3590 results about "Gene engineering" patented technology

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR / Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

The invention relates to a medicine of a bispecific monoclonal antibody, and especially to a medicine of a bispecific monoclonal antibody to human vascular endothelial growth factor (VEGF / VEGF-A) and platelet-derived growth factor receptor (PDGFR) for resistance to angiogenesis of tumor. The bispecific antibody to VEGF / PDGFR beta provided in the invention is characterized in that: a monoclonal antibody to VEGF is used as the base for the antibody and a single chain antibody to PDGFR beta is connected with the terminal of FC segment of the monoclonal antibody to VEGF to form the bispecific antibody to VEGF / PDGFR beta. The bispecific antibody related to in the invention is obtained by employing technical means like gene engineering and constructing antibody segments which identify VEGF and PDGFR beta in a same antibody molecule that can be specifically bound with the two antibody segments; the effect of the bispecific antibody on inhibiting angiogenesis of tumor issue is obviously superior to that of a single antibody to VEGF; and the bispecific antibody has good activity in resisting tumors.

The invention discloses a recombinant humanized collagen. Its amino acid sequence is as shown in SEQNO.3 and has 599 amino acids. The molecular weight is 55.0kDa. A preparation method of the recombinant humanized collagen comprises the following steps of: constructing a gene engineering strain for the expression of the recombinant humanized collagen to obtain a Pichia pastoris engineering strain; carrying out fermentation culture on the engineering strain, and realizing inducible expression of the recombinant humanized collagen to obtain a recombinant humanized collagen fermentation broth; and purifying the fermentation broth to obtain the recombinant humanized collagen. The humanized collagen gene Ge1 designed in the invention is a brand-new sequence which is greatly shorter than a natural humanized collagen gene (Kbp) in length. By the preparation method, operation simplification at the molecular level is realized, and the gene engineering strain fermentation production of the collagen is more easily realized. The humanized collagen gene Ge1 contains characteristics of a natural humanized collagen gene. And simultaneously, its expressed protein has excellent characteristics of a natural humanized collagen.

The composite spongy biological dressing contains chitosan, collagen and calcium alginate with weigh tmixing ratio o f 0.5-8:0.5-8:0.1-8. Its preparation method includes the following steps: selecting chitosan and collagen type I, adding calcium alginate, compounding and cross-linking, using buffer solution to make neutralization, emulsifying, prefreezing and one-step freeze-drying so as to obtain the invented dressing with good biological compatibility and strong adhesion property. Said invented dressing possesses active function of promoting wound healing and hemostatic action, can be combined with anti-bacterial medicine to obtain gene engineeirng dressing for curing wound surface infection, also can be combined with active growth factor or active cell to form gene engineering dressingfor curing intractable ulcer and burn wound surface.

The invention relates to the field of gene engineering, and provides a system and a method for constructing a sequencing library. The system and the method comprise a joint element and a method for applying the joint element to the construction of a sequencing library. The joint element is a bifurcate double-chain nucleic acid joint with a bifurcate region and a paired region orderly included at the 5' and the 3' ends, wherein two single chains of the bifurcate region respectively comprise at least one amplification primer binding site, and the paired region comprises at least one restrictionenzyme site. The joint element of the invention can prevent the self-connection phenomenon of the joint element and the occurrence of a non-object joint element-DNA fragment compound during the library construction.

The invention relates to a peptide for high performance inhibition of angiogenesis and method for preparing same and use, wherein high performance blood vessel production inhibiting agent RGD-ED with integration compatibility is designed, the inhibiting agent comprises polypeptide polypeptide-valine-arginine-arginine-alanine-aspartate-arginine-alanine-alanine-valine-praline, its one or two ends are connected with polypeptides containing arginine-glycine-aspartic acid sequence. The RGD-ED provided by the invention can be synthesized. The invention also discloses the expression of one RGD-ED in bacillus coli through gene engineering method, wherein the RGD-ED is prepared through the steps of inclusion body protein segregation, dissolution and renaturation, and ion-exchange chromatography segregation and purification.

A protein which inhibits osteoclast diffraction and / or maturation and a method for producing the protein. The protein is produced by human embryonic lung fibroblasts and has a molecular weight of about 60 kD and about 120 kD under non-reducing conditions and about 60 kD under reducing conditions an SDS-polyacrylamide gel electrophoresis. The protein can be isolated and purified from the culture medium of fibroblasts. Furthermore, the protein can be produced by gene engineering. The present invention includes cDNA for producing the protein by gene engineering, antibodies having specific affinity for the protein or a method for determining protein concentration using these antibodies.

