype of 1-3-5, the transformation positive rate is as high as 100%.

The invention discloses an artificial antigen presenting cell for efficiently preparing a chimeric antigen receptor T cell (CAR-T) and a construction method of the artificial antigen presenting cell, and belongs to the field of cell therapy and immunotherapy, the artificial antigen presenting cell takes a K562 cell as a platform, an anti-CD3 single-chain antibody, an anti-CD28 single-chain antibody, a membrane binding type cell factor and a costimulatory ligand are stably expressed through genetic engineering, and the chimeric antigen receptor T cell (CAR-T) is obtained. And the local concentration of the anti-CD3 antibody is improved by adopting an HOTag3 tool. The multi-signal integration strategy obviously enhances the activation and proliferation capacity of the T cells, promotes the CAR-T cells to maintain a low differentiation state, and endows the CAR-T cells with excellent anti-tumor activity and durability. The artificial antigen presenting cell provides a novel efficient tool for the field of cellular immunotherapy, and is expected to promote development of more efficient and safer CAR-T therapy to cope with challenges of various malignant tumors.

The invention relates to a preparation method of CAR-T cells and a product thereof. A method for preparing a CAR-T cell according to the present invention comprises the step of introducing a cyclic mRNA into a T cell that does not contain a chimeric antigen receptor; and, the cyclic mRNA comprises a first cyclic mRNA encoding a chimeric antigen receptor; in addition, the first annular mRNA comprises the following sequence (a) or (b): (a) a translation starting sequence and a coding sequence; and (b) translating the starting sequence, the coding sequence and the insertion element sequence. In the method, the transfection efficiency of the annular mRNA is high, and the positive rate is as high as 90%; compared with preparation methods of viruses, transposon systems and the like, the process is simple, the production cost is greatly reduced, and the safety is also improved; compared with linear mRNA, the protein expression time is greatly prolonged.

The invention discloses an improved method for screening gene knockout porcine fetal fibroblasts. The method comprises the following steps: (1) designing and synthesizing sgRNA of a target gene; (2) knocking out the Dock8 gene of the porcine fetal fibroblast; (3) carrying out flow sorting to enrich positive cells; and (4) carrying out subcloning. According to the method, the gene editing targeting efficiency of the CRISPR / Cas9 system can be improved, the positive rate of cell cloning is obviously improved, and gene editing operation can be efficiently carried out.

The invention provides an agrobacterium rhizogenes-mediated papaya hairy root genetic transformation method and a papaya transgenic positive plant, and belongs to the technical field of plant genetic engineering. The invention provides a papaya genetic transformation method independent of tissue culture, which can directly utilize agrobacterium rhizogenes to treat explants for culture to obtain papaya transgenic positive plants without a series of tedious and complicated sterile processes such as callus induction and induced differentiation rooting. The problems of long tissue culture transformation period, large manpower and material resource consumption, low positive rate and the like are avoided. The constructed agrobacterium rhizogenes mediated genetic transformation system has the characteristics of high efficiency, stability and rapidness, the agrobacterium rhizogenes K599 cultured by using the solid TY culture medium can directly infect wounds, the operation is simple, the hair root incidence rate and the positive rate are relatively high, additional experimental equipment is not needed, and the method is suitable for most laboratories.

The invention discloses an induction method for obtaining a transgenic persea americana composite plant without tissue culture, and belongs to the technical field of persea americana biology. The induction method provided by the invention comprises the following steps: germinating avocado seeds by using quartz sand to obtain avocado seedlings, infecting rootless seedlings from the avocado seedlings by adopting a vacuum infiltration method, and inserting the rootless seedlings subjected to dip dyeing into a sterilized coco coir matrix for culturing to obtain the avocado composite plant. According to the method, an efficient avocado genetic transformation system mediated by agrobacterium rhizogenes is established, tissue culture is not needed, rootless seedlings are adopted as materials, the transformation efficiency is improved, meanwhile, consumption of experimental equipment, consumables and manpower is reduced, the problem that the avocado genetic transformation system is not established yet is solved, and the method is suitable for popularization and application. The technical effect of quickly, efficiently, simply and easily obtaining the transgenic material with low consumption is achieved, and a more convenient and efficient way is provided for genetic transformation of avocados.

