247 results about "Foreign protein" patented technology

The invention relates to a protein for improving the expression yield of a foreign protein and application of the protein. Wherein the ERV29 protein and the related protein as well as the P180 protein and the related protein can be used as molecular chaperones for improving the expression yield of foreign proteins such as Brazilian sweet and the like; the amino acid sequence of the ERV29 protein is as shown in SEQ ID No. 4; the amino acid sequence of the P180 protein is as shown in SEQ ID No. 6.

The invention discloses a mammalian cell expression vector, an expression plasmid and an expression system. The expression vector sequentially comprises the following elements: (a) a promoter; (b) a Kozak sequence; (c) a polyadenine sequence, the polyadenine sequence sequentially comprises a bGH PolyA sequence and a 5 'UTR PolyA sequence, the nucleotide sequence of the bGH PolyA sequence is as shown in SEQ ID NO.5, and the nucleotide sequence of the 5' UTR PolyA sequence is as shown in SEQ ID NO.6; (d) copying the start site; and (e) screening marker genes. According to the present invention, the expression vector is optimized by the expression element, such that the exogenous gene can be conveniently, efficiently and flexibly inserted and edited by the expression vector so as to achieve the stable extracellular or intracellular expression of the exogenous protein, and the expression efficiency of the exogenous protein can be improved by carrying out the expression plasmid construction based on the expression vector; and more efficient expression and purification of the foreign protein are realized.

This invention provides an in vitro cell-free protein synthesis system, its reagent kit, and its applications, belonging to the field of protein synthesis technology. This invention improves an in vitro cell-free protein synthesis system based on Kluyveromyces lactis by adding exogenous L-arabinose, significantly enhancing the system's protein synthesis capacity. Furthermore, it provides a more efficient and higher-throughput in vitro protein synthesis reagent kit and a method for synthesizing exogenous proteins. The improvements provided by this invention can be achieved without molecular modification, are simple and convenient to operate, and save costs.

The preparation method comprises the following steps: firstly, analyzing the amino acid sequence of an alpha 1 chain of the human XVII type collagen, and selecting partial amino acid fragments which are not easily degraded by endogenous or exogenous protease in non-transmembrane regions of different functional domains of the protein; the recombinant collagen gene fragment (Col17a) n is obtained by repeatedly splicing the recombinant collagen gene fragment (Col17a) n; then introducing homologous promoters from different methylotrophic yeasts to the 5'end of a recombinant collagen gene segment, optimally synthesizing selected (Col17a) n and promoter segments, obtaining a connecting segment by utilizing PCR cloning, connecting the collagen segment (Col17a) n, the promoter segments and a pPIC9K carrier skeleton segment by virtue of a Gibson Assessment seamless connection technology, and obtaining the recombinant collagen gene. And constructing to obtain the pichia pastoris expression plasmid pPIC9K-(Col17a) n of the recombinant human source XVII type collagen.

The invention discloses a method for displaying foreign protein by utilizing salmonella and application of the method, and belongs to the technical field of biological medicine. According to the invention, the SpyCatcher and an outer membrane protein Lpp-OmpA gene are fused and are expressed in salmonella, a recombinant strain is constructed, and the SpyCatcher is displayed on the surface of bacteria; the foreign protein gene and the SpyTag gene are fused and subjected to prokaryotic expression purification, and recombinant foreign protein is obtained; after the recombinant foreign protein and the recombinant salmonella are incubated, the foreign protein can be anchored on the surface of the salmonella through a SpyCatcher-SpyTag covalent bond. When the foreign protein is an antigen protein, the foreign protein is displayed on the surface of the attenuated bacterial vector, and a live vector vaccine can be prepared. The invention provides a novel universal antigen display method, overcomes the limitation of the traditional antigen display method on the protein size and conformation, and has a good application prospect in live vector vaccine construction.

The invention discloses a preparation method of polydeoxyribonucleotide, and relates to the technical field of synthetic biology, and the preparation method comprises the following steps: S1, randomly selecting n ssDNAs from a salmon genome sequence; s2, carrying out cyclization on the ssDNA, so as to obtain cssDNA; s3, carrying out amplification on the cssDNA to obtain a repeated long single-stranded DNA (Deoxyribose Nucleic Acid); and S4, breaking the repeated long single-stranded DNA to obtain the PDRN. According to the method, a large amount of repeated long single-stranded DNA can be synthesized only by introducing a small amount of foreign proteins (T4DNA ligase and Phi29 DNA polymerase), the repeated long single-stranded DNA is broken by a physical breaking method, and a large amount of PDRN products with ideal molecular weight can be obtained in a short time.

