33results about How to "Genetic manipulation is simple" patented technology

The invention discloses a bacillus licheniformis strain which is preserved as bacillus licheniformis WX-02 with the preserving number of NO: M208065 and the preserving date of April 24th, 2008 and the preserving unit of CCTCC. The invention also discloses an application of the bacillus licheniformis strain and a method for producing poly-gamma-glutamic acid by using the bacillus licheniformis strain. Poly-gamma-glutamic acid products are produced by processes of seed solution preparation and liquid submerged fermentation, and the strain has stable genetic performance of the strain, high production efficiency and low production cost. When the bacillus licheniformis strain of the invention and the fermentation method thereof are used for preparing 3000L of poly-gamma-glutamic acid, the yield of a fermentation tank can reach 35.05g/l.

The invention discloses a ship branch pipeline path planning method. The method comprises the steps of on the basis of simplifying a device ad a pipeline model, establishing a network graph between two points referring to a visible graph method in mobile robot path planning; setting branch pipelines containing N connection points, establishing the network graph between every two points of the N connection points, and combining update data information, thereby obtaining the total network graphs among the N points; establishing a population based on a Steiner point genetic algorithm, setting genetic algorithm parameters and then starting iteration to carry out optimization; determining the equivalent lengths of the pipelines and endowing different weight coefficients to different pipelines; evaluating the fitness value of each chromosome by use of a distance heuristic algorithm; judging whether set irritation times arrives or not; outputting optimum paths, finishing path planning, updating pipeline code information in a storage document, and realizing three-dimensional visualization of the ship branch pipelines through combination of three-dimensional design software. The method has very high search efficiency and can satisfy the practical demands of ship branch pipeline path planning better. The layout problem of the branch pipelines is solved.

The invention provides a eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis, which is applied to clinical diagnosis of the bovine tuberculosis. A cfp10-esat6 fusion gene is obtained by PCR (polymerase chain reaction) amplification, a recombinant plasmid pPICZ alpha A-(cfp10-esat6) is constructed, the transfer into Pichia pastoris GS115 is performed, and the high-purity CFP10-ESAT-6 fusion protein is obtained by methanol induction and affinity chromatography. The eukaryotic expression CFP10-ESAT-6 fusion protein is used for an intradermal allergic reaction which is used for diagnosis of the bovine tuberculosis and an antigen-specific bovine IFN-gamma test as a stimulus, the former has the sensitivity of 47.4% and the specificity of 83.3% in comparison with the results of a comparison allergic reaction, and the later has the positive coincidence rate of 82.8% and the negative coincidence rate of 87.4 in comparison with a bovine IFN-gamma kit, thereby showing greater value in diagnosis of the bovine tuberculosis.

The invention relates to EV71 virus-like particles and hand-foot-and-mouth disease vaccine prepared from the EV71 virus-like particles. The EV71 virus-like particles are prepared according to the following steps: (1) constructing recombinant plasmid: respectively connecting pGAPZ alpha A plasmid with P1 protein gene of intestinal EV71 virus and 3CD protease gene to construct P1-pGAPZ alpha A and 3CD-pGAPZ alpha A recombinant plasmid; (2) transferring the recombinant plasmid to expression bacterial strains: sequentially transferring the recombinant plasmid to Pichia pastoris SMD1168 expression bacterial strains to obtain P1-pGAPZ alpha A-3CD-pGAPZ alpha A-SMD1168 recombinant expression strains; and (3) culturing thallus and purifying the EV71 virus-like particles: culturing the Pichia pastoris recombinant expression bacterial strains, centrifugalizing to separate supernate, and carrying out sucrose density gradient centrifugation on precipitation of the supernate after ultracentrifugation, thus obtaining the EV71 virus-like particles. In the invention, the EV71 virus-like particles are easy to obtain through thallus culture, have good stability, are easy to purify and suitable for preparing vaccine, and are convenient for industrial production.