The application provides an intelligent agent-based whole-process antibody drug design optimization system and method, which comprises the following steps: receiving an antibody design requirement input by a user through an intelligent agent, performing intention recognition, task classification and parameter extraction on the input by the intelligent agent, and automatically constructing an antibody design task plan; structural modeling and combination hotspot analysis; generating a brand new antibody skeleton or optimizing an existing antibody skeleton; CDR sequence filling and candidate sequence generation; structure verification and affinity evaluation; humanization design and developability evaluation; candidate antibody sorting and result output; and generating a comprehensive design report. The application is used for guiding a user to complete a complete process from a target point to a high-affinity humanized antibody candidate sequence.

The invention relates to the technical field of medical instruments, in particular to a novel puncture biopsy needle applied through an ultrasonic bronchoscope, which comprises an ultrasonic bronchoscope body, a shooting part, an excitation part and a biopsy part, the shooting part comprises a shell, a fixing clamp, an inclined convex block, a driving hammer, a thick spring, a firing pin, a delay top ring and a thin spring, the biopsy part comprises an outer sheath, an inner core and a biopsy inner needle. The driving hammer is stimulated to eject the fixing clamp open, so that the firing pin impacts the inner core to drive the biopsy inner needle to enter a septum focus beside a trachea or a bronchus of a patient, and the thin spring delays to drive the delay ejection ring to impact the sheath to cut off the focus, so that the puncture force does not need to be manually controlled or the focus is repeatedly pulled and inserted; the biopsy of the mediastinal focus beside the trachea or the bronchus can be completed by the aid of the biopsy forceps, the positive rate of obtaining effective specimens is increased, the operation difficulty of medical staff is reduced, the biopsy forceps are efficient and rapid, and complications are reduced.

The present invention belongs to the field of genetic engineering and molecular biology, and specifically relates to a simple, fast and efficient method for constructing an RNA interference vector and its application. The method specifically includes designing specific primers with homology arms and restriction sites, amplifying exogenous DNA fragments using the specific primers, and using restriction endonucleases to Bam HI and Kpn Ⅰ Enzyme cut the RNA interference empty vector, and use the homologous recombination method to connect the exogenous DNA fragment to the RNA interference empty vector to form the RNA interference final vector. This method introduces a lethal gene on the RNA interference backbone vector. Combined with the homologous recombination method, only one homologous recombination is needed to simultaneously connect two exogenous DNA fragments into the two multiple cloning sites of the RNA interference vector, which greatly shortens the RNA interference vector construction cycle, and the vector construction positive rate reaches 100%. In addition, the selection of exogenous fragments is no longer limited by the restriction site, and can be freely selected, which broadens the range of targets.

This invention discloses the application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma. Immunohistochemical experiments revealed that ABCC1 protein expression in nasopharyngeal carcinoma tumor tissues is higher than in adjacent normal tissues. The positive rate of ABCC1 staining in advanced-stage patients is significantly higher than in early-stage patients, and the proportion of patients with high ABCC1 expression is also significantly higher in patients with poor prognosis than in patients with good prognosis. Therefore, this invention further designs ABCC1 inhibitors targeting ABCC1, finding that knocking down or silencing ABCC1 significantly inhibits the proliferation, migration, and invasion of nasopharyngeal carcinoma cells; simultaneously, inhibition of ABCC1 function effectively enhances CD8... + The immune effector function of T cells. Therefore, ABCC1 can be used in the diagnosis and treatment of nasopharyngeal carcinoma.

