166 results about "Plasmid transfection" patented technology

The invention discloses a three-dimensional silk fibroin scaffold insoluble in water, and preparation and application of the three-dimensional silk fibroin scaffold. A preparation method includes the steps of injecting silk fibroin solution with the mass concentration of 0.3-30% into a mold, and obtaining a three-dimensional silk fibroin scaffold after freeze-drying; performing humid and heat crosslinking for the mold under the condition that the temperature ranges from 50 DEG C to 100 DEG C and the relative humidity ranges from 70% to 100%; and drying, removing the mold and obtaining the three-dimensional silk fibroin scaffold insoluble in water. Silk fibroin is used as a raw material, and biocompatibility is good; a preparation process is non-toxic and environment-friendly, and shape, aperture and porosity parameters of the scaffold are easy to control; the pore wall of the scaffold is nano-scale, the aperture of the scaffold is micron-scale, an extracellular matrix micro-environment is simulated, and the three-dimensional silk fibroin scaffold is used as a three-dimensional cell culture vector or a three-dimensional plasmid transfection vector when used externally; and the three-dimensional silk fibroin scaffold is used in the aspects of cartilage scaffolds, fat scaffolds, bone scaffolds, muscle scaffolds and the like when used in the internal tissue engineering field.

The invention belongs to the field of biotechnology and particularly relates to a method for constructing a stable cell strain secreting an expression protein. The method comprises the following steps: with a recombinant lentiviral vector pCDH-S-His as a skeleton, constructing a target protein gene to the recombinant lentiviral vector pCDH-S-His to obtain a recombinant plasmid pCDH-S-P-His; co-transfecting the recombinant plasmid and helper plasmids pSPAX2 and pMD2.G with 293T cells by using a PEI transfection method to obtain a recombinant lentivirus; transducing the obtained lentivirus to the 293T cells, and performing puromycin screening to obtain a plurality of monoclonal cell strains; detecting the content of target protein in each cell supernatant by an Elisa method; determining a high-expression cell strain. Compared with a traditional plasmid transfection method, the method provided by the invention has the advantages of short experimental period, high positive rate and good stability of the cell strain.

The invention provides a high-flux drug screening microfluidic chip which is composed of a cell sample introduction element, a buffer pool, a one-way drive micro-valve, a disk-like bent channel, a drug loading element, a plasmid transfection element, a liquid receiving pool and the like. The elements for cell sample introduction, transfection, cell capture, drug loading and the like are assembled on one chip, so that the invention can simultaneously detect cell integral shape and intracellular labeled protein activity variation under different drug actions on one chip in the detection process, and monitor the action effect of the drug on the actual cells, thereby greatly lowering the detection cost, enhancing the detection accuracy and detection efficiency, and achieving the goal of drug screening.

The invention discloses a method for preparing a clinic-level CAR-T cell preparation by minicircle DNA transfection T cells. The method comprises a technology of preparing the clinic-level CAR-T cell preparation by using non-viral minicircle DNA (minicircle DNA, mcDNA) vector to efficiently transfect T cells. The mcDNA transfection technology comprises the design and synthesis of a chimeric antigen receptor (CAR) carried with a target gene, the construction of target gene-CAR parent vector plasmids, the extraction of target gene-CAR-mcDNA vector plasmids, the target gene-CAR-mcDNA vector plasmid transfection T cells, and the clinic-level CAR-T cell preparation prepared by using the mcDNA transfection technology. The invention further discloses a use of the mcDNA transfection technology. The mcDNA transfection technology has the characteristics of high transfection efficiency to mammal T cells, stable expression, stable function, and no effect to cytogene feature. The prepared CAR-T cell preparation has the characteristics of strong targeting property, high killing efficiency and clinic safety. The mcDNA transfection technology can be used for preparing the clinic-level CAR-T cell preparation for treating various solid tumors and blood and lymphatic system tumors.

The invention provides a microfluidic chip for detecting early leukemia. The microfluidic chip consists of two parts, namely a basic chip and a microfluidic detector; and the microfluidic chip is characterized in that the microfluidic detector is attached to the basic chip; a cell sorting element is processed on the microfluidic detector; a detection unit is a detection channel which consists of an electric field sorting device, cell capturing elements which are communicated with one another, a plasmid transfection element and a liquid collection tank; and more than 18 detection channels can be formed in one chip. Because a plurality of detection channels are assembled on the same chip, during the detection, more than ten subtypes of common leukemia of a patient sample can be detected on the same chip, so that the detection cost is greatly lowered, and the detection accuracy and detection efficiency are increased.

The invention belongs to the technical field of biology and particularly relates to establishment of a recombinant adeno-associated virus carrier production process. The invention firstly provides a preparation method of a recombinant adeno-associated virus carrier, which includes the steps of: (1) transfecting a plasmid containing a genomic sequence of the recombinant adeno-associated virus carrier into a culture cell; (2) under proper conditions, culturing the cell obtained in the step (1) and collecting a cell culture supernatant, which refers to a mixture of the cell containing the recombinant adeno-associated virus carrier and the culture supernatant; (3) purifying and concentrating the cell culture supernatant in the step (2) to obtain the recombinant adeno-associated virus carrier.The preparation method is basically suitable for majority of rAAV serotypes and mutants. The recombinant adeno-associated virus carrier is high in titer and purity, is low in immunogenicity and is safe and high-effective.