The invention provides a method for constitutive expression of foreign protein by escherichia coli Nissle 1917, and belongs to the technical field of genetically engineered bacteria. The invention provides a method for constitutive expression of foreign protein by Escherichia coli Nissle 1917 (EcN), which is characterized in that EcN is used as a chassis strain, an engineering strain for constitutive expression of the foreign protein is constructed by transforming a covert plasmid, and high-efficiency expression of the foreign protein can be realized without induction of an inducer and antibiotic pressure. By utilizing the method disclosed by the invention, the EcN engineering bacterium for constitutive expression of the foreign protein RtSOD is successfully constructed, constitutive efficient expression of the high-stability RtSOD protein in the EcN engineering bacterium can be realized without using antibiotic pressure and inducer induction, and the EcN engineering bacterium can be used as a potential viable bacterium medicine for preventing and treating oxidative stress related diseases and has a wide application prospect. And the method has important significance on the practical application of the EcN engineering strain.

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

The invention provides application of a porcine parainfluenza virus type 5 as an expression vector, and belongs to the technical field of biological vaccines. According to the invention, a PIV5 strain separated from diarrhea pigs in Jiangsu Province in China is utilized to construct PIV5 virus infectious clone. The eGFP gene is inserted into a PIV5 infectious cloned virus genome, the recombinant PIV5 virus capable of expressing the green fluorescent protein is successfully rescued, and when the eGFP gene is inserted into a P / M gene spacer region, the expression quantity is the highest, which indicates that an exogenous gene expression cassette can influence the expression level of an exogenous gene, and the P / M gene spacer region is the optimal site for inserting the exogenous gene, so that the recombinant PIV5 virus can be successfully rescued. And a solid foundation is laid for developing a vector vaccine by utilizing the swine PIV5. The porcine parainfluenza virus type 5 can be used as an expression vector to express foreign proteins, can also be used for preparing oral vaccines, and can relieve the stress reaction of pigs in the immune process.

The invention relates to a protein combination for influencing the growth rate of yeast and the expression quantity of foreign proteins. After genes in a peptidase A1 family, a peptidase M1 family, a peptidase M18 family, a peptidase M28 family, a peptidase S54 family, a peptidase S10 family, a peptidase S16 family, a peptidase S14 family and a peptidase S8 family and protein defect related genes are knocked out as transcription activating factors, the growth rate can be increased, and the expression quantity of foreign proteins can be increased.

The invention belongs to the technical field of biological medicine, and discloses lactococcus lactis HX21473 and application thereof, the lactococcus lactis HX21473 is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, and the preservation number is CGMCC NO.33476. The invention further discloses a preparation method of the lactococcus lactis HX21473. The invention provides a strain of lactococcus lactis HX21473 capable of efficiently expressing a foreign protein. The lactococcus lactis HX21473 has strong acid resistance, can be used as an oral vaccine live carrier, and promotes research, development and application of oral vaccines.

The application provides an in-vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and application thereof, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system containing exogenous magnesium ions adopts magnesium aspartate as a novel magnesium ion source, and especially in a eukaryotic cell-free system, compared with a traditional magnesium ion source, can significantly improve the protein synthesis efficiency and protein expression amount. A more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of exogenous proteins are also provided, and the kit has the advantages of simplicity, convenience and low cost.

The invention discloses a novel plasmid vector capable of increasing CHO foreign protein expression quantity and construction and application thereof, and belongs to the technical field of gene engineering. The plasmid vector pCHO6GS belongs to a brand-new plasmid system, and the expression quantity of a common monoclonal antibody and recombinant protein (such as H5HA trimer) difficult to express in CHO can be increased by at least more than one time, so that the expression quantity of foreign protein is increased beyond expectation through the novel plasmid vector; and the method also plays an extremely important role in reducing cost and improving efficiency of various protein products taking CHO as a cell factory.

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

The invention discloses a dissolution-promoting expression tag and application thereof, and belongs to the technical field of gene engineering. The solubilizing expression tag is thioredoxin Trx1 or Trx2 from yeast, and the amino acid sequence of the solubilizing expression tag is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. Compared with other solubilizing tags from biological sources, the solubilizing expression tag provided by the invention can better promote correct folding of the exogenous fusion protein in the yeast, improve the soluble expression quantity and overcome the condition that the soluble expression of the heterologous protein in a yeast expression system is not ideal. The solubilizing expression tag is applied to heterologous expression protein of a yeast expression system, expression of heterologous protein is provided, the yield of target metabolites is further increased, and the solubilizing expression tag has a good industrial application prospect.

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The present invention belongs to the biological field, and specifically relates to a polynucleotide based on a CMV promoter and its application. The present invention constructs a plasmid containing a strong promoter for cell expression, introduces a random mutation CMV promoter through random mutation PCR technology, and screens to obtain a stronger CMV promoter sequence. The CMV promoter sequence obtained in the present invention can significantly increase the expression amount of an exogenous target protein, and provides a basis for increasing the expression amount of an exogenous protein in transient and stable transfection.