The invention discloses enterococcus faecium, a microbial inoculum as well as application and a method of the enterococcus faecium, the enterococcus faecium is enterococcus faecium TC3 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the enterococcus faecium is CCTCC NO: M 20242431. The invention also provides a microbial inoculum, wherein the microbial inoculum contains the enterococcus faecium TC3, and the enterococcus faecium TC3 can be used for preparing the microbial inoculum. Before a healthy pig is inoculated with a porcine reproductive and respiratory syndrome vaccine, the enterococcus faecium TC3 or the microbial inoculum is fed to the healthy pig, and then the healthy pig is inoculated with the porcine reproductive and respiratory syndrome vaccine, so that the effect performance of the pig inoculated with the porcine reproductive and respiratory syndrome vaccine can be improved, the antibody positive rate of the pig inoculated with the porcine reproductive and respiratory syndrome vaccine is increased, and the economic benefit of the pig is improved. The protection rate of the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is increased, the antibody dispersion of the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is reduced, and the survival rate of the pigs infected with the porcine reproductive and respiratory syndrome virus after the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is increased.

The present invention relates to the field of crop genetic breeding, and in particular to a method for inducing guava hairy roots without tissue culture. The present invention uses pretreated guava seedlings as explants, uses Agrobacterium rhizogenes carrying a reporter gene and a target gene to infect the explants, and then transfers the infected explants to a culture matrix for cultivation, thereby successfully establishing a simple, low-cost and efficient genetic transformation system for obtaining transgenic materials. Compared with traditional guava genetic transformation methods, the induction method provided by the present invention does not require tissue culture, improves transformation efficiency, shortens the transformation cycle, and also reduces consumables and manpower consumption, thereby solving the problems of complex experimental operation, large-scale equipment and consumables, long cycle and low success rate in the original guava genetic transformation technology, and provides a convenient and efficient tool for the study of guava gene functions (especially root-specific expression of gene functions).

Provided is a method for modifying a cell, which comprises the following steps: transfecting a target molecule and a screening marker molecule to a cell in a non-viral manner, so that the cell expresses the target molecule and the screening marker molecule. Also provided is a modified cell obtained using the method. The method can effectively enrich the desired cell and increase cell positivity with respect to the target molecule.

The present invention relates to a primer, probe combination and detection kit for identifying Ochrobacterium hominis. The present invention adopts a one-step fluorescence quantitative PCR detection technology with three genes and dual fluorescent group labels, which can detect and identify nucleic acid DNA of Ochrobacterium genus and Ochrobacterium hominis species and strains in pure bacteria and blood and other specimens, with high sensitivity, and can detect 3.67fg of nucleic acid DNA (Ct value is 35.38), that is, the PCR reaction system can detect as long as there is 1 nucleic acid DNA copy number. The three genes are used for simultaneous detection, which greatly improves the detection rate of nucleic acid DNA of Ochrobacterium genus and Ochrobacter hominis in blood and other specimens. The kit is simple to operate, does not require gel electrophoresis, and after the bacterial genomic DNA of the specimen is extracted, the detection and identification results can be reported in 1 hour, which is particularly suitable for the detection and identification of trace nucleic acid DNA in blood and other specimens.

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-Human CD24 monoclonal antibody as well as a preparation method and application thereof. According to the development method of the Human CD24 monoclonal antibody, the defects of a hybridoma technology and a phage display technology are avoided, the development time of a target antibody is shortened, and the prepared anti-Human CD24 monoclonal antibody is high in affinity and has good antigen-antibody binding activity on the molecular level and the cellular level.

The invention discloses a novel agrobacterium tumefaciens-mediated injection method applied to instantaneous transformation of strawberry fruits, and belongs to the technical field of genetic engineering. By optimizing the agrobacterium injection method of strawberries and selecting injection varieties, the dyeing diffusion effect and the injection effect of injected fruits are remarkably improved, and the positive rate of instantaneous transgenic strawberry fruits is remarkably increased. Jiande red strawberries with a longer ginkgo period are selected as injection strawberry varieties, the characteristics are more obvious, and material selection is easy; according to the method disclosed by the invention, injection is carried out from the fruit stems of the strawberries, and genes carried by agrobacterium are injected into the central pith of the fruits through an injector, so that the genes are diffused in the whole fruits, the direction of vascular bundles of the strawberries cultivated under normal conditions is consistent, the diffusion of the injection is facilitated, and the method has important significance for enhancing the effect of instantaneous transformation of the strawberries.

