646results about How to "Genetic stability" patented technology

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production. The invention also provides methods for increasing the effector function of monoclonal antibodies and monoclonal antibodies with increased effector function.

The invention relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

The invention discloses a method for transforming rice into fragrant rice rapidly. By means of the CRISPR / Case technology and related gene sequences in the rice fragrance metabolic process, a carrier is designed at the specific site, the element is transferred to non-fragrant rice through agrobacterium-mediated transformation, the gene is made to cause deletion mutation at the position of a fixed point, the gene is silent, fragrance cannot be normally metabolized and greatly accumulated, common rice is transformed into fragrant rice, then genetic transformation gene segments are separated from edited genes through selfing or hybridization, and the fragrant rice with the isozygoty capable of being stably inherited is rapidly obtained.

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. Cells may be selected for expression of activation-induced cytidine deaminase (AID), stimulated to produce AID, or manipulated to express AID for further enhancement of hypermutability. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production.

The invention relates to construction of a tomato transgenic material, and aims to provide a gene editing method using a CRISPR / Cas9 system to mutate a tomato SlMYB12 gene in order to transform red fruit tomato into pink fruit tomato. The method includes the following steps: designing an oligo primer corresponding to the gRNA target site of the SlMYB12 gene, and recombining and connecting an oligo dimer with a Cas9 / gRNA vector; transforming Escherichia Coli competent cells, transforming a plasmid with a correct sequencing result into Agrobacterium tumefaciens, and mediating and transforming tomato calluses to obtain a transgenic plant in order to obtain a transgenic positive strain; and carrying out gene sequencing and phenotype observation to determine a pure mutant strain which is an SlMYB12 function completely lost tomato mutant for pink fruits. The method can effectively knock out the transcriptional translation of the SlMYB12 gene, and is an ideal gene editing system for successfully transforming the tomato red fruits into the pink fruits.

The invention discloses an IL7R gene-deleted zebra fish mutant and a preparation method thereof. The construction of the IL7R gene-deleted zebra fish mutant is realized through a CRISPR / Cas9 technology. Moreover, the invention also discloses application of the IL7R gene-deleted zebra fish mutant; a mutant model provided by the invention can be used for studying an effect of IL7R in the nervous system development and myelination.

The invention discloses a genetic engineering cell line for producing an unfucosylated protein and an establishment method thereof. The invention utilizes the CRISPR / Cas9 technique to knock out the Slc35cl gene and / or the Fut8 gene in host cells, and thereby the Slc35cl gene-silencing and / or Fut8 gene-silencing stable genetic engineering cell line is obtained. By utilizing the genetic engineering cell line disclosed by the invention, the protein from which fucosylation is completely removed can be produced, moreover, the protein is still stable after 30 generations of passage, and thereby the two major problems of incomplete fucosylation removal and unstable passage in the prior art are solved. An unfucosylated antibody produced by the method disclosed by the invention shows enhanced ADCC (antibody dependent cell-mediated cytotoxicity) activity, and thereby the clinical therapeutic effect of the antibody is enhanced.

The invention relates to a breeding method for a new marine backcrossed scallop variety. The breeding method is characterized in that: a best individual is chosen from the first filial generation family or group of Argopecten irradians and Argopecten purpuratus, and is first backcrossed with Argopecten irradians to establish a backcrossed family or group, a best hermaphrodite individual is then chosen from the backcrossed family or the backcrossed group with excellent performance, at least three generations of selved families are then bred in succession, and thereby the new marine backcrossed scallop variety which has the characteristics of stable heredity, fast growth, big individual and wide temperature tolerance range is obtained. The new marine backcrossed scallop variety bred by the breeding method has the characteristics of both Argopecten irradians and Argopecten purpuratus, i.e. fast growth, big individual, wide temperature tolerance range and stable heredity, and can be cultured in northern sea areas such as the sea areas of Tsingtao and Dalian, and moreover, the current yield of the new marine backcrossed scallop variety is increased by more than 30 percent in comparison with the current yield of Argopecten irradians.

The invention discloses a preparation method of a zebrafish notch2 gene mutant. The preparation method includes: determining positions of knock-out target spots of the notch2 gene; utilizing pUC19-gRNA scaffold plasmid as a template, and performing PCR (polymerase chain reaction) amplification with primers T7-notch2-sfd and tracr rev; subjecting a PCR product to purification and in-vitro transcription to obtain gRNA (guide ribose nucleic acid); introducing the gRNA and Cas9 protein into a cell-stage embryo of zebrafish through micro-injection, and screening to obtain the notch2 gene mutant stable in inheritance. The preparation method has the advantages that by the aid of the CRISPR / Cas9 (clustered regularly interspersed short palindromic repeats, CRISPR / CRISPR-associated genes, cas gene)technology, and by means of selecting a specific section of targeting domain, the notch2 gene in the zebrafish is knocked out, other genes are protected from being 'injured accidentally', the zebrafish without the Notch2 gene is formed, experimental materials are provided for in-depth study on follow-up gene functions, and great significance is achieved on study of Notch signal channels.