The invention discloses a sugarcane streak mosaic virus infectious cloning vector as well as a construction method and application thereof, and belongs to the technical field of biology. The recombinant plasmid provided by the invention contains a whole genome of the SCSMV, and the nucleotide sequence of the whole genome is the 771 to 10583 nucleotides of SEQ ID No. 1. The recombinant plasmid disclosed by the invention is obtained by taking a pCB301 vector as a skeleton and then inserting the skeleton into a whole genome of SCSMV, and the recombinant agrobacterium tumefaciens (pCB301-SCSMV infectious clone) prepared by the recombinant plasmid can successfully infect nicotiana benthamiana and has efficient infection ability; the infectious clone of the recombinant expression plasmid prepared from the recombinant plasmid can successfully infect nicotiana benthamiana, has efficient infection ability and can express foreign proteins.

The invention relates to the fields of molecular biology and genetic engineering, and relates to a cherax quadricarinatus egg cell guide peptide, a fusion protein, a nucleic acid molecule, a recombinant expression vector, a host cell, a method for guiding a foreign protein to enter a cherax quadricarinatus egg cell and application of the cherax quadricarinatus egg cell guide peptide. The amino acid sequence of the cambarus clarkii egg cell guide peptide is as shown in SEQ ID NO. 1. According to the invention, green fluorescent protein (eGFP) is used as example protein, which proves that the polypeptide can successfully deliver foreign protein. The breakthrough can lay a technical foundation for introducing a gene editing tool (such as a CRISPR-Cas9 system) into a crayfish oocyte, and is expected to be widely applied to the fields of crayfish breeding, functional gene research, aquaculture gene improvement and the like.

The invention relates to a cytoplasm and nucleoplasm localization polypeptide used for localization in mammalian cells in vitro. The amino acid sequence of the RfA1 protein is composed of conservative motifs and non-conservative connecting fragments between the motifs, the RfA1 protein is a natural block copolymer, the amino acid sequence of block arrangement of the RfA1 protein is redesigned, the motifs and the connecting fragments are recombined, and the modular design method of the protein tag is provided. According to the method, two protein tags for respectively and strictly locating foreign proteins in cytoplasm and nucleoplasm are obtained, the method and the two polypeptide tags can locate different functional proteins in cytoplasm or assist the different functional proteins to be transferred into nucleoplasm, the target capacity of the functional proteins can be effectively improved, and the method is expected to be applied to the fields of biotechnology, biological medicine and the like.

There is provided an engineered mitochondrion comprising one or more exogenous protein binding motif / peptide expressed on the outer membrane of the mitochondrion. Also disclosed is a polynucleotide, a vector, a host cell, a cell, a composition or pharmaceutical composition, and a method of treatment thereof. Also disclosed is a method of improving mitochondrion uptake into a target cell comprising genetically modifying a host cell to express a modified mitochondrion, wherein the modified mitochondrion comprises one or more exogenous protein binding motif / peptide expressed on the outer membrane of the mitochondrion.

The invention discloses a signal peptide SEQ ID NO: 1-3 suitable for yeast, the coding gene of the signal peptide is expressed in the yeast after being fused with a foreign protein gene, the expression level of the foreign protein can be improved, and the signal peptide has popularization and application prospects.

The invention provides a gene for promoting pichia pastoris to express foreign protein, recombinant pichia pastoris, a construction method of the recombinant pichia pastoris and a method for efficiently expressing recombinant protein, and belongs to the technical field of bioengineering. The gene for promoting pichia pastoris to express foreign protein is marked as the gene PASchr30259, the gene sequence of the gene is shown as Seq No.1, the amino acid sequence coded by the gene is shown as Seq No.2, and the amino acid sequence is marked as PASchr30259 coded protein. The gene PASchr30259 is connected with a carrier and transferred into pichia pastoris cells, and experimental results show that the expression quantity of exogenous genes driven by an alcohol oxidase 1 promoter of the strain is increased by overexpression of the gene PASchr30259; according to the invention, microorganisms are modified by means of genetic engineering, so that the yield of foreign proteins expressed by the microorganisms is increased, and a foundation is laid for large-scale commercial production of the foreign proteins.

The invention belongs to the technical field of molecular biology and cells, and provides a construction method of an iPSC-sourced IL-10 protein overexpression MSC cell strain. The construction method comprises the following steps: constructing an exogenous IL-10 protein expression cassette of which an MSC specific promoter starts transcriptional activity, then stably inserting the protein expression cassette into human iPSC cells by virtue of a CRISPR / Cas 9 technology, and then inducing and differentiating the iPSC cells, so as to obtain the MSC cell strain of over-expressed IL-10 protein. The MSC for overexpressing the IL-10 protein can efficiently regulate the immune environment in the joint, and has a better treatment effect compared with the MSC from a natural source.

