198 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.

Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

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 relates to a recombinant nerve putrescence virus protein vaccine, the nucleic acid sequence coding the recombinant protein, and the use of the recombinant protein in preparing fish use vaccine product for nerve putrescence viruses resistance. Wherein the cDNA of the capsid nucleocapsid for the grouper nerve putrescence viruses is utilized to obtain the polypeptide recombinant proteins through DNA recombination process and pronucleus expression method.

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 relates to the technical field of information, provides an occluded embryo pronucleus and cleavage ball detection method based on an attention mechanism and aims to solve the problem ofmissing detection caused by mutual occlusion between an embryo pronucleus or a cleavage ball. According to the main technical schemes, the method involves a main body segmentation network used for cutting an embryo main body part from an original image, a CNN feature extraction layer combining a residual structure, a RPN network for preliminarily generating and correcting target candidate boxes, acoordinate refinement unit for refining the coordinates of the target candidate boxes predicted by the RPN network, an improved Soft-NMS algorithm for screening out redundant target candidate boxes,a shielding score network used for enhancing the characteristics of a core channel and the characteristic expressions of a target occluded part, a classification network for identifying the specific categories of targets in a fine candidate box, and a number prediction network for identifying the number of the targets.

The invention discloses human adenovirus antigen epitope chimeric protein as well as preparation and application thereof, and relates to fields of gene engineering techniques, vaccines and diagnostic reagents. The amino acid sequences of hexon protein of type 3, type 7, type 11, type 14 and type 55 of adenovirus are analyzed through computer analysis, protein fragments with good antigenicity can be screened respectively, the fragments are connected by using two glycine and one serine, then chimeric protein with multiple antigen fragments in serial connection can be formed, pronucleus preferred codons are selected and interpreted into corresponding nucleotide sequences, and full-length genes can be synthesized in a chemical manner. The chimeric protein can be obtained through expression purification by using a gene engineering technique, and the chimeric protein comprises 363 amino acids in whole length. The expressed chimeric protein can be applied to vaccine research, HAdV antibody or antigen detection and the like, and is related to fields such as gene engineering techniques, vaccines and diagnostic reagents.

The invention discloses a restructuring the fifth of the ten Heavenly Stems type hepatitis virus protein, coded sequence and usage, which is characterized by the following: the HEV virus strain to prepare restructuring the ten Heavenly Stems vaccine is the first gene type; the HEV p179 is based on popular production of the forth gene type HEV virus strain and possesses stronger placement and practical value; the HEV p179 can get highly effective expression in pronucleus system and 90% protein is nature solubility; the HEV p179-VLPs with diameter at about 20nm is constituted by 179aa; the HEV p179-VLPs is smallest restructuring HEV protein compared to now eucaryon system and HEV VLPs of pronucleus system expression.

The present invention discloses one kind of recombinant protein antigen for chicken infectious bronchitis virus (IBV) S1 and its preparation process and use. These nucleotide sequences are shown in SEQ ID No. 1~3. The present invention provides expression method of pronucleus encoding 3 truncated S1 proteins, can express the amino end 288 bp and 309 bp part segments of S1 protein of three IBV strains separately, and provides the purification method of the S1 protein expressing products of the three IBV strains. During detection with IBV positive serum, the expressed partial S1 protein has excellent antigen reaction, so that the present invention may be used as diagnosis antigen used in different detection methods for detecting IBV infection or immunologically produced antibody. In addition, the expressed partial S1 protein may be sued in preparing monoclonal antibody and polyclonal antibody for detecting IBV antigen and preparing subunit vaccine.

The invention relates to Escherichia coli genetic engineering vaccine 987P-ST-F41 for curing diarrhea of baby pigs and the constitution method, pertaining to the field of veterinary technology. The major subunit gene faeG segment of the expanded K88, the major subunit gene fanC segment of K99 and the major subunit gene ltb segment of LT linked to the pronucleus expression vector pGEX-6P-1 in series in a faeG-fanC-ltb manner; after transformation of host bacteria BL21, IPTG induction expression is conducted, and proved by SDS-PAGE and Western-blot that the objective gene is expressed. The vaccine is capable to prevent diarrhea of baby pigs, and the safety and immunogenicity are reliable.

