19 results about "High level expression" patented technology

The invention discloses bacillus subtilis for recombinant expression of a hermetia illucens HIAMP0438 gene and a construction method of the bacillus subtilis, and belongs to the technical field of gene engineering. The hermetia illucens recombinant protein used in the invention is obtained by expression of bacillus subtilis WB800N transferred into a pHT43-HIAMP0438 high-efficiency expression vector, and the pHT43-HIAMP0438 high-efficiency expression vector is obtained by a homologous recombination technology, so that high-level expression of the HIAMP0438 gene is realized, the HIAMP0438 gene can be efficiently secreted out of cells, the preparation process of the recombinant protein is greatly simplified, and the production cost is reduced. According to the present invention, the recombinant protein can significantly inhibit the growth of Escherichia coli, Salmonella and Listeria monocytogenes, and the recombinant protein can be used as the animal health product, and can be used for preventing and treating pig and chicken intestinal diarrhea caused by Escherichia coli and Salmonella and food-borne digestive tract diseases caused by Listeria monocytogenes.

The present invention relates to a method for constructing a host bacterium with high expression of human serum albumin, which method comprises: S100, designing an optimized gene encoding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector into a Pichia pastoris host bacterium. The optimization design of a full-length sequence of a target gene is performed at the molecular level to obtain a target gene with the most suitable expression in Pichia pastoris; at the cellular level, the latest international Pichia pastoris expression system host bacterium CBS7435 is introduced for the gene expression of human serum albumin; and Pichia pastoris transformants harboring high-copy-number exogenous genes are selected by means of using a G418 resistance gene for the construction of an engineering bacterium with high expression of recombinant human serum albumin.

The application discloses a recombinant pseudorabies virus strain expressing classical swine fever virus E2 protein and application, and relates to the technical field of biology.The recombinant E2 protein provided by the application successfully realizes high-level expression of the E2 protein by removing the transmembrane region of the E2 protein and using the 18aa signal peptide of the E2 protein itself and a pig albumin signal peptide.The recombinant PRV expressing the classical swine fever virus (CSFV) E2 protein can induce E2 antibodies and cellular immunity after immunization of mice.The recombinant pseudorabies virus strain provided by the application can be used as a bivalent vaccine for preventing CSFV and PRV infection.

The present application relates to the field of microorganisms and genetic engineering, and specifically discloses an Escherichia coli engineering bacterium for efficiently producing plastic depolymerase and application thereof. The present application uses Escherichia coli as a host, and through synergistic optimization of multiple key dimensions such as signal peptide, promoter, solubility-promoting tag and co-expression of molecular chaperones, a multi-dimensional engineering system for efficiently expressing plastic depolymerase is constructed. The construction method provided by the present application includes a rational design and a screening strategy for combination and assembly of the multiple elements. The engineering bacterium can realize extracellular high-level expression of plastic depolymerase during induced fermentation, and the enzyme preparation prepared can be directly used for catalyzing efficient depolymerization of polyester plastics. The present application provides an Escherichia coli cell factory with stable high yield for industrialization and low-cost production of plastic depolymerase, and has important industrial application prospects for promoting large-scale biological degradation and resource recycling of waste plastics.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The invention discloses a xylanase mutant and application thereof. The amino acid sequence of the xylanase mutant comprises SEQ ID NO: 7. The xylanase mutant MUT-1 provided by the invention has higher specific enzyme activity compared with a parent P1, the specific enzyme activity of the xylanase mutant MUT-1 is improved by 41% compared with that of the parent P1, and the xylanase mutant MUT-1 also has good acid resistance and heat resistance. The invention also provides an expression vector and a host cell of the xylanase mutant, high-level expression of xylanase can be realized by using engineering bacteria, and the production cost is greatly reduced.

The invention discloses an application of a targeted ABC transporter PbABCB25 in prevention and control of clubroot of rape. ABC transporter protein ABCB25 (abc transporter b family meber 25, ABCB25) with high-level expression in each stage is screened out from a plasmodiophora brassicae ZJ-1 transcriptome, then an RNAi carrier of the ABC transporter protein ABCB25 is constructed through an HIGS technology, the RNAi carrier is transformed into wild type arabidopsis thaliana, then the resistance of transgenic arabidopsis thaliana to clubroot is tested, the result shows that PbABCB25 has remarkable resistance to plasmodiophora brassicae, and the PbABCB25 can be used for preparing the plasmodiophora brassicae. The result shows that the ABCB25 in the plasmodiophora brassicae can be used as a gene resource for resisting the clubroot of the cruciferae.