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing foreign protein based on an NC048599.1 site in a CHO cell genome. A site for stably expressing a protein in a CHO cell genome is located in a range of 69195000 to 69199000 of a CHO cell gene NC048599.1 with a sequence as shown in SEQ ID NO: 1 (Sequence Identifier Number 1), and the sequence of the CHO cell gene NC048599.1 is shown in SEQ ID NO: 1. Different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

Provided are a Salmonella engineered cell-penetrating bacteriophage, and a construction method therefor and a use thereof. The Salmonella engineered cell-penetrating bacteriophage comprises a bacteriophage and a cell-penetrating peptide, the surface of the bacteriophage has a surface protein, the surface protein is fused with the cell-penetrating peptide, and the surface protein is a protein containing an Ig-like domain. By means of a bioinformatics method, proteins which are suitable for fusion expression with foreign proteins, namely proteins containing Ig-like domains, are screened out in a high-throughput manner from structural proteins of a Salmonella typhimurium bacteriophage, and then, fusion expression of a cell-penetrating peptide is implemented on the surface of the Salmonella typhimurium bacteriophage to obtain an engineered bacteriophage capable of efficiently killing Salmonella typhimurium in cells, thereby increasing the titer of the Salmonella typhimurium bacteriophage entering cells, and extending phage therapy to treatment of intracellular infections.

The invention provides a preparation method of a recombinant cell, the recombinant cell and application of the recombinant cell, and relates to the technical field of biology. A target protein expressed by the recombinant cell contains one, two, three or four polypeptide chains; the preparation method comprises the following steps: integrating exogenous polynucleotides for coding a target protein into a genetic material of a starting cell in a site-specific manner, wherein each exogenous polynucleotide for site-specific integration contains coding genes of all polypeptide chains of the target protein; each exogenous polynucleotide contains at least four expression cassettes, and each polypeptide chain is encoded by at least one expression cassette. The preparation method is simple to operate, coding genes of all polypeptide chains can be integrated into genetic materials of starting cells at a time, and the expression quantity of foreign proteins of the recombinant cells obtained through the preparation method is high.

The invention discloses an Escherichia coli culture medium and application thereof in preparation of cell extracts required by cell-free protein synthesis, the culture medium comprises an initial culture medium or an initial culture medium and a supplementary culture medium, the initial culture medium comprises fructose, lactose, magnesium sulfate, ammonium sulfate, dipotassium phosphate, monopotassium phosphate, citric acid, trace elements, a vitamin solution and a nitrogen source; the fed-batch culture medium comprises a carbon source and a nitrogen source, the carbon source is fructose and a magnesium sulfate solution, and the nitrogen source comprises a nitrogen source solution added with yeast powder and yeast peptone. According to the culture medium disclosed by the invention, the escherichia coli can maintain logarithmic phase growth and reach relatively high thallus density (OD600 is greater than or equal to 60), and the prepared escherichia coli cell extract is used for cell-free protein synthesis, has high activity consistent with that of a cell extract obtained by low-density fermentation, and can be used for expressing various foreign proteins. According to the invention, the problem of low thallus density during preparation of the cell-free protein synthetic cell extract is solved, and the expression yield of the cell-free protein is effectively improved.

The invention discloses a non-fried instant noodle formula capable of improving amino acid score and a preparation method thereof, and the formula comprises the following raw materials in percentage by mass: 40% of chickpea powder, 8% of vital gluten, 44% of wheat flour, 8% of foreign protein, 1% of salt and 30% of distilled water. Wherein the foreign protein is one or a composition of egg white protein and soybean protein isolate. The protein content, the essential amino acid content and the protein digestibility are effectively improved, and the amino acid score is corrected. When the addition amount of the two exogenous high-quality proteins is 2% of egg white protein and 6% of soybean protein isolate, compared with a blank sample, the content of protein in a group added with the high-quality proteins is increased by 136.24%, the content of essential amino acid is increased by 56.85%, and the PDCAAS value is increased by 72.09%.

The invention relates to the technical field of genetic engineering, in particular to recombinant human collagen as well as a preparation method and application thereof. The recombinant human collagen provided by the invention is a polypeptide containing an amino acid sequence such as SEQ ID No.1. The recombinant human collagen disclosed by the invention is expressed at a high level outside cells, the culture is easy to amplify, the target protein can be produced at high yield by utilizing a high-density fermentation mode, secretory expression is beneficial to separation and purification of an expression product and reduction of production cost, and the recombinant human collagen has a certain post-translational modification function, such as glycosylation and the like, on the expressed foreign protein.