The application belongs to the technical field of biology and specifically relates to an anti-CD25 monoclonal antibody, a preparation method thereof and application thereof. The anti-CD25 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region VH comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO: 1-3. The light chain variable region VL also comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO: 4-6. The application further provides a preparation method of the anti-CD25 monoclonal antibody. The monoclonal antibody development method has the advantages of short time consumption, high throughput and high positive rate.

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-human TIGIT monoclonal antibody and application thereof. The anti-human TIGIT monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 of which the amino acid sequences are as shown in SEQ ID NO: 1-3; the light chain variable region comprises a VLCDR1, a VLCDR2 and a VLCDR3 of which the amino acid sequences are shown as SEQ ID NO: 4-6. The invention also provides an application of the anti-human TIGIT monoclonal antibody in tumor immunotherapy, preparation of a reagent for detecting the TIGIT protein or preparation of a product combined with the human TIGIT protein. The anti-human TIGIT monoclonal antibody has the technical effects that the novel anti-human TIGIT monoclonal antibody is obtained through screening, and the antigen binding activity of the anti-human TIGIT monoclonal antibody is better than that of the existing anti-human TIGIT monoclonal antibody. According to the antibody development method provided by the invention, the development time of the target antibody is shortened, the flux is high, the positive rate is high, and the antigen binding activity is better.

The present invention belongs to the field of molecular biology technology, and specifically relates to an anti-human CD24 monoclonal antibody, a preparation method thereof and an application thereof. The development method of the human CD24 monoclonal antibody provided by the present invention avoids the shortcomings of hybridoma technology and phage display technology, shortens the time-consuming development of the target antibody, and the prepared anti-human CD24 monoclonal antibody has strong affinity and good antigen-antibody binding activity at both the molecular and cellular levels.

The present invention relates to the field of genetic engineering technology, and in particular to a method for constructing a porcine BECN1 gene knockout cell and its application. First, a guide RNA with a restriction site for the BECN1 gene and its complementary sequence are designed and synthesized, and the sgRNA and its complementary sequence are connected into a double-stranded sgRNA fragment using the principle of base complementary pairing. A recombinant CRISPR / Cas9 plasmid is constructed by molecular cloning and recombination technology, and the recombinant plasmid is transfected into porcine lung epithelial cells. An sgRNA verification primer is designed to verify the sgRNA activity, and then a positive monoclonal cell line is screened by puromycin drug, and DNA and protein of the monoclonal cell line are extracted, and BECN1 gene sequencing and protein expression detection are performed, and finally PPE cells with BECN1 gene knockout are obtained. This method is simple and efficient, and the constructed knockout cell line can be widely used in the study of BECN1 gene function and related pathways.

The application provides a Lemna australis endogenous strong promoter, a recombinant vector containing the Lemna australis endogenous strong promoter, and application of the Lemna australis endogenous strong promoter and the recombinant vector in starting expression of a target gene in monocotyledonous plants and dicotyledonous plants. The Lemna australis endogenous strong promoter provided by the application has high activity in monocotyledonous plants and dicotyledonous plants, can maintain high activity under conditions of different concentrations of screening agents, and basically does not affect expression of adjacent genes under conditions of different concentrations of screening agents. The application can solve problems of the 35S promoter widely used in the prior art, such as difficulty in further improving activity, low activity in monocotyledonous plants, and influence on expression of adjacent genes.