The invention relates to the preparation of a functional area of a sea purse blood vessel growth inhibition factor 1 and the use of the functional area of the sea purse blood vessel growth inhibition factor 1 in medicaments for preventing and curing tumors. In the invention, a gene engineering technique is used to realize the cloning, expression and recombination of the sea purse blood vessel growth inhibition factor 1; the recombinant functional fragment of the sea purse blood vessel growth inhibition factor 1 has the bioactivities for resisting the growth of blood vessels, tumor growth, tumor transplantation, acute toxicity tests and stability tests; and the functional area of the sea purse blood vessel growth inhibition factor 1 can be mixed with or dissolved in pharmaceutically acceptable carriers to prepare the medicaments for curing various tumors. The functional area of the sea purse blood vessel growth inhibition factor 1 has high action specificity, has the characteristics of easy expression, low degradation rate and the like of gene engineering medicaments and has the effects of inhibiting blood vessel growth, tumor growth and tumor transplantation; the provided gene engineering technique can realize the industrial production of the functional area of the sea purse blood vessel growth inhibition factor 1; and the functional area of the sea purse blood vessel growth inhibition factor 1 prepared by the gene engineering technique can be used in the preparation of medicaments for inhibiting blood vessel growth and preventing and curing tumors.

The invention belongs to the field of gene engineering applications and particularly relates to a sgRNA targeting sequence of a specific target human ABCB1 gene and an application. The sgRNA targeting sequence of the specific target human ABCB1 gene is Sg1 or Sg2; the nucleotide sequences of Sg1 and Sg2 are as follows: Sg1: 5'-TTGGACTGTCAGCTGCTGTC-3'; Sg2: 5'-GACTGTCAGCTGCTGTCGTC-3'. Two single-stranded oligo sequences are designed and synthesized according to the sgRNA targeting sequence, double strands are formed through annealing and then connected with a Cas9 carrier, sgRNA and a CRISPR system are introduced into a target cell through the Cas9 carrier, Cas9 protein can find a matched DNA sequence under the guidance of sgRNA, cutting is performed, and the ABCB1 gene is knocked out.

The invention discloses a biological production method of tanshinol, which comprises the following steps: synthesizing p-hydroxyphenylpyruvic acid into 3,4-dihydroxyphenylpyruvic acid under the catalytic action of a p-hydroxyphenylacetic acid meta-position hydroxylation enzyme, and then, synthesizing to obtain the tanshinol under the catalytic action of a D-lactate dehydrogenase; or synthesizing p-hydroxyphenylpyruvic acid into p-hydroxyphenyllactic acid under the catalytic action of a D-lactate dehydrogenase, and then, synthesizing to obtain the tanshinol under the catalytic action of a p-hydroxyphenylacetic acid meta-position hydroxylation enzyme. According to the invention, gene engineering glutamic acid corynebacteria and Escherichia coli are used to produce the tanshinol through fermentation, and the tanshinol can be synthesized without adding a substrate, thereby realizing de novo synthesis of the tanshinol and solving the problem on the source of the tanshinol; and meanwhile, the production cost is lowered to the greatest extent. Thus, the biological production method is beneficial to industrial production.

The invention belongs to the technical field of gene engineering, and concretely relates to a CRISPR / Cas9 single transcription unit directionally modified backbone vector and an application thereof. Technical problems to be solved in the invention comprise low species versatility and difficult realization of Cas9 protein expression and gRNA transcription cooperation of present CRISPR / Cas9 genome editing systems. A technical scheme in the invention is characterized in that a CRISPR / Cas9 single transcription unit backbone vector is constructed, and transcription of Cas9 and a guide RNA core unit are regulated by a promoter. The invention also provides a method for constructing a target site specifically modified Cas9-gRNA recombinant vector by adopting the CRISPR / Cas9 single transcription unit backbone vector. The efficient CRISPR / Cas9 single transcription unit backbone vector can effectively realize Pol II promoter driving-based Cas9 and gRNA unit cooperative transcription, and also can realize simple, fast and efficient genome directional heredity modification of various eukaryotes.

