194 results about "Pronucleus" patented technology

The invention discloses a method for producing transgenic mice through a homologous recombination technology. The method comprises the following steps: replacing mouse NGF genes on mouse chromosomes by human NGF genes through a Cas-9/CRISPR gene knock-in technology, and obtaining the genes with homozygous human NGF genes through breeding and knocking the genes in mice, thus obtaining filial generation mice with salivary glands capable of secreting human NGF. Compared with the conventional targeting technology utilizing positive and negative double screening homologous recombination genes, the method disclosed by the invention is simple to operate, capable of being realized only by transfecting mouse embryonic stem cells by three plasmid DNAs or carrying out pronucleus injection on the zygotes of mice, and high in success rate which is up to 2-5% and remarkably higher than the mouse embryonic stem cell positive rate of 0.1% of the common gene targeting technology.

The invention discloses a monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and a method for detecting the nonstructural protein (NSP) antibody of a foot-and-mouth disease virus (FMDV) (FMD NSP B-ELISA); the kit comprises ELISA reaction plates, serum diluent, 25 times concentrated detergent, substrate solution, 100* concentrated ELISA detecting antibody, stop buffer, positive control serum and negative control serum; the ELISA reaction plates are two 96-pore high-affinity ELISA reaction plates, firstly 6* groups of amino acid monoclonal antibody or NSP 2C polyclonal antibody, and then FMDV 3ABC or 2C3AB NSP which is expressed by pronucleus and is provided with 6* groups of amino acid labels is captured through the monoclonal antibody or the polyclonal antibody; and compared with other similar kits, the method has higher coincidence rate and higher positive serum detection rate, and is applicable to detecting the serum of cattle, sheep, pigs and other susceptible animals.

The invention discloses soluble programmed dead protein-1-IgV and a preparation process. The protein sequence of is showed as followed: PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQ DSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEV PTAHPSP. The invention obtains gene of Hpd-1-IgV through applying artificial synthesis according to the structure of human PD-1, target gene is cloned into pQE-30 pronucleus expression carrier to transform colibacillus and is expressed in colibacillus with high efficiency, bacterioclasis shows that target protein is existed in inclusion body form, and purified soluble programmed dead protein-1-IgV is finally obtained through inclusion body scouring, affinity chromatography, gel column chromatography and dialysis renaturation. The soluble programmed dead protein-1-IgV can respectively combine with mPD-L1/Fc and hPD-L1/Fc in extra-corporal shPD-1IgV and on cell level and molecular level, and has certain tumor killing effect in internal shPD-1IgV. Therefore, shPD-1IgV has excellent internal and external biological activity, provides experiment basis for curing tumor, establishes experiment foundation, and has potential application prospect.

The invention discloses a method for establishing an immunodeficiency mouse model. The method comprises the following steps of: (1) constructing DNA (deoxyribonucleic acid) which enables functions of an immune related gene in a mouse cell to be lost; (2) purifying mRNA (messenger ribonucleic acid), and cryopreserving in a super-purified water of RNAase free; (4) feeding a pregnant female mouse and a pseudopregnant female mouse under an SPF (specific pathogen free) level feeding condition; (5) taking a fallopian tube from the abdominal cavity of the pregnant female mouse in the step (4), and collecting fertilized ovum; (6) injecting the mRNA obtained in the step (3) into the pronucleus of the fertilized ovum in the step (5) by using a microinjection method; (7) transplanting the fertilized ovum obtained in the step (6) into the body of the pseudopregnant female mouse obtained in the step (4), and enabling the pseudopregnant female mouse to culture the second generation; (8) establishing a stable deficient mouse strain. The method has the advantages that a mouse model which completely does not have immunological rejection to allotransplantation can be obtained for testing.

The invention discloses double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting an avian leukosis group specific antigen. The kit comprises an enzyme plate coated by a monoclonal antibody which is secreted by a hybridoma cell strain the preservation serial number of which is CGMCC (china general microbiological culture collection center) No.5961. The invention also discloses a double-antibody sandwich ELISA method which is established by utilizing the monoclonal antibody and is capable of rapidly and effectively detecting an ALV (avian leukosis virus). In the double-antibody sandwich ELISA method, the monoclonal antibody prepared by pronucleus expressive HLJ09mdj-1p27 albumen is utilized as a peridium antibody, and antibodies prepared by p27 are utilized as a detection antibody. According to the invention, the minimum detection amount of the p27 is 1.25 ng/ml, the method is not reacted with the common virus of birds, and the specificity is good. The method is utilized to detect egg white and an anus swab sample, and the coincidence rate is respectively 96.5% and 88.9% compared with a PCR (polymerase chain reaction) method; and the result proves that the method has the advantages of convenience, celerity, differentia, sensitivity and the like, and is useful for the detection and population purification of the ALV.