The invention belongs to the technical field of medicine and biology, and particularly discloses application of a fibroblast BALB / c-3T3 overexpressing specific protein in preparation of hybridoma. On one hand, the invention provides an application of the fibroblast BALB / c-3T3 in preparation of a hybridoma cell strain; on the other hand, the invention provides an application of the fibroblast BALB / c-3T3 overexpressing the specific protein in a hybridoma cell strain; the invention further provides a preparation method of the hybridoma based on the fibroblast BALB / c-3T3. According to the present invention, the target hybridoma can be directly obtained by using the specific protein overexpressed fibroblast BALB / c-3T3 immunization, the high positive rate is provided, and the hybridoma cell strain can be prepared without the protein + adjuvant immunization mode by singly using the target hybridoma cell strain, such that the specific protein overexpressed fibroblast BALB / c-3T3 immunization can be easily achieved; different target hybridomas can be prepared by changing the types of overexpressed target proteins according to research requirements, and in addition, the comprehensive cost can be reduced to half of the original cost.

This invention discloses a nucleic acid molecule encoding a modified retroviral Gag protein and its applications. The Gag protein contains an engineered acylated tag sequence at its N-terminus, comprising: a glycine residue at the second position after the initiating methionine for myristylation modification; and / or a cysteine ​​residue in the N-terminal region for palmitoylation modification. The tag sequence is selected from SEQ ID NO:1, 3-9, or derivative amino acid sequences having at least 80% identity and retaining function. Preferably, the modification involves replacing the first 9 amino acids at the N-terminus of the wild-type HIV-1 Gag protein with the aforementioned tag to enhance membrane anchoring and improve viral packaging efficiency. This invention also provides packaging plasmids containing this molecule, host cells, packaging systems, and preparation methods. This invention can significantly improve viral yield and titer, and is particularly suitable for overcoming the efficiency bottleneck in packaging large-load lentiviruses of 7-10 kb.

The utility model discloses a multi-range gynecological cell sampler and a sample box, and belongs to the field of cervix sampling. The bottom plate is arranged at the head end of the holding rod; the insertion rod is arranged on the bottom plate; two inserting shafts are arranged on the outer wall of the top end of the inserting rod; the side sampling piece and the upper sampling piece are detachably arranged on the insertion rod respectively; the upper sampling piece comprises an upper brush head which is crown-shaped, two wings of the upper brush head protrude towards two sides, and bristles in the middle of the upper brush head are longer than the two wings. According to the multi-range gynecological cell sampler and the sample box, through the arrangement of the side sampling piece and the upper brush head, the cervical orifice, the fornix beside the cervical orifice and the vagina can be sampled at the same time, and compared with the prior art, the material consumption is reduced, the sampling process is greatly simplified, and the sampling efficiency is improved. Meanwhile, as the two wings of the upper brush head protrude towards the two sides and the middle bristles are longer than the two wings, the two protruding wings can reduce the contact probability of the middle bristles (cervical orifice samples) and the vagina wall, and therefore the positive rate of certain TCT can be increased.

The invention discloses a detection method for improving the positive rate of human papilloma virus (HPV) nucleic acid detection, which is characterized in that on the premise of keeping the morphology and nucleic acid completeness of cervical exfoliated cells, red blood cells are completely dissolved, mucus is dispersed, CC points (monitoring hybridization process) and PC points (monitoring extraction and amplification process) of a sample are colored, the success rate of an experiment is ensured, and the detection accuracy is improved. According to comparison experiment results, the processed HPV nucleic acid detection experiment is high in success rate and convenient and simple to operate, the pain of resampling of a patient is reduced, the waste of repeated test reagents is avoided, the cost is saved, the generation of false negative results is avoided, and the accuracy of HPV nucleic acid detection results is improved, namely, the positive rate of the HPV nucleic acid detection results is improved.

