42results about How to "Easy genetic manipulation" patented technology

The invention provides mesenchymal stem cell injection liquid and a preparation method, which aim to solve the problem of how to use a mesenchymal stem cell to efficiently repair and improve damaged skin and fundamentally delay and stop skin cell aging. The injection liquid provided by the invention is prepared from the mesenchymal stem cell, DMSO, EGF, glutathione, vitamin C, human serum albumin,a ginkgo biloba extract and a menstruum. The mesenchymal stem cell is obtained through carrying out hungry culture on P2 to P5-generation mesenchymal stem cells and stimulating through an epidermal growth factor. The obtained mesenchymal stem cell has a good differentiative capacity and a good proliferation capacity, has a better differentiation potential, and is synergized with the DMSO, the EGF, the glutathione, the vitamin C, the human serum albumin and the ginkgo biloba extract, so that skin wrinkles and hyperpigmentation can be remarkably reduced.

The invention discloses a method for efficiently acquiring inductive pluripotent stem cells (iPSCs). Adipose-derived stem cells (ADSCs) are induced and reprogrammed to form the iPSCs by using the method. The method concretely comprises the following steps: (1) inducing cDNA (Complementary Desoxvribose Nucleic Acid) of a multifunctional factor to the ADSCs; (2) culturing the ADSCs by using a serum-free culture medium in a feed-layer-free system, wherein the ADSCs is obtained in the step (1); (3) after cloning sample cells of embryonic stem cells (ESCs), further culturing by using a culture medium with an MEK (Methyl Ethyl Ketone) and GSK3 (Glaxo Smith Klein) signal channel inhibitor, selecting cells to clone, and enlarging culture; and (4) authenticating the cell cloning pluripotency. In the method, the ADSCs are low in cost and easily available on a large scale, and the reprogramming speed and efficiency are very high; the feed-layer-free system can be used for increasing the cloning purity of positive iPSCs; the serum-free culture medium can be used for avoiding unstable factors brought by undefined components in serum and batch difference; the signal channel inhibitor can be used for promoting cells to be up to the sufficient reprogramming state and improving the cloning quality of the positive iPSCs.

The invention discloses a bacillus gene traceless knockout / knockin plasmid and method, and a kit. The genome of the bacillus gene traceless knockout / knockin plasmid contains only one kind of resistance gene marked with a positive selection marker, a replicon capable of being duplicated in escherichia coli, a thermo-sensitive type replicon capable of being duplicated in bacillus, at least one I-Scel enzyme cutting site and only one kind of chromogenic protein gene, wherein promoters in front of the resistance gene and the chromogenic protein gene are both constitutive promoters in bacillus. The invention further provides a helper plasmid used for improving the knockout / knockin efficiency of the bacillus gene traceless knockout / knockin plasmid, and resistance gene and chromogenic protein gene in the genome of the helper plasmid are different from those of the knockout / knockin plasmid. By the adoption of the two plasmids, one or more target DNA sequences in the genome of bacillus can be modified continuously or iteratively, and the plasmids can be applied to multiple fields including bacillus genetic modification, metabolic process researching, functional genome researching and industrial application researching.

A method for converting Chinese ephedra total DNA into yeasts, the transfer gene yeasts thereof, a culture medium for separating the transfer gene yeasts and liquid culture medium for transfer gene yeasts are disclosed.

The invention discloses a recombination blue-green alga for producing a lactic acid as well as a preparation method and applications thereof. The method disclosed by the invention comprises the following steps of: modifying a metabolic pathway, and importing a coding gene of D-lactic dehydrogenase into a genome of the blue-green alga to obtain the recombination blue-green alga for producing an optical pure D-lactic acid. An amino acid sequence of the D-lactic dehydrogenase is a sequence 3 in a sequence table. Proved by experiments, the recombination blue-green alga disclosed by the invention is constructed through a metabolic engineering modification method, and carbon dioxide (CO2) can be directly converted into organic matter by utilizing solar energy by utilizing the characteristic that the blue-green alga is photoautotrophic; and the recombination blue-green alga can be directly synthetized into the optical pure D-lactic acid by utilizing the solar energy and the CO2 by utilizing the characteristic that the blue-green alga can grow in seawater and sewage and the advantage that genetic manipulation can be easily performed on the blue-green alga.

The invention discloses a method for preparing strain which can generate protoplast fusion by using marked plasmid. The invention has the advantages that marks can be easily added and removed, thereby being convenient for performing breeding and improvement of the strain based on the gene shuffling technology. The invention also has the advantages that the operation is simple, the cost is low, the requirement for equipment is low, and the chromosome of the strain can not be damaged.

