93results about How to "Efficient and stable expression" patented technology

The invention discloses single guide ribonucleic acid (sgRNA) capable of effectively editing a pig ROSA26 gene, and application of the sgRNA. Based on the sgRNA capable of specifically identifying the pig ROSA26 gene in a pig genome, a cell line of enhanced green fluorescent protein (EGFP) site-specific integrated porcine fetal fibroblasts is successfully established by using a CRISPR / Cas9-mediated gene knock-in technique; the results show that the cell line can stably and efficiently express an EGFP gene; furthermore, in the preparation process of the cell line, exogenous promoter genes and positive and negative screening marker genes are not introduced into the cell line. Therefore, the sgRNA greatly improves the safety of transgenic pigs, and has important significance for eliminating the biological and food potential safety hazards of agricultural products of the transgenic pigs.

The invention employs a CRISPR-Cas9 system to complete mediation of a goat Tbeta4 gene for site-directed knock-in. A gRNA expression vector and a Tbeta 4 homologous recombinant vector based on the CRISPR-Cas9 system are constructed according to a CCR5 gene sequence of the goat. An optimized CRISPR-Cas9 vector and a constructed gRNA expression vector and the Tbeta 4 homologous recombinant vector are transferred to skeletal muscle satellite cells together, in order to obtain cells with the site-directed Tbeta4 gene. A targeting vector constructed based on the CRISPR-Cas9 system provides a simple, fast and safe pathway for site-directed knock-in of the goat Tbeta4 gene. Any screening marker genes are not related in a cell line screening process, safety of transgenic animals is greatly improved, and the method has important value for genetic breeding of goats and research of gene function.

The invention discloses a pig ROSA26 specific integration site and an application thereof. The nucleotide sequence of the site is represented by SEQ ID NO.2. A targeting vector derived from the site is represented by SEQ ID NO.1. The ROSA26 specific site adopted by the invention is not subjected to immune-mediated exclusion and epigenetic modification in genomes of pigs or higher mammals. The site can mediate stable and efficient expression of exogenous genes. With the ROSA26 specific site targeting vector, targeting can be realized with high efficiency.

The invention provides an insect-resistant gene Cry1F-t and application thereof. A Cry1F gene is completely modified to obtain the Cry1F-t insect-resistant gene as shown by SEQ ID NO: 1; an AdhI enhancer is added in the front of the Cry1F-t insect-resistant gene; the Cry1F-t insect-resistant gene is put on an expression vector including a strong promoter 35S; and the Cry1F-t gene is transferred into a corn genome through an agrobacterium-mediated genetic transformation method to obtain a transgenic material including the Cry1F-t. Proved by an experiment, the Bt toxalbumin modified and synthesized by the invention has an obvious insect killing effect on corn borers; and the Cry1F-t and a target protein expressed by the Cry1F-t can be stably and efficiently expressed in a corn, so that the Cry1F-t and the target protein are used for culturing a Cry1F-t transgenic insect-resistant corn. The Bt insect-resistant gene Cry1F-t can be also applied in the improvement of the insect resistance of other farm crops, fruit trees or vegetables and the like, and an effective approach is provided for solving insect pests in the production of plants such as the corn.

The invention discloses a porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application. An antigen fusion gene shown as SEQ ID No. 1. is obtained by connecting an antigen locus of porcine epidemic diarrhea virus (PEDV) S glycoprotein and a cell wall anchoring sequence. The invention further constructs a secretion expression vector containing the antigen fusion gene, and secretion expression vector is converted into the bacillus megaterium to express recombinant protein on the cell wall or surface of the bacillus megaterium. Immunoblotting experiments indicate that the expressed recombinant protein can react with PEDV immune serum and has the same antigenicity as PEDV natural antigens. Immunofluorescent tests of live bacteria which are subjected to induced expression indicate that the expressed recombinant protein is positioned to the surfaces of the bacteria. Experiment results indicate that the recombinant protein can be prepared into safe and effective mucous immune live vaccines for preventing and treating porcine epidemic diarrhea.

The invention provides an insect-resistant gene Cry1Ab-Material. The synthesis of the gene is as follows: according to the amino acid sequence of the N-terminal of the original Bacillus thuringiensis (Bt) protein (Cry1Ab), the preferred codons of monocotyledon (corn) is used to perform human reformation and synthetize a new Cry1Ab DNA sequence; and the prokaryotic expression vector and plant expression vector are constructed, and transformation is performed in the host cells. Vitro tests prove that the transformed and synthetized Bt gene toxic protein has obvious insecticidal effect on Ostrinia nubilalis. The expression of the insect-resistant gene Cry1Ab-Ma in monocotyledon is stable and efficient, thus the gene can be used to produce insect-resistant transgenic plant.