The invention relates to a fusion protein of Exendin-4 tandem polypeptide and human serum albumin, a preparation method and application thereof. The fusion protein of the Exendin-4 tandem polypeptide and the human serum albumin is expressed by a genetic engineering method. The fusion protein shows long-acting controlling blood sugar activity in a body, solves the problem that the half life period of the Exendin-4 tandem polypeptide in the body is short and has the advantage of convenient preparation. The fusion protein can be used for preparing diabetes medicaments and weight-losing products.

The invention provides arthrobacter for producing cyclic adenosine monophosphate through fermentation and application thereof. With a preservation number of CGMCC No.3584, the arthrobacter of the invention has a A302 strain which is three times higher than an original strain in terms of the cyclic adenosine monophosphate output. Undergoing over 10 generations of passage, the A302 strain of the invention is stable in producing cyclic adenosine monophosphate.

The invention relates to a genetically engineered bacterium for producing 2'- fucosyllactose and application of the genetically engineered bacterium, and belongs to metabolic engineering and food fermentation technologies. The expression levels of phosphomannomutase, mannose-1-phosphoguanine transferase, GDP-mannose-6-dehydrogenase, GDP-fucosyl synthase, alpha-1,2-fucosyl transferase and lactose permease in a metabolic pathway are regulated and controlled by changing the expression of a transcription regulation factor in escherichia coli, so that an optimal plasmid combination is obtained; beta-galactosidase and UDP-glucose lipid carrier transferase in an escherichia coli synthetic route are knocked out through a CRISPR-Cas9 gene editing system, and therefore, the accumulated amount of 2'-fucosyllactose is increased; and the recombinant escherichia coli obtained by the invention can be used for synthesizing 2'-fucosyllactose by utilizing glycerol or glucose, is stable in heredity and high in expression level, and has obvious industrial production potential.

The invention relates to a slaughter type high-quality chilling fresh chicken breeding method. The breeding method includes: selecting recessive white-feather chickens (B line) having green shins and yellow skins as first male parents, and selecting large cockscomb high yield chickens (C line) having yellow shins and yellow skins as first female parents; hybridizing the B line and the C line to acquire parental generations (BC system) to be as final female parents according to the heredity principle that a green shin gene is sex-linked and an autosome controls a skin color gene, wherein parental chickens can be distinguished through the shin color, the shins of the female chickens are green, and the shins of the male chickens are yellow. using yellow-feather fast-growing chickens (A line) having green shins, yellow skins, and large cockscombs as final male parents, producing commodity chickens (ABC) by A line male X (B line male X C line female) female mating hybrid, wherein commodity male and female chickens have green shins, yellow skins, large cockscombs, and wide chests. The commodity chickens can be sold at the age of 63-65 days, the average weight of the female chickens is 1.6-1.65 Kg, the average weight of the male chickens is 1.8-1.85 Kg, the average uniformity can reach more than 96%; the dressing percentage, the whole net carcass rate, and the chest muscle rate can exceed 85%, 65%, and 18%, and the carcass character has significant advantages.

The invention discloses a method for efficient selective breeding of a new odor type rice blast resisting rice variety. The method is characterized in that eurytopic and high-quality odor type rice restorer fengxianghui No. 1 is taken as a receptor parent, a rice blast resisting rice material containing rice blast resisting genes is taken as a donor parent, the conventional breeding technique is adopted, the high-throughput molecular marker-assisted selection technique, the rice anther in vitro culture technique and the human nose fragrance smelling and resistance field identification means are comprehensively utilized, a resistant improved odor type rice fengxianghui No. 1 restorer with stable fragrance, high rice blast resistance and excellent agronomic trait is prepared, and the odor type rice blast resisting rice variety is obtained through efficient selective breeding by means of combination and measuring matching of the resistant improved odor type rice fengxianghui No. 1 restorer and a sterile line. The method is efficient, quick and low in cost. Meanwhile, the traditional breeding technique and the modern molecular biological technique are effectively combined, the popularization and application values of the variety obtained through selective breeding are high, and the method can meet the requirement of enterprises for commercial breeding.