The application relates to the technical field of medicines, in particular to application of a VER155008 compound in preparation of a coronavirus-dependent RNA polymerase antagonist. Through construction of a reliable screening model of SARS-CoV-2 RdRp inhibitors, the application tests the inhibiting effect of a plurality of nucleoside analogs on SARS-CoV-2 RdRp, finds that the VER155008 compound has obvious inhibiting effect on SARS-CoV-2 RdRp, the VER155008 compound has SARS-CoV-2 exonuclease nsp14 resistance, and can effectively inhibit replication of coronaviruses HCoV-OC43 and HCoV-NL63. Therefore, the application provides key data for research and development of a novel anti-new coronavirus lead compound, and provides a theoretical basis for research and development of an effective coronavirus treatment drug.

The invention provides an immunodetection solution for preventing fluorescence leakage, a kit and application of the immunodetection solution, and the immunodetection solution is characterized by comprising 5-100mM of a buffer solution; 0.1%-10% (W / V) of cyclodextrin or a derivative thereof; and 0.05% to 0.2% (W / V) of a surfactant. The invention also provides an application of the immunodetection liquid for preventing fluorescence leakage in droplet immunodetection. The immunodetection liquid has the beneficial effects that the cyclodextrin or the derivative thereof is added into the immunodetection liquid, so that the leakage of resorufin is effectively controlled, and the accuracy of a single-molecule droplet photographing immunodetection result is guaranteed; by controlling the concentration of magnesium ions in the immunodetection liquid, the positive rate is remarkably increased, and then the sensitivity of a liquid drop system is improved; the immunodetection liquid is stable and can be stored at room temperature, and related temperature control components on an instrument are prevented from being used; the immunodetection solution is simple in preparation process and controllable in cost.

The invention belongs to the technical field of nano antibody libraries, and particularly relates to a random combinatorial library, an establishment method thereof and application of the random combinatorial library in screening of H9 subtype avian influenza virus nano antibodies. The library is obtained by connecting a fragment containing a CDR1 region, a fragment containing a CDR2 region and a fragment containing a CDR3 region which are respectively amplified by three pairs of PCR amplification primers, and the sequences of the three pairs of PCR amplification primers are respectively SEQ ID NO: 1-SEQ ID NO: 6. The positive rate of the library is 95.8%, and the capacity of the library is 2.7 * 10 < 9 >. In the library, the positive rate of the nano antibody capable of being combined with the H9 subtype avian influenza virus is 60%. According to the invention, the blank of the random combinatorial library aiming at the H9 subtype avian influenza virus nano antibody is filled, and a new way and strategy are provided for the establishment of the random combinatorial library aiming at the H9 subtype avian influenza virus nano antibody.

The invention belongs to the technical field of medical instruments, and particularly discloses a miniature fluorescence microscopic imaging and automatic counting system for onychomycosis detection. The system comprises an integrated handheld sampling imaging terminal, a cloud intelligent analysis server and a user interaction terminal. The terminal integrates sampling, fluorescence labeling and microscopic imaging functions, automatically completes sample processing and image acquisition, and uploads the sample processing and image acquisition to the cloud. And the server performs automatic identification, segmentation and counting of fungal targets on the image through a deep learning algorithm to generate a detection report. According to the invention, mechanical sampling, microfluidic fluorescence labeling and miniature fluorescence microscopic imaging are highly integrated in the handheld terminal, a closed and automatic sample processing and imaging assembly line is constructed, and the problem of leak detection caused by insufficient sampling depth and inconsistent dyeing operation in a traditional method is solved.

The present invention relates to a large fragment DNA in vitro cloning method, a kit and related uses mediated by CRISPR / Cas12a system. The mechanism of the DNA cloning is that Cas12a / crRNA is used to perform specific cutting at both ends of the target DNA to obtain a target DNA sequence, a cloning vector is constructed by cloning the DNA sequences at both ends of the target DNA, and a linearized vector is obtained by cutting the cloning vector using Cas12a / crRNA, and the sticky ends formed after cutting are connected by DNA ligase, so as to achieve cloning of the target DNA. The large fragment DNA cloning method of the present invention, the kit and its related uses can not only save time and cost when cloning a large fragment DNA (up to 140kb) with high GC content (e.g., more than 70%), but also have a high positive rate and the potential for market application.