The invention discloses the application of a β-N-acetylhexosaminidase (HaHex74) from Haloferula sp. in the synthesis of human milk oligosaccharides. The invention provides the use of HaHex74 protein or related biological materials thereof in any one of the following: synthesizing human milk oligosaccharides; synthesizing Lacto-N-triose II and / or Lacto-N-neotetraose; the HaHex74 protein having the amino acid sequence shown in SEQ ID No. 2 is derived from Haloferula sp. The β-N-acetylhexosaminidase HaHex74 disclosed by the invention possesses high-level expression, excellent hydrolysis properties and transglycosylation activity, which may make it potentially useful in the production of human milk oligosaccharides.

The invention relates to the technical field of biology, in particular to yarrowia lipolytica engineering bacteria with an optimized metabolic pathway and an itaconic acid production process of the yarrowia lipolytica engineering bacteria. The engineering bacterium is constructed by taking yarrowia lipolytica as a host strain through genome multi-site modification, and the optimization of a metabolic pathway of the engineering bacterium comprises the following steps: introducing itaconic acid to synthesize a key enzyme gene: integrating a cis-aconitic acid decarboxylase gene CadA (SEQIDNO: 1) derived from Aspergillus in a strain genome, driving high-level expression through a strong promoter TEF1 and a terminator XPR2, and carrying out high-level expression through a high-level expression vector; and catalyzing the decarboxylation of intracellular cis-aconitic acid to generate itaconic acid. The metabolic bottleneck is obviously reduced, and the carbon flow continuity in the pathway is improved, so that the synthesis rate and the product cumulant of itaconic acid are effectively improved, and the problem of low acid production efficiency in a single gene expression system is solved.

Improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The process also provides an optimized autoinduction media, enabling routine batch production at various culture volumes where cells densities routinely reach ˜5-7 g cell dry weight per liter and offer protein titers above 2 g / L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

The present invention relates to an IFITM scaffold enabling high-efficiency engineering of extracellular vesicles. In the present invention, active substances to be expressed in extracellular vesicles are linked to the IFITM scaffold so that the active substances can be expressed in the vesicles at a high level. In particular, the active substance linked to the C-terminus of the IFITM scaffold is present on the surface of the vesicles to exhibit the intrinsic activity. In addition, the present invention provides a U-shaped IFITM scaffold, which enables two types of active substances linked to the N-terminus and the C-terminus of the IFITM scaffold to be present on the surface of extracellular vesicles at a 1:1 ratio.

The invention belongs to the field of biopesticides, and relates to application of a metabotropic glutamate receptor PbMGR in prevention and control of rape clubroot. The application of the metabotropic glutamate receptor PbMGR gene in improving the clubroot resistance of plants and / or cultivating clubroot-resistant plants is characterized in that the CDS sequence of the gene is as shown in SEQ ID NO.1. The invention also discloses an application of the metabotropic glutamate receptor PbMGR gene in improving the clubroot resistance of plants and / or cultivating clubroot-resistant plants. According to the invention, G protein coupled receptors (metabotropic glutamate receptors, PbMGR) which are expressed at a high level in each stage are screened from a plasmodiophora brassicae ZJ-1 transcriptome, an RNAi carrier of the gene is constructed, corresponding transgenic plants are obtained, and disease resistance tests of plasmodiophora brassicae show that two PbMGR-RNAi transgenic strains both show good disease resistance phenotypes, and the two PbMGR-RNAi transgenic strains show good disease resistance phenotypes. Therefore, the G protein coupled receptor PbMGR of plasmodiophora brassicae can be used as a gene resource for resisting clubroot.

The present invention relates to a recombinant expression vector for preparing highly expressed human serum albumin, a host strain and a use thereof. By means of optimized design of a target gene, a Pichia pastoris engineered strain containing a high-copy-number exogenous gene and expressing the exogenous gene at a high level is constructed; by means of optimization of a pilot-scale fermentation process, the expression level of recombinant human serum albumin from the gene reaches an ultra-high level of 25.82 g / L; and by means of fermentation broth pretreatment and a purification process using three-step column chromatography, the purity of the target protein reaches 99% or more, and the recovery rate reaches 60% or more.