The invention discloses a method for producing a body interleukins29 and restructuring the engineering bacteria IL-29, which comprises artificial synthetic primers, PCR augmenting mature peptides of IL-29 coding areas, the construction of IL-29 pronucleus expression carrier pET44-IL-29, transforming into colibacillus to restructure engineering bacteria IL-29, culturing the engineering bacteria to highly effective express IL-29, and purifying IL-29. In the invention, adopting a less dependent interconnection reaction clone process (LIC) to precise and quick-speed get the mature peptides coding areas of IL-29, adopting the terminator codon of colibacillus preference to increase the expression efficiency, one-step purifying using a S protein affinity chromatography column to get the very purified production.

The invention relates to a preparation of an ELISA kit of human tissue kallikrein. The technical scheme is achieved as follows: the ELISA kit for testing the content of human tissue kallikrein is characterized in that: (a) the method adopted by the kit is a double antibody sandwich method; (b) a biotin-aviden amplification system is added; (c) the standard substance is pronucleus-expressed and purified human tissue kallikrein; (d) the coated antibody is a polyclonal antibody for resisting the human tissue kallikrein, and a detection antibody is a monoclonal antibody of biotinylation; and (e) the sensitivity is 0.3ng/ml to 500ng/ml. The kit has the characteristics of high sensitivity, strong specificity and favorable repeatability. By adopting the expressed fusion protein as the standard substance, the correlation coefficient of a standard curve is as high as 0.99, and by adopting a batch of human plasma as samples to be tested, both the difference in a group and the difference among groups are lower than 10 percent.

The atmospheric cold plasma microbe mutagenesis method belongs to the field of microbe mutagenesis technology, and features the cold plasma mutagenesis of microbe strain to denature. The microbe strain may be pronucleus microbe or eukaryotic microbe; the atmospheric cold plasma may have discharging medium of air, nitrogen or helium with discharge period of 10 s to 10 min, applied voltage of 1-100 kV and frequency of 0-15 MHz; and mutagenesed microbe may be in suspension or plate. Compared with other physical and chemical mutagenesis method, the present invention has the advantages of simple apparatus, low cost, operation safety and high mutagenesis effect, and may be applied widely in industrial microbe breeding.

The invention discloses a procaryotic cell non-merge solubility pressing system, which comprises the following steps: building one type auxiliary protein gene or two type auxiliary protein gene and pre-expressing exogenesis goal gene in a transcription unit; proceeding intracellular co-express through each self-contained translational control system of auxiliary protein gene and pre-expressing exogenesis goal gene in the same pronucleus expression host cell; utilizing the auxiliary protein to realize non-merge solubility expression especially to realize non-merge solubility expression with multiple disulfide bond peptide chain. This system can make the expressing quantity of procaryotic cell occupy above 105 of thallus total protein under the normal condition.

The invention relates to a kit for quickly detecting a swine fever antibody. In the method, a pair of specific primers is designed, a relatively conservative gene sequence is cloned, an antigen aiming at a swine fever E2 protein is expressed through a pronucleus expression technology, and, on the basis, the kit containing an enzyme linked plate coated with a high-purity and high activity swine fever virus specificity antigen, an enzyme conjugate of rabbit-anti-swine monoclonal antibody containing an HRP (Horseradish Peroxidase) marker, a TMB (Tetramethylbenzidine) color developing liquid and the like is prepared. The kit can quickly detect the swine fever antibody in blood serum or blood plasma and has strong specificity and high sensitivity.

The invention provides a preparation method of a European American type pig propagation and syndrome virus antibody differential diagnosis Kit. The method constructs the pronucleus expression vector pGEX-6P-1-N aimed at American type PRRSV N protein regarding the gene deficiency of the conserved epitopes 25aa-30aa, 37aa-52aa and 50aa-66aa of European and American type strain in an ORF7 gene of a PRRSV VR-2332 strain. Regarding the amino acid sequence of the European type PRRSV N protein epitopes 2aa-12aa, 40aa-46aa and 67aa-128aa, the method establishes the pronucleus recombination expression vector PQE30-N of the European type PRRSV N protein. The recombination plasmid expresses successfully in the bacillus coli, and an indirect ELISA diagnosis Kit for PRRS antibody detection is prepared through using the expression depurated North American type N protein. The coincidence rates of the antibody Kit and an IDEXX Kit both reach 90 percent, and the depurated European type protein serves as enveloping antigen, thereby optimizing the European type PRRSV antibody detection indirection ELISA method, and laying a foundation for PRRS antibody typing detection.

The invention belongs to the technical field of biomedicine, relating to a clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and a nucleic acid vaccine thereof. The codon optimized gene sequence gives consideration to use preferences of mammalian cells and Esherichia coli codon, and the related clostridium difficile vaccine is composed of exotoxin A carboxy-terminal gene sequence with optimized codon and eukaryon expression vector pJW4303. The clostridium difficile exotoxin A carboxy-terminal gene segment optimized by codon can not only be used for constructing nucleic acid vaccines, effectively stimulating host immune system after immunizing mammals, generating better humoral immunity reaction, being applicable to conduct pronucleus expression in esherichia coli, and laying a foundation for acquiring protein in great quantities.