The invention discloses a method for establishment of a hybrid strain between bluntsnout bream and topmouth culter. The bluntsnout bream is used as a female parent, the topmouth culter is used as a male parent, the hybrid strain F1 of the bluntsnout bream and the topmouth culter is obtained through distant hybridization and is then bred and tested to obtain a bisexual fertile F1; then F1 is cultured as an object, selfing of the bisexual fertile F1 is performed, and a diploid F2 of the bluntsnout bream and the topmouth culter is obtained through incubation and breeding; and the ploidy and sterility of F2 are tested to obtain a bisexual fertile F2, and thus the heterologous bream and culter stain with genetic stability and bisexual sterility is obtained. The invention also discloses a breeding method of topmouth bream. The bisexual fertile diploid hybrid strain F1 of the bluntsnout bream and the topmouth culter and the bisexual fertile F2 of the bluntsnout bream and the topmouth culter are obtained by the establishment method, and backcross breeding is performed with the bluntsnout bream to obtain the hybrid topmouth bream. By adopting the breeding method, the novel hybrid topmouth bream with characteristics of high growth speed, high resistance, high meat quality, attractive shape and the like can be prepared.

The invention discloses gene recombination bacillus amyloliquefaciens and a microbial inoculum preparation method. A bacillus amyloliquefaciens FZB42 strain is taken as a host bacterium to construct the gene recombination bacillus amyloliquefaciens XLV09-1 with transparent vitreoscilla hemoglobin which is regulated and controlled by a P43 strong promoter by contrasting a gene recombination vector. The produced spore number of microbial inoculum prepared by fermenting the bacillus amyloliquefaciens can reach more than 100 hundred millions per milliliter, the overall yield of antibacterial lipopeptid reaches more than 1 gram per liter and prevention effect on various fungal plant diseases is improved by over 30 percent. The microbial inoculum is a green microecological preparation, of no toxic residue, safe to human and livestock, and favorable for protecting the ecological environment.

The invention discloses an induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots. The induction method comprises the following steps: cutting off a root cap from the root tip of the catharanthus roseus, performing inoculating to an induction culture medium, performing culturing in dark, and proliferating and growing a stem cell layer inan explant to form a cell cluster; transferring the layered stem cell layer to a subculture medium for culturing for 10-15 days, and performing complete culturing in dark; and performing transferringto a new subculture medium, and performing culturing for 10-15 days under the same condition to obtain a catharanthus roseus stem cell line with stable cell morphology. A catharanthus roseus stem cell culture system is established by inducing apical meristem of the catharanthus roseus roots, stable subculture can be performed under certain conditions, and epigenetic variation is avoided. The catharanthus roseus stem cell culture system established by the invention has the characteristics of being high in growth speed, high in genetic stability and the like, and culture with a 10L bioreactor is preliminarily realized.

The invention discloses a genotype VII Newcastle disease virus marker vaccine strain and an application thereof, and belongs to the field of genotype VII Newcastle disease virus marker vaccine strain rescue and application. A built Newcastle disease virus reverse genetic operating platform is utilized for enabling NP protein of a G7 strain to miss 18 amino acids and conducting mutation on F-protein cleavage loci, and the highly-weak virulence and high-virus titer genotype VII Newcastle disease virus marker vaccine strain MG7-NPdelta18+Fmut is rescued through screening. The microbial preservation serial number is CCTCC NO: V201505. The marker vaccine strain has the biological characteristics of high growth titer and low virulence in chick embryos and is genetically stable. The immune protection test result shows that the marker vaccine strain is good in immunogenicity, capable of inducing high-level protective antibodies, and capable of completely protecting immunized chicken, can be used for preventing and controlling a currently-popular genotype VII Newcastle disease virus and lays the foundation of identifying vaccine immunity and wild virus infection.

The invention discloses saccharomyces cerevisiae for producing ribonucleic acid by fermentation. The saccharomyces cerevisiae has a strain number of TKZZY-06 and is preserved in China General Microbiological Culture Collection Center, China Committee for Culture Collection of Microorganisms, on April 26th, 2013, with a preservation number of CGMCC No. 7522. The invention also discloses an application of the saccharomyces cerevisiae. RNA content of the TKZY-06 strain thallus can reach 17.5% that of dry weight of the thallus and is increased by 9.5% than RNA yield of an original strain. The strain is subcultured by over 10 generations; and the performance for producing the ribonucleic acid can be kept stable.