The invention discloses a PEDV S gene major antigen epitope serial connection recombination gene, and a preparation method and an application thereof, and belongs to the field of a PEDV S protein major neutralizing epitope region efficient expression method. The PEDV S gene major antigen epitope serial connection recombination gene is obtained through connecting three major antigen epitopes of a PEDV S gene in series; every two major antigen epitopes are connected through a flexible amino acid base sequence; the name is S123; and the sequence of the base sequence is shown as SEQ ID NO:1. The recombination gene contains most PEDV antigen epitopes; the selected fragments avoid a high-variation region; the expression quantity is efficient and stable; the immunogenicity is high; when the application of PEDV S gene major antigen epitope serial connection recombination protein to a PEDV indirect ELISA detection kit is used, the PEDV can be fast diagnosed; and the swinery neutralization protection can be well monitored and evaluated.

The invention discloses a method for the expression and production of FGF (fibroblast growth factor)-20. By the method of bioinformatics for optimization design, full length FGF-20 nucleotide sequence is synthesized, and is connected with pET series carriers, the expression carrier connection is introduced into proper escherichia coli host cells, a high expression quantity and stable inheritance engineering strain is obtained through screening, a high density fermentation method, an inclusion body expression FGF-20 purification method and a corresponding quality detection method are established, and mass production of rhFGF-20 is possible. On the basis, clinical application of the FGF-20 in tissue repair and degenerative diseases of the nervous system and effect of the FGF-20 in occurrence, development and migration of tumors can be further explored.

The invention provides a method for expressing and producing pseudoplectania nigrella mature peptide recombinant proteins in pichia pastoris cells in a constitutive type mode, which comprises the following steps of: 1) optimizing pseudoplectania nigrella mature peptide genes; 2) obtaining pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter DNA sequences by adopting methods such as molecular cloning and the like, and placing the pseudoplectania nigrella mature peptide genes under the regulation and control of the glyceraldehyde-3-phosphate dehydrogenase promoters to connect with plasmids to obtain new, constitutive and secretory type pichia pastoris efficient expression vectors; 3) converting the pichia pastoris by using the vectors and obtaining the recombinant pichia pastoris strains to obtain the pseudoplectania nigrella mature peptide with bioactivity; and 4) optimizing the high-density fermentation conditions of the engineering strains and the expression and purification of the recombinant protein. The pseudoplectania nigrella mature peptide prepared by the method can be applied in the fields of medicinal treatment, food, feed, scientific research and the like and is suitable for the scale production of pseudoplectania nigrella protein.

The invention discloses a glyphosate resistant protein, and a coding gene and application thereof. The protein provided by the invention is (a) or (b) or (c) as follows: (a) a protein composed of an amino acid sequence shown as 73rd-504th site of a sequence 1 in the sequence table; (b) a protein composed of the amino acid sequence shown in the sequence 1 in the sequence table; and (c) a protein, derived from the sequence 1 and with glyphosate resistance, obtained by substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence of (a) or (b). The GmEPSPS protein and the coding gene thereof provided by the present invention have significant resistance effect on a glyphosate herbicide. Experiments prove that GmEPSPS transgenic tobacco and GmEPSPS transgenic maize can still grow normally under 0.15% of glyphosate, but non-transgenic tobacco and maize are obviously suppressed in growth, and have leaves yellowing and withered. It shows that GmEPSPS can stably and efficiently express in plants, and be further used in production of glyphosate resistant transgenic plants.

The invention relates to a novel EPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase) gene, an expression product of the EPSPS gene, a herbicide-resistant gene CTP2-EPSPS (chloroplast transit peptide 2-EPSPS), an expression product and an expression vector of the herbicide-resistant gene CTP2-EPSPS and application of the expression product and the expression vector of the herbicide-resistant gene CTP2-EPSPS. Nucleotide sequences of the EPSPS gene are shown as SEQ ID No.2, amino acid sequences of encoding proteins of the expression product of the EPSPS gene are shown as SEQ ID No.3, the herbicide-resistant gene CTP2-EPSPS contains chloroplast transit peptide CTP2 and the EPSPS gene, nucleotide sequences of the herbicide-resistant gene CTP2-EPSPS are shown as SEQ ID No.5, and the expression product and the expression vector of the herbicide-resistant gene CTP2-EPSPS can be applied to cultivating glyphosate-resistant transgenic corn. The EPSPS gene, the expression product of the EPSPS gene, the herbicide-resistant gene CTP2-EPSPS, the expression product and the application vector of the herbicide-resistant gene CTP2-EPSPS and the application have the advantages that the herbicide-resistant gene can be stably expressed in crop and is high in expression amount, good herbicide-resistant effects can be realized, the corn or other plants can be transited, accordingly, herbicide-resistant characteristics of plants such as the corn can be improved, and an effective way can be provided for improving the herbicide-resistant characteristics of the plants such as the corn during production.