The invention provides a method for the heterologous expression of a secondary metabolite encoded by a biosynthetic pathway. Also provided is a method for introducing a large sized DNA molecule into the chromosome of a heterologous host using a transposable element. Novel myxochromide S derivatives are also provided.

A polymorphous Hanson yeast expressed recombinant cholera toxin B subunit gene (CTB) is disclosed. In order to increase the expression of CTB in Hanson yeast, the coding gene of CTB is redesigned according to the application method of codon for the high-expression gene of Hanson yeast. The expression product can be used for preventing the diarrhea caused by cholera ribro and as the immune vaccine.

The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.

The invention belongs to the field of medical chemistry, and aims at providing a method for synthesizing trans-4-hydroxy-L-proline. According to the technical scheme, by virtue of genetiuc engineeringmodified escherichia coli, [alpha]-ketoglutaric acid, which is generated from autologous TCA circulation of a catalysis host, is converted into an L-proline gene, and the L-proline gene undergoes tandem and over-expression, so that the yield of L-proline is improved; and prolyl hydroxylase is introduced, so that a reaction that the L-proline is converted into the trans-4-hydroxy-L-proline and a reaction that the [alpha]-ketoglutaric acid is converted into succinic acid are coupled; therefore, the efficient synthesis of the trans-4-hydroxy-L-proline under a condition of not adding exogenous L-proline is achieved. The synthesis method of the trans-4-hydroxy-L-proline provided by the invention is optimized, so that the yield of the target product (the trans-4-hydroxy-L-proline) is greatly improved.

A transferred hepatitis B surface antigen (HBsAg) gene halophila is prepared by recombining the exogenous gene HBsAg into the cells of eucaryotic monocell halophila, so obtaining the transgenic halophila used to prepare the oral vaccine is hepatitis B. Its advantages are high reproduction speed, simple culture condition, low cost, and rich nutrients contained by halophila cells.

The invention discloses a genetically engineered bacterium for high yield of farnesene and a construction method and application thereof, and belongs to the technical field of microorganisms. In orderto solve the problem that in the process of synthesizing farnesene from escherichia coli, the catalysis efficiency of a heterologous MVA downstream path is low, and the farnesene yield is low, plasmids pACYC-mvaE-mvaS-ispA-AaFS, pTrcLow-delta IDI, pTrcLower-AaIDI and pET28a-AaFS-ispA-SlIDI/AaIDI (IDI genes are respectively from tomatoes and artemisia apiacea) are constructed; after the plasmids are combined, escherichia coli is transformed, culture conditions are improved, the prepared genetically engineered bacterium can remarkably improve the farnesene synthesis yield, and the industrialization process of synthesizing farnesene by a biological method is promoted.

The present invention discloses a new method of obtaining rabbit embryonic stem cells (rbES), which adopts a serum free culture system to establish an rbES cell line which can proliferate under undifferentiated state for a long time (more than 1.5 years) and maintain the normal karyotype. The rbES expresses a pluripotent marker Oct4 and a plurality of other molecules, including EBAF2, FGF4, and TDGF1, which are expressed in stem cells. The stem cells can form an embryoid body in vitro and form a teratoma in immunodeficient mice. The rbES exhibits high colony forming efficiency and can be used as a nuclear donor to produce a clone rabbit, and genetic modifications of the rbES can be performed. The present invention also discloses potential applications of the rbES.

The invention discloses construction of a cargo plasmid in an anabaena 7120 triparental transformation system and application method of the cargo plasmid. The plasmid pPT27 is formed by recombining nucleic acid elements in one step through a sequence and linkage independent cloning (SLIC) method. After a target nucleic acid sequence is connected, the cargo plasmid is generated, and the cargo plasmid and helping and combination plasmids are used for transferring an exogenous DNA (Deoxyribonucleic Acid) sequence into a host cell; a target mutant strain is screened through a marker gene. The invention further discloses the application method of the pPT27 plasmid, and green fluorescent protein (GFP) and fused protein (FdnifK) are transferred in. The cargo plasmid (pPT27) constructed by the invention can be used for functional analysis and protein expression of a target DNA segment; the cargo plasmid can be rapidly and efficiently constructed in a time-saving manner by applying the constructed plasmid and limitation on a DNA sequence can be avoided; the cargo plasmid has very important meanings in the aspect of researches of gene functions.