The invention discloses an antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris (Pichia pastoris FZM2009, CCTCC NO:M209259), and a preparation method and applications thereof. The method comprises the steps: A, the construction of expression cassette PAOX1-alpha Factor-IFN alpha-6xHis-bufforin II-Zeocinr; B the construction of expression engineering bacteria Pichia pastoris FZM 2009, wherein the engineering bacteria is resulted from the screening of antibiotics Zeocin and PCR identification; C, the preparation of fusion protein, which comprises the steps: 1) the recovery is implemented on a YPD plate containing 100mug / ml of Zeocin; 2) single clone is inoculated to a YPD-containing culture medium for vibration and culture; 3) 1 volume of culture is inoculated into 10 volumes of a fermentation culture medium for culture; 4) supernatant is obtained by centrifugation; 5) loading buffer with double volume is added into the supernatant, and electrophoresis detection is performed; 6) the supernatant is subject to a molecular sieve gel column to collect components; 7) the collected components are lyophilized to result in fusion protein; 8) buforin II and IFN alpha are obtained by the enzyme digestion of enterokinase on the fusion protein. The fusion protein buforin II and IFN alpha in fusion expression are applied to pig feeds. The method has great feasibility, simple and convenient operation, high expression, low cost and strong bioactivity.

The invention relates to a maltose inducible trehalose synthase synthesis engineering bacterium, a method for preparing the same and application. The maltose inducible trehalose synthase synthesis engineering bacterium is characterized in that maltose inducible promoters are inserted in the fronts of BamHI cleavage sites of PHT01 plasmids of recombinant plasmid vectors instead of Pgrac promoters on the PHT01 plasmids, expression genes of Tat type signal peptides are inserted in the fronts of the BamHI cleavage sites, and expression genes of trehalose synthase are inserted in the rears of the BamHI cleavage sites. The maltose inducible trehalose synthase synthesis engineering bacterium, the method and the application have the advantage that expression effects realized after the maltose inducible promoters and the trehalose synthase are fused with one another are obviously superior to other inducible expression effects.

The invention discloses a bacillus subtilis for highly producing D-ribose and co-producing acetoin, which is preserved in a China general microbiological culture collection center with CGMCC No.3720 and the preservation data of April eighth, 2010. The invention also discloses a method for producing the D-ribose and co-producing the acetoin by fermenting the bacillus subtilis. The selected bacterial strains can produce produce the D-ribose and co-produce the acetoin by using the fermentation of a plurality of types of carbon and nitrogen sources, the operation is convenient and simple, the conditions of culture are extensive, and the industrialization is easy. In the invention, an organic acid is added to a fermentation medium, therefore, the yield of the D-ribose is improved by 64.5g / L compared with the original strains, and the acetoin is improved by 17.3g / L. The bacillus subtilis has higher capability for synthetizing the D-ribose and the acetoin, and the yield of reduced by-products of 2,3-butanediol of the acetoin is low.

The invention discloses a micropropagation method of fraxinus rhynchophylla and relates to the micropropagation method. The problems of long reproductive cycle, low reproduction efficiency and poor offspring genetic stability in nursery stock propagation of fraxinus rhynchophylla are solved. The method comprises the following steps of: 1, sterilizing fraxinus rhynchophylla seeds, and culturing single cotyledons of zygotic embryos to obtain cotyledonal cell embryos; 2, performing maturation cultivation on the cotyledonal cell embryos; 3, performing sprouting cultivation on the cotyledonal cellembryos subjected to maturation cultivation to obtain regenerated plants; and 4, transplanting the regenerated plants into a culture medium to culture until new leaves are completely unfolded, and removing a covered plastic thin film. The micropropagation method has the advantages of short nursery stock reproductive cycle, high reproductive rate (culturing for 60 to 70 days) and high reproductionefficiency (each explant can generate 15 to 20 somatic embryos); the germination rate of the somatic embryos is up to 87 to 89.55 percent; the transplanting survival rate of the regenerated plants isup to 75 to 80 percent; and the regeneration plants with the same good characteristics as female parents can be generated in mass.

The invention discloses a corn transformation event 'double resistance 12-5' and a specificity identification method thereof. The corn transformation event 'double resistance 12-5' is characterized in that a nucleotide sequence shown in SEQ ID No.1 is taken as a left side wing region of a foreign gene and a nucleotide sequence shown in SEQ ID No.3 is taken as a right side wing region of the foreign gene. The transformation event '12-5' provided by the invention is good, the foreign gene can be specifically introduced into a corn line, and insect resistant and glyphosate resistant capability is given to a corn acceptor; an insect-resistant and glyphosate-resistant gene can be stably inherited in the corn acceptor; and expression of the insect-resistant and glyphosate-resistant gene can not produce adverse effects on agronomic characters of the corn acceptor.