The invention relates to the field of microorganisms and genetic engineering, and particularly discloses an escherichia coli engineering bacterium for efficiently producing plastic depolymerases and application of the escherichia coli engineering bacterium. According to the invention, escherichia coli is taken as a host, and a multi-dimensional engineering system for efficiently expressing the plastic depolymerases is constructed by synergistically optimizing a plurality of key dimensions such as signal peptide, a promoter, a dissolution promoting tag and a co-expression molecular chaperone. The construction method provided by the invention comprises rational design of the plurality of elements and a screening strategy of combination and assembly. The engineering bacterium can realize extracellular high-level expression of the plastic depolymerization enzyme in the induced fermentation process, and the prepared enzyme preparation can be directly used for catalyzing efficient depolymerization of polyester plastics. The invention provides a stable and high-yield escherichia coli cell factory for industrial and low-cost production of the plastic depolymerases, and has important industrial application prospects for promoting large-scale biodegradation and resource circulation of waste plastics.

The invention belongs to the technical field of gene engineering, and discloses an application of an OsDUF3778-1 gene in promoting crop growth. The OsDUF3778-1 gene regulates the ROS dynamic level in the rice body by directly or indirectly activating RBOH and antioxidant enzyme activity, and further participates in rice growth and development regulation. Researches find that agronomic trait indexes such as fresh weight of rice seedlings, yield of rice plants, grain density, pollen viability and the like are in a trend of increasing and then decreasing along with the increase of the relative expression level of the OsDUF3778-1 gene and the ROS accumulation level, the OsDUF3778-1 is slightly higher than the basic expression level to promote the growth and development of rice, and an inhibition effect is generated if the expression level is too high. The relative expression quantity of the OsDUF3778-1 gene is up-regulated by 50-80 times, so that the growth of crops can be obviously promoted.

This invention discloses a high-expression promoter PETS3 in plants and its applications. The sequence of the high-expression promoter PETS3 is shown in SEQ ID NO.1. This invention successfully cloned a meristem-high expression level promoter PETS3 from Brassica napus. This promoter drives the expression level of the heterologous gene Cas9 in the stem meristem of Brassica napus to be more than 18 times higher than that driven by pro35S. Furthermore, this promoter can also drive high-level expression of the gene in roots and callus tissues. This invention solves the problem of the lack of strong meristem promoters in existing rapeseed gene editing applications. Using this promoter in gene editing practice has the advantages of short cycle time, low workload, and high efficiency, and has a promising application prospect in the fields of plant genetics and breeding and genetic engineering.

Provided are methods for determining that a candidate polypeptide in a library of polypeptides has editing activity comprising expressing in a plurality of cells: a library of first expression cassettes each comprising a nucleic acid encoding a polypeptide wherein each polypeptide in the library comprises, a first editing domain, a first linker, a nuclease-deficient RNA guided endonuclease domain, a second linker, and a second editing domain; and a second expression cassette comprising a nucleic acid encoding a guide RNA directed to a target of interest, wherein said first and second expression cassettes are expressed in the plurality of cells; separating the plurality of cells based on the expression level of the target of interest; extracting genomic DNA (gDNA) from the plurality of cells; and sequencing the gDNA of cells that have a high level expression or a low level of expression of the target of interest.

This invention relates to an engineered *Escherichia coli* strain and a method for preparing gene 3 knockout helper phages. The genome of the engineered *E. coli* strain integrates a DNA module containing elements such as T7 Lysozyme, araC, ParaBAD, T7 RNA polymerase, PT7-lacO, and Ff gene 3. Through dual induction with L-arabinose and IPTG, tight regulation and high-level expression of the g3p protein can be achieved. Its property of strictly limiting leakage expression effectively reduces g3p protein degradation and misfolding. Using this strain to produce gene 3 knockout helper phages significantly improves the packaging efficiency and yield of the helper phages, reaching ≥ 8.5 × 10⁻⁶. 14 The pfu / L concentration is at least 600 times higher than existing technologies, significantly reducing production costs. This invention has significant application value in the field of phage display technology.