The invention discloses a method for transforming schizochytrium limacinum by agrobacterium tumefaciens and application, and the transformation method comprises the following steps: introducing an exogenous gene into the agrobacterium tumefaciens, and introducing the exogenous gene into the schizochytrium limacinum by utilizing the agrobacterium tumefaciens, so that the schizochytrium limacinum obtains the function of the related exogenous gene. According to the invention, the agrobacterium AGL-1 transferred into a G418 resistance gene expression cassette and the schizochytrium limacinum HX-308 are co-cultured, so that an exogenous gene is successfully transformed into the schizochytrium limacinum HX-308, and fixed-point knockout of the gene is realized. Experimental operation is very simple, time and cost are effectively saved, Agrobacterium tumefaciens can be converted into schizochytrium limacinum, more importantly, gene knockout can be achieved, and a set of complete genetic operating system can be constructed in the schizochytrium limacinum through gene knockout. According to the invention, site-specific integration can be completed by using a short homologous arm, and a 2-3kb homologous arm is possibly needed in general fungal transformation, so that a foundation is laid for simplifying genetic manipulation of schizochytrium limacinum.

The invention relates to the technical field of disease animal models, in particular to a specific thymus lymphocyte injury and regeneration model and a construction method thereof. The construction method of the specific thymus lymphocyte injury and regeneration model sequentially comprises the following steps: culturing fertilized eggs of transgenic zebrafish to obtain juvenile fish to be treated; the transgenic zebrafish is used for expressing nitroreductase, and the expression of the gene of the nitroreductase is regulated and controlled by a promoter of the coro1a gene; 2 mM metronidazole is used for treating juvenile fish to be treated, and juvenile fish treated by metronidazole is obtained; and cleaning the metronidazole treated juvenile fish to obtain the specific thymus lymphocyte injury and regeneration model. According to the scheme, the technical problem that a thymus injury model animal model for research on lymphocyte regeneration and immune system recovery is lacked in the prior art is solved, and conditions are created for research on immune system recovery conditions and related drug screening under acquired immune system injury.

The invention discloses a cloud workflow scheduling optimization method using a two-dimensional fixed-length coding intelligent computing algorithm. The method comprises the following steps: acquiringinformation required by scheduling optimization; calculating a sorting value rank of the tasks; initializing a contemporary population and carrying out decoding improvement on the contemporary population; performing crossover operation to form a new population; performing mutation operation on the new population; performing decoding improvement on the new population; forming a new contemporary population by the contemporary population and the new population until an evolutionary termination condition is satisfied; outputting a scheduling optimization scheme. According to the method, a two-dimensional fixed-length coding method is adopted, global search can be realized, a serial individual decoding method in an insertion mode and a load balancing individual improvement strategy consideringtransmission time are adopted in evolution, a set of simple and effective crossover mutation method is designed, and the optimization capability and the search efficiency are improved.

The invention discloses a CMV virus-like particle for producing a VLP recombinant vaccine and a preparation method thereof, the CMV virus-like particle is obtained by cloning a C-terminal G4SLPETG modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting escherichia coli BL21 (DE3) with the recombinant expression vector, and expressing by the recombinant escherichia coli BL21 (DE3); the amino acid sequence of the C-terminal G4SLPETG modified CMV is shown as SEQ ID NO. 1. Tests prove that the recombinant strain constructed by the invention is stable in exogenous protein expression. The recombinant protein expressed by the invention is used for preparing virus-like particles for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, no pathogenicity is caused to animals such as mice and the like, and the safety evaluation is easy to pass.