158 results about "Foreign protein" patented technology

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 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 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 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 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 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.

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.

To provide a new method for efficiently purifying an exogenous protein expressed in a plant.SOLUTION: A method for purifying a foreign protein expressed in a plant, comprising the step of adding an acid to an extract of a tissue of a plant in which a foreign protein is expressed to degrade contaminating proteins derived from the plant.SELECTED DRAWING: None

The invention discloses a PVX expression vector pWX for efficiently expressing foreign protein and application of the PVX expression vector pWX. In the construction process of optimizing potato X virus infectious clone, after a single base mutation NruI-gene sequence (CCGCGA) is inserted into an upstream regulation region of a PVX coat protein subgene promoter, a novel PVX expression vector capable of promoting foreign protein expression is obtained, and the novel PVX expression vector is named as pWX. Experiments prove that compared with a control vector, the accumulation amount of GFP (Green Fluorescent Protein) generated by the pWX vector through transient expression of agrobacterium in tobacco is obviously increased. The invention further applies a pWX vector to successfully express an important target protein HPV16L1 researched and developed by an HPV vaccine, and the expression quantity of the important target protein HPV16L1 in tobacco is 5.25 micrograms per gram of fresh heavy leaves. The invention provides a novel vector based on a PVX genome for high-efficiency expression of a plant source target protein, and the vector has a good application prospect in preparation of plant source vaccines.

The invention relates to a Sebastes schlegeli endogenous retrovirus envelope protein Penv, a lentiviral vector and application thereof, and belongs to the field of genetic breeding of molecular biology, and the amino acid sequence of the envelope protein Penv is as shown in SEQ ID NO.1. The invention further provides an in-vitro assembly system, a transformant and a kit containing membrane fusion protein particles and lentiviral particles of the envelope protein Penv and application of the envelope protein Penv. VSVG protein is replaced with Penv protein from sclerobone fish, so that the transduction efficiency of the lentiviral particles to sclerobone fish cells is effectively improved, and the transduction efficiency of the lentiviral particles to the sclerobone fish cells is improved. And efficient gene delivery is realized. Meanwhile, as the endogenous Env protein, the Env protein overcomes the immunological rejection of a host to the exogenous Env protein, and also has the potential of in-vivo application.

The present invention discloses a VHH chain of an anti-APP nanobody and its application. The anti-APP nanobody of the present invention has the following beneficial effects: (1) small molecule and drug delivery. (2) easy to manufacture and express. (3) high affinity and strong specificity. (4) stable performance and good plasticity. (5) low immunogenicity and good metabolic characteristics. The APP nanobody of the present invention retains the ability of the VVH chain antibody and avoids the introduction of non-human exogenous proteins as much as possible, taking into account the function of effectively binding to anti-APP and the lowest possible heterology. In addition, the reduction of heterology can not only reduce hypersensitivity reactions but also prolong its half-life in the body and improve metabolic characteristics.

The invention provides an in-vitro cell-free synthesis method and kit of foreign protein, and belongs to the technical field of protein synthesis. According to the in-vitro cell-free synthesis method of the foreign protein, a water-phase in-vitro cell-free protein synthesis system and a nucleic acid template for coding the foreign protein are provided, a hydrophobic interface in surface contact with the water-phase in-vitro cell-free protein synthesis system is provided, incubation reaction is carried out, the foreign protein is synthesized, and the in-vitro cell-free synthesis method of the foreign protein is obtained. The protein synthesis efficiency and the protein expression quantity of the cell-free system are improved. The invention further provides an in-vitro protein synthesis kit which is more efficient and higher in throughput. The invention provides a simple and convenient hydrophobic interface optimization method which is simple and convenient to operate and saves cost.

The invention relates to a combined additive, a CHO cell culture medium, a preparation method and a culture method. The combined additive comprises cyclohexyl alanine and 3-(2-naphthyl)-L-alanine. The CHO cell culture medium comprises amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates and a combined additive, the preparation method is used for preparing the CHO cell culture medium. The culture method uses a CHO cell culture medium to culture CHO cells. The combined additive is obtained through structural domain virtual docking and hydrophobicity regulation and control screening, G1 / S conversion can be mildly delayed, cell apoptosis can be inhibited, and an expression window can be prolonged. The culture medium contains amino acids, vitamins, trace elements and the like, and preferably selects a functional additive to replace serum and foreign protein functions. According to the application, high proliferation of CHO cells and efficient target protein expression can be realized under the conditions of no serum and no foreign protein, meanwhile, the accumulation of lactic acid and ammonia is reduced, the cell caking phenomenon is reduced, and the application has remarkable technical advantages.

The invention belongs to the technical field of biological genetic transformation, and relates to a method for preparing recombinant foreign protein based on transient expression of fruits of cucurbitaceae crops. The efficient expression of the foreign protein is realized through the processes of constructing an instantaneous transformation vector, infecting fruits with agrobacterium and purifying the protein. The method overcomes the defects of high cost and low purity of a traditional tobacco transient expression system, has the advantages of easily available raw materials, large biomass, edibility, high safety and the like, and provides a new way for low-cost large-scale production of recombinant proteins.