The present invention discloses a method of expressing recombinant odorant binding protein (OBP) of insect in prokaryotic cell, and vector containing target nucleic acid sequence is transformed to competent prokaryotic cell. The present invention also discloses new OBP-coding nucleic acid sequence obtained from Locusta migratoria antenna.

The invention offers recombination staphylococcus aureus enterotoxin C2(SEC2) making method. It can realize by the following steps: amplifying and preparing SEC2 gene; connecting SEC2 gene and pronucleus expression plasmid; transforming recombination plasmid to expression host to efficiently express; purifying recombination SEC2. The method efficiently expresses recombination SEC2; and it is solubility, and has no occlusion body. Its advantages are simple steps, and quick purifying speed. Compared with corresponding nature albumen, the gained recombination has the same super antigen and immunology activity.

The invention relates to a construction method and application of a humanized mouse model, in particular to a construction method and application of a transgenic mouse model of a human Nbs1<c.657del5> gene. The method includes: constructing a 5bp deletion mutation-containing BAC carrier in the human Nbs1 gene, conducting pronuclei microinjection, and performing screening to obtain 3 stable transgenic lines for high expression and low expression of the human Nbs1 gene. The transgenic mouse involved in the invention has the phenotypes of delayed puberty, uniform shortening of body length and bone dysplasia at certain proportion in one of the lines, and a new mouse model is established for Nijmegen breakage syndrome diseases. At the same time, as the Nbs1 gene function impairment is closely related to cancers, the transgenic model can be applied to short-term carcinogenic tests in drug pre-clinical safety evaluation, thus providing a potential substitution model for traditional biennium carcinogenic tests and also providing an effective tool for research of carcinogenesis mechanisms.

The invention discloses a method to prepare antigen protein of abortion brucellosis subunit vaccine and usage, which comprises the following steps: abstracting bacteria total DNA from abortion brucellosis; using the total DNA as mold; adopting property primer; proceeding polyose chain reaction; cloning; getting OMP25 protein gene; transferring into pronucleus expressing carrier; getting restructuring express carrier; leading-in bacillus coli; getting reconstructing large intestine Escherichia; proceeding inducing expression; culturing reconstructing large intestine Escherichia; preparing antigen protein. This protein can be used to prepare abortion brucellosis subunit vaccine.

The invention relates to the genetic engineering field, in particular to the construction, expression, purification and optimal copy strain screening technologies of a domestic silkworm antibacterial peptide CM4 multi-copy expression vector. The technical proposal comprises the following steps of: using a DNA recombination technology to construct the multi-copy orthokinetic series connection expression vector pET32a-nCM4 (n is equal to 1, 2, 3...8) of the antibacterial peptide CM4, and then using a nickel column affinity chromatographic technology to purify a multi-copy antibacterial CM4 fusion protein; and using hydroxylamine to cut a TrxA-n CM4 fusion protein to obtain an antibacterial CM4 monomer. The invention adopts the genetic engineering technologies for expressing the recombination antibacterial peptide CM4 with high efficiency in a pronucleus host cell, separation and purification are simple, amplification is easy and the cost is low, thereby laying a foundation for the mass production, study and application of the antibacterial peptide.

The invention discloses a multi-epitope vaccine YL66, the pronucleus expression carrier pGEX-4T-2-YL66 and the eukaryon expression carrier pCDNA3.1-YL66 are built on the basis of the multi-epitope vaccine YL66, and the fusion protein GST-YL66 is obtained through the pronucleus expression of the carrier pGEX-4T-2-YL66. Through the in-vitro and in-vivo ELISPOT experiment and the LDH experiment on the peripheral blood of a healthy supplier HLA-A2 and the transgenosis mouse HLA-A2.1/Kb, a research is performed on immunological competence of the fusion protein GST-YL66 and the DNA vaccine pCDNA3.1-YL66. The research finds that a certain kill rate for the tumour cell EC-9706 by the specificity CTL induced in-vivo mouse and in-vitro human body is indicated according to the tumour-resistant multi-epitome vaccine, meanwhile the induced CTL has a certain specificity of antigen, and can be applied in a further research on the strategy and the application of building tumour multi-epitome vaccine.

The invention relates to a process for preparing high activity thrombin inhibitors, which comprises subjecting hirudin, the natural specific inhibitor of thrombin to DNA family restructuring, carrying out restructuring to hirudin HV1, HV2, HV3 genes, constructing reorganization library for hirudin bacteriophage display, using thrombin as coating antigen, subjecting bacteriophage reorganization library to three rounds of enriched elutriation including adsorption, elution and expansion, obtaining a plurality of bacteriophage monoclons with high affinity with thrombin through ELISA screening, carrying out pronucleus induced expression to these clones, finally detecting the antithrombogenic activity of the purification expression product.