The invention provides a preparation and application of a targeting exosome. The preparation method includes: covalently binding a PSP1 polypeptide with optional one targeting peptide or oligopeptidewith an immunity promoting function to form a PSP1 targeting peptide, and binding the PSP1 targeting peptide with an exosome, wherein the amino acid sequence of the PSP1 polypeptide is CLSYYPSYC or apolypeptide with more than 80% homological sequence of PSP1. The preparation method has the advantages that through a large number of tests and experiments, the PSP1 polypeptide is bound with the targeting peptide or the oligopeptide with an immunity promoting function for the first time, and the formed bidirectional-targeting polypeptide is bound with the exosome; the method solves the problem that existing surface modification methods such as plasmid transfection are low in transfection efficiency and increases the surface modification efficiency of the exosome, large-scale preparation of the targeting exosome is achieved, and the method is quite significant to the researches and application of the exosome serving as the transport vector.

The invention discloses an absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, an absolute fluorescent quantitative PCR probe and an absolute fluorescent quantitative PCR method for determining the growth titer of mycoplasma hyopneumoniae. The sequences of the absolute fluorescent quantitative PCR primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2. The sequence of the absolute fluorescent quantitative PCR probe is shown as SEQ ID NO.3. The absolute fluorescent quantitative PCR method for determining the growth titer of the mycoplasma hyopneumoniae comprises the following steps: (1) extracting the deoxyribose nucleic acid (DNA) of the mycoplasma hyopneumoniae; (2) performing common PCR amplification to obtain a target fragment, and designing the absolute fluorescent quantitative PCR primer pair and the absolute fluorescent quantitative PCR probe in the middle of the target fragment; (3) preparing plasmids by connecting the target fragment and a PMD-18-T carrier, transfecting competent cells, and extracting plasmids of a positive bacteria solution; (4) calculating an initial plasmid copy number by using the plasmids extracted in the step (3) as standard substances; and (5) performing fluorescent quantitative PCR. According to the absolute fluorescent quantitative PCR method for determining the growth titer of the mycoplasma hyopneumoniae, the copy number of target genes, namely the growth titer of the mycoplasma hyopneumoniae, can be quickly and accurately determined.

Belonging to the technical field of immunodetection, the invention provides an LRP4 antibody cell coated microporous plate, a preparation method and a human LRP4 antibody cell immunofluorescence detection kit. The human LRP4 antibody cell immunofluorescence detection kit comprises the LRP4 cell coated microporous plate, a sample diluent, a positive reference substance, a secondary antibody and a blocking solution. The LRP4 antibody cell coated microporous plate provided by the invention is directly coated with LRP4-GFP-HEK293T recombinant cell and EGFP-HEK293T recombinant cell. Repetition of plasmid transfection, cell inoculation, cell culture and other processes within a short period of time can be avoided, and then the cost of every detection is reduced, and the detection time is shortened.

Chitosan delivers a plasmid encoding Glucagon-Like Peptide 1 (GLP-1) to cells in a patient for gene therapy of diabetes. Chitosan is optimized for plasmid transfection by modulating three of its physico-chemical properties: degree of deacetylation (DDA), molecular weight (MW), and ratio of amines on chitosan to phosphates on DNA (N:P ratio), Chitosan 92-10-5 (DDA-MW-N:P) is more efficient than chitosans 80-10-10 and 80-80-5 in delivering a plasmid encoding luciferase or GLP-1(7-37) to cells. In the Zucker Diabetic Fatty (ZDF) rat model of diabetes, chitosan-delivered pVax plasmid encoding GLP-1 lowers glucose levels, increases insulin production and reduces weight gain.

The invention provides a preparation method and application of a target exosome which can effectively improve the modifying efficiency of an exosome so as to realize large-scale preparation, a drug delivery system and a drug. The preparation method of the target exosome comprises the following steps S1, performing sulfhydrylation treatment on a target antibody so as to obtain a sulfhydrylation target antibody; and S2, enabling the sulfhydrylation target antibody to be connected with the exosome through a protein cross-linking agent. The target antibody is subjected to sulfhydrylation modification, so that an amino on the antibody is converted into a sulfydryl; and then the exosome and the antibody are in cross-linking combination so that the target exosome is obtained. Compared with a method of obtaining the target exosome through modification in a plasmid transfection manner in the prior art, the preparation method provided by the invention has the advantages that the problems that the transfection efficiency is low in the plasmid process and large-scale preparation and secretion of mother cells through genetic engineering plasmid transfection are difficult to perform can be solved, industrialization preparation of the target exosome can be realized, and the preparation method has important significance in research and application of the exosome as a delivery carrier and a drug carrier.