The invention belongs to the technical field of chemiluminescence detection, and particularly relates to a kit for detecting a bovine leukemia virus IgG antibody as well as a detection method and application of the kit. A sample treatment solution in the kit is prepared from Protein A, disodium hydrogen phosphate dodecahydrate, monopotassium phosphate, potassium chloride, sodium chloride, casein, mercaptoethanol, Tween-20 and Proclin; and the mark of the magnetic bead is sealed by combining PEG4000 and ethanolamine. Compared with traditional single protein sealing, the sealing mode provided by the invention has the advantages that interference of foreign protein is removed, non-specific binding in a sample is reduced, the background is reduced, and the stability, sensitivity and specificity of detection are improved; compared with a traditional large-proportion diluted sample, the sample treatment liquid has the advantages that excessive IgG in the sample is removed, dilution treatment of an equipment dilution module is not needed in the detection process, manual pre-dilution treatment is also not needed, and the detection efficiency is improved.

The invention belongs to the technical field of biological medicine, and discloses lactococcus lactis HX9302 for expressing foreign protein and application, the lactococcus lactis HX9302 is preserved in China General Microbiological Culture Collection Center on January 20, 2025, and the preservation number is CGMCC NO.33477. The lactococcus lactis HX9302 is preserved in China General Microbiological Culture Collection Center on January 20, 2025; the lactococcus lactis has good acid resistance and digestive enzyme tolerance, can be used as an oral delivery live carrier of helicobacter pylori vaccines, and promotes research, development and application of oral vaccines.

The invention relates to the technical field of tag peptides, and particularly discloses a tag peptide combined on the surface of corynebacterium, and the amino acid sequence of the tag peptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The tag peptide provided by the invention enriches the types of tag peptides bound to the surfaces of current bacteria, and corynebacterium bacteria bound with the tag peptide have wider adaptability to pH and can be compatible with the optimal activity conditions of more foreign proteins; the molecular weight of the tag peptide is only 3.5 kDa, so that the interference to the structure or function of an exogenous protein can be remarkably reduced; in addition, the tag peptide can be fused at the N end or the C end of the target protein, the binding efficiency is not affected, simultaneous display of multiple proteins is supported, and a basis is provided for multi-enzyme cascade reaction and multivalent vaccine development.

The present invention discloses a recombinant Vibrio natriegens and a method for promoting the secretion and expression of exogenous proteins. The recombinant Vibrio natriegens is constructed by using the Vibrio natriegens with the preservation number of CICC 10908 as the chassis cell, knocking out the deoxyribonuclease gene on its genome, and inserting a combined expression cassette of T7 RNA polymerase and aminoglycoside adenyltransferase. The recombinant plasmid carrying the exogenous protein is transformed into the above-mentioned recombinant Vibrio natriegens, and the seed liquid is inoculated into the fermentation medium. After culturing to the mid-logarithmic growth phase, IPTG and NaCl are added, and then the culture is continued to obtain a fermentation broth containing the exogenous protein. The present invention solves the problem of secreting and expressing exogenous proteins by Vibrio natriegens at the fermentation level for the first time, with a short culture time, low cost, and certain universality, providing technical guidance for the research of Vibrio natriegens engineering bacteria in the fields of protein engineering and fermentation engineering.

The invention particularly relates to a sebastes schlegeli endogenous retrovirus envelope protein percomORF, a lentiviral vector and application of the sebastes schlegeli endogenous retrovirus envelope protein percomORF and the lentiviral vector, and belongs to the field of genetic breeding of molecular biology, and the envelope protein percomORF has an amino acid sequence as shown in SEQ ID NO.1. The invention also covers a membrane fusion protein particle constructed based on the protein, a system for in-vitro assembly of lentivirus particles, a corresponding transformant and a matched kit. By replacing conventional VSVG protein with percomORF protein from sclerobone fish, the infection and transduction efficiency of lentivirus on sclerobone fish cells can be effectively enhanced, and more efficient gene delivery is realized. Besides, the protein belongs to host endogenous Env protein and is beneficial to avoiding immunological rejection caused by introduction of foreign protein, so that the protein has good potential in in-vivo gene delivery application.

The invention belongs to the field of biology, and discloses preparation and application of a non-methanol-induced expression H5 subtype avian influenza virus subunit antigen and a vaccine thereof, PDAK is applied to pichia pastoris expression of avian influenza virus H5 subtype HA protein, an HA protein multiple-insertion recombinant strain with a PDAK promoter is successfully constructed, and in the research process, it is found that the H5 subtype avian influenza virus subunit antigen and the vaccine thereof have the advantages that the H5 subtype avian influenza virus subunit antigen and the vaccine thereof can be used for preparing the H5 subtype avian influenza virus subunit antigen; by adopting the recombinant plasmid disclosed by the invention, multiple insertions can be generated in a pichia pastoris strain, the target protein expression quantity can be obviously improved, and the foreign protein expression efficiency of the recombinant plasmid is superior to that of a PAOX1 promoter and a PGAP promoter; according to the invention, the HA protein of the PDAK promoter is multiply inserted into the recombinant strain to express the HA protein to prepare the subunit vaccine, after the vaccine is immunized, a higher hemagglutination inhibition antibody level can be generated aiming at homologous strains, and the survival rate of experimental chickens after challenge is 100%.