The invention discloses a CRISPR Cas9 system for Bacillus subtilis genome edition and an establishment method thereof, belonging to the technical field of gene engineering. A knock-out plasmid PHY300dsrf is established; and the plasmid comprises sgRNA and cas9 genes of the specific targeted target gene, a homologous restoration arm, and replication initial points and tolerance screening markers of Escherichia coli and Bacillus subtilis. The sgRNA of the specific targeted srfA-C gene can guide the cas9 protein to cut double strands at the specific site of the srfA-C gene, and can be used for accurate homologous recombination on the genome under the guide of the homologous restoration arm, thereby introducing the XhoI enzyme digestion site at the rupture. When the tank feed fermentation culture is carried out on the Bacillus subtilis of which the srfA-C gene is successfully knocked out, the foam is obviously reduced, which proves that the CRISPR / Cas9 gene editing system can effectively perform gene edition on the Bacillus subtilis genome.

The invention belongs to the field of gene engineering, and particularly relates to a method for specifically knocking out CCR5 genes of human bodies through CRISPR / SaCas9 and sgRNA used for specifically targeting CCR5 genes. The invention provides the method for specifically knocking out the CCR5 genes of the human bodies through CRISPR / SaCas9 and sgRNA used for specifically targeting the CCR5 genes and relates to a CRISPR / SaCas9 recombinant lentivirus and adeno-associated virus vector system and application. CRISPR / SaCas9 recombinant lentivirus and adeno-associated virus vectors can express SaCas9-RNA aiming at the CCR5 target spot of one same gene of the human bodies and rhesus monkeys, therefore, the application range is wider, and the efficiency is higher. The preparation method is easy to construct, the targeting efficiency is high, and a novel technology is provided for the gene therapy of HIV.

The invention relates to the field of gene engineering and particularly discloses a tool for efficient site-specific transposition of genes. The tool comprises or can produce a gRNA-Cas9 protein (without incision enzyme activity)-PB transposase compound, and gRNA targets the specific site of a genome; the Cas9 protein without incision enzyme activity is double-mutant Cas9 protein with 10th-site amino acid residue D mutating into A and 840th-site amino acid residue H mutating into A. Through design of gRNA (guide RNA) targeting the animal genome, under the coexpression condition of gRNA and site-specific transposase, PB transposase is targeted to a specific position of the genome along with a CRISPR / cas9 and gRNA compound, the PB transposase realizes a transposition function at the specific site, accordingly, donor plasmids (carriers containing exogenous target genes) are simultaneously co-transfected, the exogenous target carriers can be transposed to the specific site in the genome through transposition, and site-specific transposition of the genes is realized.

The invention provides a targeted sgRNA for editing a pig APN gene and a modified carrier as well as a preparation method and application thereof, and relates to the technical field of gene engineering. The sgRNA and the gene modified carrier provided by the invention are strong in speciality, and effectively edit the pig APN gene on cell level by a CRISPR / Cas9n system, and positive cells are edited by the obtained APN gene into donor cells to carry out somatic cell cloning and embryo transferring, so that the obtained APN gene edited cloning pig has ability of resisting piglet diarrhea. According to the preparation method of the piglet diarrhea resistant pig provided by the invention, a receptor of a virus causing the piglet diarrhea is destroyed, any other exogenous genes cannot be introduced except edition of a targeted gene APN, non-speciality edition on a non-APN gene area on a gene group cannot be carried out, a genetic background is clean and clear, and a late safety assessment work of transgenesis is greatly reduced.

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

The invention provides an antibody detection double-antigen sandwich method using nanoparticles. A nanoparticle label and a label between the labeled antigens implement an indirect labeling by combining the label on an antigen and a ligand which is labeled on the nanoparticle label and can specifically recognize the label, wherein the labeled antigen is a gene engineering recombined antigen. Withthe indirect labeling manner, the sensitivity and the specificity of the method can be remarkably improved.