The invention discloses construction of modified porcine alpha-interferon gene and an expression vector as well as a method for making protein of the modified porcine alpha-interferon gene. A sequence table of the modified porcine alpha-interferon gene is shown in SEQ ID NO.1. The invention discloses a method for making the protein of the modified porcine alpha-interferon gene at the same time, relating to the steps of reformation of the porcine alpha-interferon codon, clone of the gene and high expression in pichia, in particular to improvements on operation methods relevant to matching and reformation of yeast codon preferendum by porcine alpha-interferon codon; a culture method for effectively producing porcine alpha-interferon; measures for preventing pollution; the control of determining quantity and the time of adding methanol and the time of protein expression, namely protein harvest time; asepsis examination; the test of protein content and activity, and a method for enlarging culture condition. The method is easy to operate with low cost, thereby realizing the stable and effective expression of the porcine alpha-interferon in pichia, and improving as much as 300 times of an antiviral activity of the interferon.

The invention discloses a low oxygen induction factor low interruption RNA SiRNA-HIF-1 alpha particle. The particle could be high efficiently expressed in cell and could strongly restrict the expression of independent gene in mammal cell.

The invention relates to a method for producing hydroxylated triple helical proteins in yeast host cells by introducing to a suitable yeast host cell, DNA sequences encoding the triple helical protein as well as prolyl 4-hydroxylase (PH4), in a manner wherein the introduced DNA sequences are replicated, stably retained and segregated by the yeast host cells.

The invention discloses a method for synthesizing rebaudioside-A through high-density fermentation. The method comprises the steps that firstly, genes encoded with a cell wall synthetase system is knocked out, and fermentation synthesis of the rebaudioside-A is achieved while the permeability of a cell wall is improved; secondly, thalluses obtained through fermentation are applied to a whole-cellcatalysis reaction, and the high recovery ratio of the rebaudioside-A is achieved; in the high-density fermentation process, when the thalluses grow to the amount of (10-25) g / L (DCW), a culture medium is subjected to fed-batch in a mode of constant dissolved oxygen index feeding, and when cells grow to the stable phase, the density OD<600> of the thalluses reaches to 100-150; the thalluses grow to the stable phase, the culture medium is subjected to fed-batch in a mode of constant speed feeding, and the content of the rebaudioside-A in fermentation liquid after fermentation is completed is 13.7+ / -1.6 g / L; and the thalluses collected in fermentation are subjected to buffer cleaning treatment by using potassium phosphate and then used for a 50 L whole-cell catalysis reaction, and the concentration of the rebaudioside-A in the system is 28.2+ / -4.9 g / L. According to the method, the problem that the conversion rate of stevioside in cell fermentation is low is solved well, operation is easy, production is easy to enlarge, and the good rebaudioside-A preparing prospects are achieved.

The invention relates to an encephalitis b virus non-structural protein NS1 truncated mutant as well as a coding gene and application thereof, which belongs to the technical field of viruses. According to the encephalitis b virus non-structural protein NS1 truncated mutant, on the basis of original NS1, two strong hydrophobic zones on an end C are deleted, a truncated mutant NS1 delta63 gene with63 amino acids on the end C being deleted, then the truncated mutant NS1 delta63 gene is cloned to a prokaryotic expression vector pET-28a(+), a recombinant expression vector pET28a-NS1delta63 is obtained, escherichia coli is converted, and by virtue of the induction of IPTG, the NS1 delta63 can be efficiently and stably expressed in the escherichia coli. The NS1 delta63 protein expressed in the Escherichia coli purifies and immunizes a mouse, and the NS1 anti-serum titer of the immunized mouse reaches 1:12150 by virtue of ELISA detection. The protection capability test of the immunized mouseproves that the NS1 delta63 has the protection activity. The NS1 delta63 can lay a foundation for later research of the virus vaccine and diagnosis kit.

The invention provides an insecticidal protein Cryl A. 301 which has 1) an amino acid sequence shown by SEQ ID No.2; or 2) an amino acid sequence which is obtained by substituting the amino acid sequence shown by SEQ ID No.2 or decreasing and or increasing one or more amino acid(s) of the amino acid sequence shown by SEQ ID No.2, and has the same function. The experiment in vitro proves that the reformed and synthesized Bt gene toxin production protein has remarkable insecticidal effect for european corn borer. The insecticidal protein Cryl A. 301 can be efficiently expressed in monocotyledons, thus being used for producing insect-resistant transgenic plants.