The invention belongs to the field of genetic engineering and particularly relates to a method for knocking out a pseudorabies virus TK gene by using dual sgRNA and application of the method. According to the method, a dual-locus knockout method is employed, two sgRNA sequences are designed on the TK gene, two loci are cut in a pointed manner, and thus, the TK gene can be efficiently knocked out. The method for knocking out the pseudorabies virus TK gene mainly comprises the procedures of sgRNA designing, Cas9 vector-sgRNA expression vector constructing, donor vector constructing, plasmid transfection, cre enzyme vector transfection and recombinant virus purification. Compared with the traditional methods, the method disclosed by the invention has the advantages of high accuracy and low target missing effect.

The invention provides a pair of TALENs for efficiently editing a rice WAXY gene, and an identification targeting site and an application thereof, and concretely provides a transcription activator-like effect nuclease (TALEN) gene for efficiently targeting the rice endogenous WAXY gene, and a method using a recombinant plasmid containing the gene to carry out oriented targeting. The TALENs contain a pair of TALE proteins and DNA endonuclease catalytic subunits respectively fused with the TALE proteins, and can respectively identify and cut two adjacent sites of the rice endogenous WAXY gene. Synthesis of a nucleotide sequence for encoding the TALENs and construction of a vector including the nucleotide sequence are realized on the basis of designing the amino acid sequence of the TALEs, and the cell targeting efficiency is greatly improved through plasmid transfection of cells by the TALENs.

The invention discloses a construction method of an HDAC8 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC8 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC8, and performing separation to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method. After genome DNA is extracted, PCR amplification and sequencing are carried out, and cell clones of which two HDAC8 genes are subjected to homozygous frameshift mutation are successfully identified. In the HDAC8 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC8 knockout has no obvious influence on the cell growth rate, which shows that the HDAC8 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. A foundation is laid for further knocking out HDAC8 from suspension culture type BHK-21 cells and directly applying HDAC8 to production of foot-and-mouth disease vaccines.

The invention discloses a construction method of an HDAC3 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC3 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC3, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing, and successfully identifying the cell clones of which two HDAC3 genes are subjected to homozygous frameshift mutation. In the HDAC3 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC3 knockout has no obvious influence on the cell growth rate, so that the HDAC3 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC3 from suspension culture type BHK-21 cells and directly applying HDAC3 to foot-and-mouth disease vaccine production.

The invention provides a method for establishing a stable transgenic flounder embryo cell strain. The method for establishing stable transgenic flounder embryo cell strain comprises the following steps: transfecting flounder embryo cells by using plasmids which can widely express enhanced green fluorescent protein promoted by a beta-actin promoter in flounders and can express G418 resistant protein; after 24h, observing expression of the EGFP (express enhanced green fluorescent protein) under a fluorescent microscope, and through G418 screening, obtaining the embryo cells with stable genetic expression. By the method, an exogenous gene widely expressed in the flounders and promoted by the beta-actin can be effectively transfected into the flounder embryo cells, and can be genetically expressed in the flounder embryo cells stably, so that a novel method is provided for researching for flounder genes, the problem that difficult cellular-level gene function analysis and research caused by low transient transfection efficiency of the flounder cells is solved, and a novel technical means is provided for a molecular-level genetic breeding work of the flounders, and a transgenic method for stably inheriting a marine fish cell line is provided.

The invention discloses an autophagy monitoring method for fat cells. Due to the adoption of a special structure, mature fat cells are full of plenty of lipid droplets, so that organelle and nucleus are displaced towards the periphery, and a typical autophagosome is hard to be seen under an electron microscope. Proved by a plurality of experiments, the method disclosed by the invention finally and successfully observes autophagy structures in various autophagy stages in mature 3T3-L1 fat cells by continuously improving the experiment condition. Besides, mature fat has a characteristic of being not easy to be transfected, so common plasmid transfection is hard to play an expectant effect. Therefore, the method disclosed by the invention utilizes a GFP-LC3 lentivirus method to infect 3T3-L1 fat cells, and the infection efficiency is as high as 80% or greater by observation under a fluorescence microscope. At nutrition and hunger condition, by the method disclosed by the invention, GFP-LC3 point-like aggregation phenomenon is successfully observed. The establishment of the autophagy monitoring method for mature fat cells lays the foundation of research on the relationship between autophagy and fat metabolism for us.

The invention discloses a construction method of an HDAC5 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC5 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC5, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing and successfully identifying the cell cloning of which one HDAC5 gene is subjected to homozygous frameshift mutation, wherein the HDAC5-KO-A2 has deletion of 13 basic groups at a Cas9 predetermined cutting position. In the HDAC5 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC5 knockout has no obvious influence on the cell growth rate, so that the HDAC5 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC5 from suspension culture type BHK-21 cells and directly applying HDAC5 to production of foot-and-mouth disease vaccines.

The invention discloses a plasmid expressed by prostate-specific antigen (PSA) promoter mediated firefly luciferase gene. The plasmid is formed by connecting a PSA promoter pPSA-ZsGreen dominant skeleton with a firefly luciferase gene fragment. The plasmid is applied to the transfection of prostate cancer cells to lay a foundation for constructing a prostate PSA fluorescence reporter gene animal model. The part of the tumor in the prostate cancer animal model is positioned by use of fluorescence, and then a reliable animal model can be provided for the treatment of the prostate cancer and the development of new drugs.