The invention belongs to the field of gene engineering and discloses a target sequence identified by a CRISPR-Cas9 system from neisseria meningitidis. The target sequence is as shown in the nth-24th sites of optional one of SEQ ID No. 1-20, and n=1-9. The invention further discloses sgRNA and coding DNA molecules thereof, the sequence of the sgRNA is 5'-identificiation sequence-sequence recruiting Cas9 protein-3', and the DNA sequence corresponding to the identification sequence is identical with the target sequence. The invention also discloses the CRISPR-Cas9 system which comprises the Cas9 protein, the sgRNA and / or a coding sequence carrying the Cas9 protein and a vector of the coding sequence of the sgRNA. The invention also discloses application of the CRISPR-Cas9 system to editing of CCR5 genes and preparation of medicine for HIV infection. By the CRISPR-Cas9 system, the CCR5 genes can be edited, and cells cannot be infected by HIV.

The invention discloses a method for rapidly establishing a Cas9 dual-element expression carrier library of paired sg RNA, and relates to a method for rapidly establishing a dual-element carrier in the gene engineering field. According to the method, on the basis of establishing a CRISPR-Cas9 dual-element expression carrier, corresponding positive and negative primers are designed according to a specific gene design target sequence and have two BsaI restriction enzyme cutting sites; through one-step PCR, the target sequence and target segments of two BsaI sites are obtained, and the segments enter the dual-element expression carrier with a Cas9 gene through a simple restriction enzyme cutting and connecting mode to form the Cas9 carrier of the paired sg RNA. A random library and a non-random library can be efficiently established and can be used for large-scale screening of gene functions in plants and meanwhile can be used for establishment and research of a gene interoperation network among multiple genes; it is proved through experimental results that the Cas9 dual-element expression carrier library of the randomly-paired sg RNA of 14 target spots (from 14 genes) is successfullyand efficiently established through the method.

The invention provides a targeting sgRNA for pig CD163 gene editing, a modification vector, and a preparation method and application thereof, which relate to the technical field of gene engineering. The sgRNA and the gene modification vector provided by the invention have high specificity, and can be used for efficiently editing a pig CD163 gene on a cell level through a CRISPR / Cas9n system. According to the preparation method of a blue-eared disease resistant pig provided by the invention, a blue-eared disease virus receptor is damaged, the target gene CD163 is edited, no other exogenous gene is intruded, no non-specific editing is carried out on a non-CD163 gene area on a genome, a genetic background is clean and clear, and the work of later transgene safety evaluation is greatly reduced. The blue-eared disease resistant pig obtained by utilizing the invention can be ensured to survive normally, and resists the infection of a blue-eared disease virus, so that the economic loss caused by the blue-eared disease in pig husbandry is greatly reduced.

The invention belongs to the field of gene engineering, and relates to chimeric antigen receptor of anti-human CD19 antigen and application thereof. The chimeric antigen receptor of anti-human CD19 antigen comprises polypeptide (scFv) for recognizing human CD19 antigen, a hinge region, a transmembrane region and an intracellular signal domain which are connected in sequence, wherein an amino acid sequence of the polypeptide (scFv) for recognizing human CD19 antigen is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. The chimeric antigen receptor can be more stably expressed in T lymphocyte, has the capacity for properly removing cancer cell, can not only maintain the positive rate of CD19 targeted chimeric antigen receptor during the cell culture of a patient, but also enhance the capacity for CAR-T multiplication and tumor killing, does not have toxic and / or side effects on antigen-negative cell, and can be applied to the targeted therapy of tumor.

The invention relates to a DNA transmethylase defective CHO (Chinese hamster ovary) cell line and a preparation method and application thereof and belongs to the technical field of gene engineering. DNA Transmethylase Dnmt3a gene of CHO cells is knocked off by means of CRISPR / Cas9 gene editing technique, and screening and identifying are performed to obtain DNA transmethylase Dnmt3a defective CHOcells; the cells are transfected with a eukaryotic expression vector, stably expressed recombinant CHO cell strains are screened, and accordingly a novel CHO cell expression system based on DNA transmethylase deficiency is established. The recombinant gene CHO cell expression system is established via host CHO cell genetic modifications, expression level of recombinant proteins can be significantly increased, the problem of recombinant protein expression instability is solved, and recombinant protein expression stability is improved.