The invention discloses a new function of cyanobacteria. Under irradiation of specific wave length lasers, and through photosynthesis, oxygen is generated, the condition of oxygen deficiency caused bytumors can be improved, and the problem of reverse tolerance of chemicals, such as adriamycin can be effectively solved. The cyanobacteria is subjected to photosynthesis in light to generate oxygen,and under the aerobic condition, hypoxia factors are reduced for tumor cells, so that multidrug resistance genes can be restrained, expression of P-glycoprotein is restrained to restrain exocytosis ofcancer cells on adriamycin, the reverse tolerance of the cancer cells on the reverse tolerance is overcome, and the antitumor treatment effect can be increased.

The invention belongs to the technical field of biological pharmacy, in particular to a chimeric antigen receptor (CAR) targeting BCMA and an application thereof. According to the chimeric antigen receptor, a single-domain antibody targeting BCMA is used as an antigen binding domain; in the process of preparing the CAR-T cells, optimization is carried out by adjusting the proportion of CD4<+> andCD8<+> T cells, the obtained novel CAR-T cells can effectively kill tumor cells, cell factors such as IFN-gamma, IL-10 and IL-6 are controlled within an acceptable range, and the product has good clinical application prospects.

The invention features soluble FGF decoy polypeptides and fusion polypeptides comprising an FGF decoy polypeptide linked to a heterologous polypeptide, such as an aggrecan binding protein. Both soluble FGF decoy polypeptides and fusion polypeptides can be used to prevent or treat skeletal disorders, such as achondroplasia.

The invention provides a method and application for realizing one-step purification and immobilization of a foreign protein through a magnetic induction protein serving as a purification label, and a universal vector. The method comprises the following steps: (1) by taking pET-28a(+) as a vector plasmid, inserting a magnetic induction protein gene into the vector plasmid, thus building the universal vector; (2) inserting a foreign protein gene into the universal vector, thus building a recombinant expression plasmid, and introducing the recombinant expression plasmid into a host cell, thus obtaining a recombinant strain; (3) cultivating the recombinant strain, inducing foreign protein expression, collecting a product, and feeding nano iron beads into the product, thus realizing one-step purification and immobilization of the foreign protein. Sites from 5' to 3' of the universal vector comprise the following elements in sequence: a promoter T7, the magnetic induction protein gene, a thrombin cleavage site, a linker, a multiple-cloning site and a terminator T7. The invention provides the method and the application for realizing one-step foreign protein purification and immobilization, which are easy to operate and have the advantages of improving the efficiency and reducing the cost.

The invention discloses a recombinant plasmid containing a tetH promoter, a firefly luciferase gene luc, a chloramphenicol resistance gene cat, a replicator oriV, a junction transfer gene mob, replication protein genes repA, repB and repC and a Kanamycin resistance gene Kanr element, and the nucleotide sequence is shown in SEQ ID No.1. The invention further discloses an application of the recombinant plasmid in identification of gene expression of thiobacillus aeruginosa, luciferase activity of recombinant bacteria is detected after establishment, and the intensity of gene expression of thiobacillus aeruginosa is determined according to the size of the fluorescence value. The method has the advantages of low background, high sensitivity, simple operation, high repeatability and accuracy; establishment of engineering bacteria with stress resistance and high efficiency leaching, for realizing the screening of the efficient promoters and the identification of the gene expression intensity in the thiobacillus autotrophic bacteria has the theoretical guiding significance and the practical application value.

The invention relates to a new streptomyces secretion expression plasmid and application thereof. The new streptomyces secretionexpression plasmid is particularly characterized by being cloned with a colon bacillus replication original region ori, a streptomyces conjugational-transfer essential region oriT, a penbritin resistance gene bla, an apramycin resistance gene aac (3) IV, a promoter ermE*p, a lidamycin prosthetic-group protein gene promoter cagAp, a lidamycin prosthetic-group protein gene mutation type signal peptide SPcagA (CTG) and multiple cloning sites by taking a minimum replicon of a pSGL1 plasmid naturally existing in streptomyces globisporus C-1027 as a framework. The new streptomyces secretory expression plasmid can be used for efficiently secreting and expressing various heterologous proteins in streptomyces.

The invention discloses a transfer plasmid for transforming Acinetobacter baumannii, which comprises, in clockwise order, a replicon ori, an ampicillin resistance gene AmpR, a transposon sequence, a junction transfer initiation site oriT, the transposon sequence contains a base sequence capable of autonomously emitting Acinetobacter baumannii and a resistance gene, and both ends of the resistancegene are provided with DifR and DifL sequences; also disclosed is an autonomously illuminated Acinetobacter baumannii without a resistance marker, the gene of Acinetobacter baumannii containing a genecapable of expressing autonomous light-emitting protein, and no resistance screening gene is in the genome of Acinetobacter baumannii. In the present invention, the autonomously illuminated Acinetobacter baumannii, which is constructed by the transfer plasmid pUC18T-mini-Tn7T-lux-Ab-dif-Apr and the auxiliary plasmid pTNS3, can emit light without adding any substrate; and luminescent colonies canbe seen with the naked eye in a dark environment.