The application 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, the recombinant expression vector is transfected into E. coli BL21 (DE3), and expression is obtained through the recombinant E. coli BL21 (DE3); and the amino acid sequence of the C-terminal G4SLPETG modified CMV is SEQ ID NO. 1. Test proves that the recombinant strain constructed by the application is stable to expression of an exogenous protein. The recombinant protein expressed by the application is used for preparing a virus-like particle for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, the virus-like particle is not pathogenic to mice and other animals, and the safety evaluation is easy.

The invention discloses an inflammation regulation sequence of dairy cow mastitis and application thereof, and belongs to the technical field of biology, the inflammation regulation sequence is a regulation sequence responding to a gram-negative bacterium inflammation signal, the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 1, and the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 2. A regulatory sequence responding to a gram-positive bacterium inflammation signal, which is a promoter partial sequence of the NLRP3 gene and is as shown in SEQ ID NO: 2; the regulatory sequence responding to a gram-positive bacterium inflammation signal is a promoter reverse complementary sequence of the TLR2 gene and is shown as SEQ ID NO: 3; the endogenous inflammation regulation sequence provided by the invention is composed of genome elements of the dairy cow, and no exogenous coding gene is introduced, so that potential risks caused by exogenous proteins are avoided.

The invention discloses a site for stably expressing a protein in an HEK293T cell gene NG027973.1 and an application of the site for stably expressing the protein in the HEK293T cell gene NG027973.1. According to the present invention, the obtained stable expression site is located at the upstream 69 bp and the downstream 134bp of the 135153 base in the HEK293T cell gene NG027973.1, such that the 135084-135287 base can be integrated, and the stable expression can be performed; according to the invention, a target gene is integrated to a stable expression region in a site-specific integration manner. The site information provided by the invention is clear, site-specific integration of foreign protein genes can be realized at the site, the protein can be stably expressed, the screening process and time in the cell construction process can be greatly shortened, and the research and development cost can be reduced.

The invention relates to the technical field of saccharomyces cerevisiae expression foreign protein, and discloses a saccharomyces cerevisiae strain with ASFV p14.5 displayed on the surface and a construction method and application thereof, and the method comprises the following steps: (1) synthesizing a delta site homologous arm sequence; (2) seamlessly connecting the homologous arm sequence of the delta site to a carrier pET23a, connecting a selective marker tryptophan biosynthetic gene TRP1 to the 5'end of the upstream homologous arm sequence of the delta site, and sequentially connecting an Aga2 gene, an E120R gene, a T2A peptide gene and an mCherry gene to the 5 'end; (3) carrying out enzyme digestion on two sides of the delta site through restriction endonuclease salI, and recovering a linearized fragment through agarose gel; and (4) integrating the recovered fragments into a host saccharomyces cerevisiae genome through a chemical conversion method, and culturing until bacterial colonies grow. The surface display type saccharomyces cerevisiae strain obtained by the invention not only has the stability of integrative saccharomyces cerevisiae for expressing foreign protein, but also can be used for more easily and more intuitively screening a high-expression quantity saccharomyces cerevisiae strain by combining the T2A peptide with mCherry.

The invention relates to the technical field of bioengineering, and discloses a saccharomyces cerevisiae self-replication system and application thereof.The system comprises a recombinant plasmid, and the recombinant plasmid comprises upstream and downstream homologous arms of a saccharomyces cerevisiae delta site, a selection marker TRP1, an autonomous replication sequence ARS310, a G418 resistance gene and a resistance gene promoter; wherein the nucleotide sequence of the autonomous replication sequence ARS310 is as shown in SEQ ID NO. 3; the nucleotide sequence of the G418 resistance gene is as shown in SEQ ID NO. 4; the nucleotide sequence of the selection marker TRP1 is as shown in SEQ ID NO. 5. The invention provides a pressure-driven saccharomyces cerevisiae self-replicating system for high-efficiency expression of foreign protein (target gene), and the expression of the foreign target gene with high conversion efficiency, high stability and high copy number is realized by utilizing natural multiple copies of a delta site, the self-replicating capability of ARS and the screening pressure of a G418 resistance gene.

The application discloses a silk material with a cell proliferation function and application thereof, the functional material is expressed by a human type I collagen truncated fragment in a middle silk gland of a domestic silkworm, a sericin promoter is used as a tissue-specific promoter to drive specific expression of an exogenous gene in the middle silk gland, and finally the exogenous gene is combined with sericin protein and is secreted to outside of the body, the exogenous protein can be separated after a certain treatment of a cocoon, and the expression product can keep biological activity. The silk material is used to prepare a hydrogel, has a function of promoting wound healing, and can be applied to aspects of promoting skin regeneration and repair, bone graft substitutes, cartilage reconstruction, artificial skin and the like.