The invention belongs to the field of gene engineering and discloses a target sequence identified by a CRISPR-Cas9 system from neisseria meningitidis. The target sequence is as shown in the nth-24th sites of optional one of SEQ ID No. 1-15, and n=1-9. The invention further discloses sgRNA and coding DNA molecules thereof, the sequence of the sgRNA is 5'-identificiation sequence-sequence recruiting Cas9 protein-3', and the DNA sequence corresponding to the identification sequence is identical with the target sequence. The invention also discloses the CRISPR-Cas9 system which comprises the Cas9 protein, the sgRNA and / or a coding sequence carrying the Cas9 protein and a vector of the coding sequence of the sgRNA. The invention also discloses application of the CRISPR-Cas9 system to editing of CXCR4 genes and preparation of medicine for HIV infection. By the CRISPR-Cas9 system, the CXCR4 genes can be edited, and cells cannot be infected by HIV.

The invention is related to a recombinant single-chain tri-specific antibody made from anti-Tumor Associated Antigen (TAA) antibody, FC interlinker, anti-CD3 antibody, HSA interlinker and anti-CD28 antibody in turn. Particularly, the invention relates to an anti-CEA, anti-CD3, anti-CD28 recombinant single-chain tri-specific antibody, CEA-scTsAb, which was constructed with three tandem antibody fragments (anti-CEA scFv, anti-CD3 scFv and anti-CD28 single-domain antibody) linked by two interlinkers (FC interlinker, HSA interlinker), and could be appended by C myc tag or histidine tag ((His)6-tag) at the C terminal. It also concerns a method for construction, expression and purification of the antibody. It also offers the encoded DNA sequence of the antibody, expression vectors and host cells for the vectors.

The invention relates to the technical field of gene engineering, and discloses a recombinant yeast strain, and a construction method and application thereof. In the recombinant yeast strain, gal1, gal7, gal10 and ypl062w genes are knocked out, and the recombinant yeast strain comprises 9 gene segments which are integrated to the genome by yeast homologous recombination. The construction method comprises the following steps: constructing a four-knock-out yeast strain to provide an optimized host cell for producing beta-carotin, selecting lycopene cyclases crtY from different sources, and carrying out modular design to integrate functional genes crtE crtB and crtI of the specific-source synthetic lycopene, specific yeast endogenous genes and the like to the four-knock-out yeast strain genome, thereby obtaining a brand-new recombinant strain capable of producing beta-carotin at high yield.

The invention relates to an application of CRISPR / Cas 9 technology in obtaining of bombyx zinc finger protein gene mutation, and belongs to the technical field of bombyx breeding and gene engineering. In the invention, the CRISPR / Cas 9 gene editing technology is applied to obtain bombyx zinc finger protein gene mutation, a set of method for performing gene edition on zinc finger protein and exploring the gene function is realized, and thus a set of effective method for the batch research and transcription factors in future is provided.

The invention discloses application of an OsKTN80b gene in terms of reducing rice plant height, belonging to the field of rice gene engineering. A plant with reduced plant height is finally obtained mainly by performing gene editing on the OsKTN80b gene through a CRISPR / Cas9 system, so as to obtain a transgenic plant subjected to OsKTN80b gene knockout, and selecting a homozygous mutant. The invention further discloses two target spot sequences SG1 or SG2 used for the OsKTN80b gene knockout; the target spot sequences are respectively formed by nucleotide sequences shown as SEQ ID No. 4 and SEQ ID No. 5. Compared with wild type rice, the plant height of the transgenic plant subjected to the OsKTN80b gene knockout is reduced by 13.56 to 16.33 percent, and the effect of reducing the rice plant height is good; secondly, a method provided by the invention is not limited to a genetic background, and the problems linked with mal-characters do not exist.

The invention relates to a method for biosynthesis preparation of human glucagon-like peptide-1 (GLP-1) and an analogue thereof. With adopting of a gene engineering technology, a recombinant escherichia coli expressed GLP-1 fusion protein is constructed, and a protein enzyme digestion site is designed in the fusion protein; a fusion gene has a gene sequence with a form of A-B-C structure, wherein A is a chaperonin gene, B is a nucleotide sequence encoding a connection peptide containing the enzyme digestion site, and C is a gene encoding the GLP-1 or the analogue thereof. After recombinant engineering bacteria is subjected to induced expression, the fusion protein is purified and subjected to enzyme digestion, and then the GLP-1 and the analogue thereof are obtained and are detected to have biological activity. The preparation method of the GLP-1 and the analogue thereof provided by the invention is simple and quick, the production conditions are mild, the product is convenient to separate and extract, the process is simple, and the industrialization prospect is good.