A method for converting Chinese ephedra total DNA into yeasts, the transfer gene yeasts thereof, a culture medium for separating the transfer gene yeasts and liquid culture medium for transfer gene yeasts are disclosed.

The invention discloses a method for producing isoprene by adopting a cell fusion technology and a fusant constructed by the same. The method comprises the following steps: screening by utilizing a protoplast fusion technology, taking escherichia coli and ralstonia eutropha as parents and taking lipid as a unique carbon source and a resistance maker plasmid to obtain the reconstituted cell of high-efficiency transformed lipid; and fermenting by utilizing the fusant to produce the isoprene. The method disclosed by the invention can increase the velocity of lipid metabolism, enhance the yield of the isoprene and reduce the fermentation cost.

The invention discloses a method for biologically synthesizing fatty alcohol from fatty acyl ACP (acyl carrier protein) reductase. According to the method, fatty acyl carrier protein reductase genes are guided into a host cell body, and the genes are expressed in the host cell body, so the fatty acid metabolism pathway in heterotrophic microorganisms or heterotrophic cell bodies is transformed for producing the fatty alcohol. The method also further improves the fatty alcohol yield through metabolism transformation. The method has the advantages that the fatty alcohol synthesis in micro-organisms by a biosynthesis method is realized, the technology is a renewable low-consumption technology, in addition, the consumption of scarce resources of petroleum can be reduced in the environment-friendly production mode, the chemical modification is also not needed, the fatty alcohol can be directly produced, and great industrial application prospects are realized.

The invention discloses a target detection method based on an evolutionary neural network under a constraint condition, and the method comprises the steps: constructing a plurality of structural blocks and a population composed of a plurality of individuals, and carrying out the coding of each individual through a variable-length coding mode, thereby completing the initialization of the population; performing training updating on each individual according to the training data set; evaluating the individuals on the verification data set, and calculating the accuracy and complexity of the individuals to obtain the fitness of the individuals; according to a preset constraint quantity, adjusting the individual fitness by using a constraint control method, and adjusting the individual framework of which the accuracy exceeds a threshold value; selecting male parents from the population according to the adjusted fitness, generating first-level offspring through male parent crossing, and generating second-level offspring through probabilistic variation of the first-level offspring; and selecting the parent, the first-level filial generation and the second-level filial generation to generate a new population, and performing iterative evolution. According to the invention, a light-weight structure unit is designed, a constraint method is utilized, and an optimized target detection result is achieved without artificial experience.

The invention discloses a recombinant herpes simplex virus carrying fluorescent Timer gene capable of discoloring, a preparation method and an application thereof. The recombinant herpes simplex virusprepared by the invention changes the fluorescence timer from green to red in time during the HSV cross multisynaptic loop tracing, which provides important consideration in the time dimension for the real-time study of viral transsynaptic tracing, so that neurons infected with virus at different times can be distinguished by the fluorescent color differences, so that the link series of labeled neural network and the initial infected area can be indirectly obtained. The recombinant herpes simplex virus carrying the fluorescent Timer gene capable of discoloring has a wide range of applicationson neural loop marker, virus-infected cell process, animal infection model establishment, virus replication and pathogenic mechanism analysis, and antiviral drug screening in the time-space dimension.

The invention relates to the technical field of gene engineering, in particular to a construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae. The klebsiella pneumoniae is constructed from transfer plasmids pTXR and auxiliary plasmids pUCTns. The method comprises the following steps: S1, preparing the transfer plasmids and the auxiliary plasmids; S2, transferring the auxiliary plasmids for expressing transposase genes into klebsiella pneumoniae competent cells, and coating a kanamycin-resistant LB culture medium plate with the auxiliary plasmids; and S3, preparing the klebsiella pneumoniae containing the auxiliary plasmids into a competent state, then transferring the transfer plasmids into the klebsiella pneumoniae competent cells containing the auxiliary plasmids, and coating an APR-resistant LB culture medium plate with the klebsiella pneumoniae competent cells containing the auxiliary plasmids to obtain the self-luminous klebsiella pneumoniae. According to the construction method disclosed by the invention, exogenous dissociation enzyme does not need to be artificially expressed, the operation is simple, convenient and rapid, and the constructed SfAlKp can emit strong light without adding any substrate; and moreover, the SfAlKp has no resistance genes, so that potential side effects on screening and evaluation of drugs due to cross drug resistance caused by the resistance genes can be avoided.