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 field of medical biotechnology, and relates to two series major vault protein of glucagon-like peptide-2 (GLP-2) of people and a preparation method of the two series major vault protein. A second place of an amino acid fragment of the glucagon-like peptide-2 of people is mutated, two segments are connected in series through connecting peptide, a dimmer structure of the GLP-2 is simulated, encoding gene of the two series major vault protein of the GLP-2 is obtained by a synthetic method, the encoding gene is cloned to a pET22b(+) pronucleus so as to express a vector, Escherichia coli BL21 is transformed, the encoding gene is expressed in the Escherichia coli efficiently, the purified reconstructed purpose protein is obtained through salt fractionation of ammonium sulfate in fractional and chromatography of anion exchange columns. The reconstructed purpose protein can stimulate a Caco-2 cell, multiplication of the Caco-2 cell can be promoted notably, the two series major vault protein of GLP-2 of people has natural biological activities, and therefore a foundation is established for further study and extensive use of the GLP-2 of people.

The invention discloses an embryo division process analysis and pregnancy rate intelligent prediction method and system. The method comprises the following steps: collecting embryo images within D1 toD6 periods; inputting the embryo image into a prokaryotic number prediction network model, a blastomere number prediction network model, a fragment proportion prediction network model, a blastocyst cavity and inner cell mass grade prediction network model and a trophoblast grade prediction network model; calculating and outputting a predicted prokaryotic number, a predicted blastomere number, a predicted fragment proportion, a predicted blastocyst cavity proportion, a predicted intracellular mass grade and a predicted trophoblast grade of the embryo image; and inputting the same into an embryo pregnancy rate state prediction machine learning model to calculate and output an embryo pregnancy rate prediction result. According to the intelligent prediction method and system, the whole embryodevelopment process is monitored, the embryo pregnancy rate is obtained through calculation by means of the comprehensive scoring function, manual intervention is not needed in the prediction process, and doctors can be helped to quickly and accurately judge embryo scores.

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 invention discloses a Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier which comprises a pronucleus replicon pUC Ori sequence for plasmid replication, an amicillin resistance gene AmpR containing sequence for mass amplification of target strain, virus replicon SV40Ori sequence for enhancing replication in eukaryocyte, a lentivirus packaging cis-element for lentivirus packaging, a ZsGreen1 green fluorescent protein for green fluorescence expression of eukaryocyte, an IRES ribosome combination sequence for joint transcriptional expression of protein, a human EF1(alpha) promoter for the eukaryotic transcription of chimeric antigen receptor gene, a chimeric antigen receptor for forming a second-generation CAR or third-generation CAR integrating identification, transfer and start, and an eWPRE element for improving the transgenic expression efficiency. Moreover, the invention also discloses an establishment method and application of the carrier. In the invention, the secretion of cell factors and the in-vitro killing effect of CAR-T cells can be remarkably enhanced, and the effect of clinical treatment of malignant pleural mesothelioma and pancreatic cancer is outstanding.

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 discloses a biological preparation method of chitin deacetylase derived from racemomucor, which comprises the following steps: (a) preparing a recombinant expression vector of the chitindeacetylase containing a gene sequence such as SEQ ID NO.1; (b) preparing a transformant containing the recombinant expression vector in the step (a); and (c) expressing and purifying the chitin deacetylase. In the invention, a gene specificity degenerate primer is designed, a reverse transcription and polymerase chain reaction and a rapid amplification technology of cDNA segment ends are adopted, and escherichia coli strains which can generate the chitin deacetylase are obtained by combining the DNA recombination method and utilizing the known pronucleus express vector. The invention provides a reliable source for biologically preparing the chitin deacetylase and has the advantages of short production period, low extraction cost, higher product enzyme yield, higher output, and the like.

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 obtains a full length coding region cDNA sequence of a QSOX protein through an RT-PCR method. Then fixed point mutation is carried out; wild type genes and mutant genes are respectively constructed into pET series, pGEX series and pTYB series of vectors of pronucleus expression vectors and yeast expression series vectors so as to convert Escherichia coli and yeast for inductive expression and carry out the purification and function study on an expressed product.

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.