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production. The invention also provides methods for increasing the affinity of monoclonal antibodies and monoclonal antibodies with increased affinity.

High yield antibiotics producing fungus strain, preparation method and use thereof are provided. The fungus strain is a mutant derived from Glarea lozoyensis, and deposited in CGMCC with the accession number of CGMCC 2933. The preparation method concludes following steps: (a) mixing the culture media of Glarea lozoyensis strain ATCC 20957 with nitrosoguanidine, and obtaining mixture a; (b) mixing lywallzyme with the mixture a, and obtaining protoplasts; (c) regenerating the protoplasts, and obtaining single clones; and (d) culturing the single clones, then obtaining the mutant strain. This fungus strain has stable genetic and producing property, produces little impurities in fermentation, and is suitable to be used in industry.

The invention relates to a method for establishing tissue culture regeneration system of tea tree. The method comprises the steps of inducing callus; performing callus subculture and proliferation; inducing adventitious bud; proliferating adventitious bud; hardening seedlings; rooting and transplantation. The method includes two proliferation stages, i.e., callus proliferation and adventitious bud proliferation. The callus proliferation manner has the characteristics of simple proliferation manner and stable inheritance. After subculture for 4 years, cell activity and differentiation ability have no obvious change. The callus with low phenols content can be taken as genetic transformation object. The technology establishes solid experimental foundation for establishment of genetic transformation system of tea tree.

The present invention relates to an Aspergillus niger strain ANUV90 with high stable resistance to carbendazim obtained by mutagenesis, and the resistance of the strain to carbendazim is 1000 times higher than that of the original starting strain AN. Aspergillus niger ANUV90 can still grow normally under the treatment concentration of active ingredient carbendazim up to 1000 μg / mL, and the fermentation broth of this strain also has nematicidal activity. After testing, the strains before and after mutagenesis have no significant difference in colony morphology and other characteristics. At the same time, they have certain biological activity against tomato root-knot nematode. The preliminary research results of the analysis of the molecular mechanism of drug resistance of the mutant strains show that the gene encoding β-tubulin has a site-directed mutation , leading to the generation of drug resistance in biological characteristics of ANUV90. The bacterial strains specifically involved in the present invention have been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on October 30, 2007, with the preservation number CGMCC No.2234, and the classification name: Aspergillus niger. This feature of the strain allows it to be used synergistically with most commonly used pesticides to control a variety of harmful organisms, providing a way to solve the problem of unstable control effects of biocontrol strains in the field, and has a good development and application prospect.

The invention discloses a breeding method for amphiprotic fertile autotetraploid crucian carps and an establishment method for strains of the crucian carps. The breeding method includes the following steps that allotetraploid crucian carps are selected from crucian carp hybridization F1 offspring with red crucian carps as female parents and megalobrama amblycephala as male parents, female individuals capable of generating ova of different sizes and male individuals capable of generating watery semen are selected from the allotetraploid crucian carps for hasten parturition, insemination is carried out manually through a dry method, running water incubation is conducted on fertilized ova, then the fertilized ova are bred, and the amphiprotic fertile autotetraploid crucian carps are selected from selfing offspring. Selfing is carried out on the male individuals and the female individuals in the obtained autotetraploid crucian carps, and the strains of the amphiprotic fertile autotetraploid crucian carps can be established and obtained through multi-offspring breeding. Ploid hybridization is conducted on the autotetraploid crucian carp colonies obtained through the breeding method and diploid crucian carps, so allotriploid fishes can be bred on a large scale. The strains of the crucian carps obtained through the breeding method are excellent in character, stable in heredity and high in fertilization rate and hatchability, and the breeding method has a great significance in genetic research of fishes.

The invention provides a coxsackie virus A16-type virus strain. The collection number of the coxsackie virus A16-type virus strain is CGMCC No.5372, wherein CGMCC refers to China General Microbiological Culture Collection Center. The virus is a 20-face stereoscopic symmetrical sphere under observation through an electron microscope, and the diameter of the virus is 23-30nm. VP1 conserved region sequence analysis and mass spectrum analysis are respectively performed on the virus strain, and a result shows a CA16 virus. The CA16 virus can be efficiently proliferated in Vero cells (African green monkey kidney cells), and the virus titer can reach 6.61g CCID50 / ml. Moreover, the virus strain has no external pollution, better immunogenicity and a good effect.