The invention discloses a genome integration method and an application. Specifically, the invention discloses an escherichia coli large fragment genomic integration vector and an application of the vector in synthesis of carotenoids. The escherichia coli genomic integration vector includes a replicon, a target exogenous metabolic pathway, a resistance gene and an integration site, wherein the integration site is an IS sequence; and engineering bacteria include escherichia coli and the above integration vector integrated into the escherichia coli genome. The method uses the IS sequence with a large number of copies in the escherichia coli genome as the integration site to integrate the vector with the genome, not only the integration method is simple, but also the genetic integration of thetarget gene integrated into the genome is stable, so that the problem of unstable separation when plasmid is used as an expression vector is solved, a current integration technology is simplified, and the problem of cumbersome and time-consuming operation of the current large fragment integration technology is solved.

The invention discloses a method for efficiently acquiring inductive pluripotent stem cells (iPSCs). Adipose-derived stem cells (ADSCs) are induced and reprogrammed to form the iPSCs by using the method. The method concretely comprises the following steps: (1) inducing cDNA (Complementary Desoxvribose Nucleic Acid) of a multifunctional factor to the ADSCs; (2) culturing the ADSCs by using a serum-free culture medium in a feed-layer-free system, wherein the ADSCs is obtained in the step (1); (3) after cloning sample cells of embryonic stem cells (ESCs), further culturing by using a culture medium with an MEK (Methyl Ethyl Ketone) and GSK3 (Glaxo Smith Klein) signal channel inhibitor, selecting cells to clone, and enlarging culture; and (4) authenticating the cell cloning pluripotency. In the method, the ADSCs are low in cost and easily available on a large scale, and the reprogramming speed and efficiency are very high; the feed-layer-free system can be used for increasing the cloning purity of positive iPSCs; the serum-free culture medium can be used for avoiding unstable factors brought by undefined components in serum and batch difference; the signal channel inhibitor can be used for promoting cells to be up to the sufficient reprogramming state and improving the cloning quality of the positive iPSCs.

The present invention relates to a recombinant herpes simplex virus type 1 (HSV-1), a recombinant HSV-1 vector, and a method for preparing same. The recombinant HSV-1 according to one aspect enables easy genetic modification, as a relatively large foreign gene can be inserted or various foreign genes can be inserted simultaneously, as ICP6 and an IR region are deleted simultaneously. In addition,the present invention can be variously utilized in the cancer treatment field as an oncolytic virus that is safe, while having an excellent effect for killing cancer cells.

The invention discloses a method for preparing strain which can generate protoplast fusion by using marked plasmid. The invention has the advantages that marks can be easily added and removed, thereby being convenient for performing breeding and improvement of the strain based on the gene shuffling technology. The invention also has the advantages that the operation is simple, the cost is low, the requirement for equipment is low, and the chromosome of the strain can not be damaged.

The invention discloses a genome integration method and application. Specifically disclosed is an Escherichia coli large-segment genome integration vector and its application in synthesizing carotenoids. The E. coli genome integration vector includes a replicon, a target exogenous metabolic pathway, a resistance gene, and an integration site. The integration site is the IS sequence; the engineering bacterium includes Escherichia coli and the above-mentioned integration vector integrated into the genome of Escherichia coli. The present invention uses the IS sequence with a large copy number in the Escherichia coli genome as an integration site to integrate the vector and the genome. Not only the integration method is simple, but also the target gene integrated into the genome is genetically stable, which solves the problem of using the plasmid as an expression vector. When the separation is unstable, it simplifies the existing integration technology, and solves the cumbersome and time-consuming problems of the existing large-segment integration technology.

The invention discloses a bacillus gene traceless knockout / knockin plasmid and method, and a kit. The genome of the bacillus gene traceless knockout / knockin plasmid contains only one kind of resistance gene marked with a positive selection marker, a replicon capable of being duplicated in escherichia coli, a thermo-sensitive type replicon capable of being duplicated in bacillus, at least one I-Scel enzyme cutting site and only one kind of chromogenic protein gene, wherein promoters in front of the resistance gene and the chromogenic protein gene are both constitutive promoters in bacillus. The invention further provides a helper plasmid used for improving the knockout / knockin efficiency of the bacillus gene traceless knockout / knockin plasmid, and resistance gene and chromogenic protein gene in the genome of the helper plasmid are different from those of the knockout / knockin plasmid. By the adoption of the two plasmids, one or more target DNA sequences in the genome of bacillus can be modified continuously or iteratively, and the plasmids can be applied to multiple fields including bacillus genetic modification, metabolic process researching, functional genome researching and industrial application researching.

The invention relates to a nano antibody of specific identification heavy metal Cr and an application of the nano antibody. A nano antibody for the heavy metal Cr is disclosed, and the nano antibody has good specificity for the heavy metal Cr, and very low cross reactivity with other metals. By utilizing the nano antibody, a kit for conveniently, rapidly and accurately detecting the heavy metal Cr can be prepared.