The invention belongs to the field of biotechnology and botany, and particularly provides a construction method of a rolling-circle replication recombinant vector for heterologous protein expression,the recombinant vector and an application of the recombinant vector. According to the construction method provided by the invention, the high-efficiency rolling-circle replication transient expressionvector mainly based on the sequence of a sweet potato leaf curl virus (SPLCV) is constructed, so that duration time and expression abundance of heterologous nucleic acids and proteins on plants are remarkably improved, and the results of the vector have high repeatability and reliability. The recombinant vector provided by the invention has a molecular tool vector which has an extremely long expression time to heterologous proteins (longer than 20 day compared with the expression time of a common transient expression T vector) and has an extremely high expression abundance (larger than 30-fold expression compared with a common transient expression T vector), and the recombinant vector endows different plants (tobaccos, sweet potatoes and morning glory) with heterologous protein expressioncapability, and makes up the blank of related plants for utilizing transient expression to study gene functions.

The invention discloses a method of establishing a hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus and belongs to the technical field of cell modification. The method comprises the following steps: S1, preparing an hNTCP recombinant lentivirus concentrated liquid; S2, preparing liver tissues which are fully digested; S3, preparing the porcine primary hepatocyte; S4, preparing a porcine primary hepatocyte single cell suspension; S5, preparing a cell suspension for paving a plate; S6, paving cell plate and culturing; S7, carrying out hNTCP recombinant lentivirus infection on the porcine primary hepatocyte; and S8, establishing the hepatitis B virus infection cell model. According to the invention, the recombinant lentivirus containing an hNTCP gene is constructed in vitro, the hNTCP gene is integrated into the primary hepatocyte genome through the lentivirus with high efficiency of infection to achieve stable overexpression of hNTCP, so that the primary hepatocyte supports hepatitis B virus infection.

The invention provides an anti-insect protein Cry1A.401, which is characterized by comprising: (1) an amino acid sequence shown as SEQ ID No.2, or (2) an amino acid sequence which is obtained by performing substitution, deletion and / or addition of one or more amino acids on the amino acid sequence shown as SEQ ID No.2 and has equal function. As proved by an in-vitro experiment, a modified and synthesized Bt gene toxoggenic protein has a remarkable insecticidal effect on corn borers. The anti-insect protein Cry1A.401 can be stably and efficiently expressed in monocotyledons, and is further applied to production of anti-insect transgenic plants.

The invention discloses a modified dog alpha1-interferon gene and a preparation method of dog alpha1-interferon. The modified dog alpha1-interferon gene has the nucleotide sequence as shown in SEQ ID NO.1 and the amino acid sequence coded by the gene is shown in SEQ ID NO.2. The invention concretely relates to matching and modification for the predilection of a dog alpha1-interferon codon to a yeast codon as well as optimal control on a culture method for efficiently producing dog alpha1-interferon, pollution avoiding measures and operation methods of methanol adding amount and time determination, protein expression time control, namely protein harvest time, sterility test, protein content and activity measurement, culture condition expansion and the like. The method disclosed by the invention is simple and easy to realize, relatively low in cost, and capable of realizing stable and efficient expression of the dog alpha1-interferon in pichia pastoris, and the expressed dog alpha1-interferon has a remarkable effect on treating dog viral infectious diseases and can enhance the immune effect of a rabies vaccine.

The invention discloses an anti-glyphosate protein as well as an encoding gene and application of the anti-glyphosate protein. The protein, provided by the invention, is the following (a), (b) or (c): (a) the protein consists of amino acid sequences shown at 56th-532th position of the sequence 1 in a sequence table; (b) the protein consists of amino acid sequences shown by the sequence 1 in a sequence table; and (c) the protein with glyphosate resistance is derived from the sequence 1 and subjects the amino acid sequences of (a) or (b) to substitution and / or deletion and / or addition of one or more amino acid residues. The GmEPSPS-2 protein and the encoding gene of the GmEPSPS-2 protein, provided by the invention, have remarkable resistance effect on glyphosate phytocide; experiments prove that GmEPSPS-2 transgenic tobaccos and GmEPSPS-2 transgenic corns can grow normally under the action of 0.15% glyphosate, while growth of the non-transgenic tobaccos or corns are obviously inhibited, leaves of the non-transgenic tobaccos or corns become yellow and wither, which shows that GmEPSPS-2 can be stably and efficiently expressed in plants, and is further used for producing anti-glyphosate transgenic plants.

The invention belongs to the field of molecular biology, and relates to a transposase for efficiently mediating exogenous gene integration, and a use thereof. The transposase is a fusion protein, and fuses a human c-myc pyrenoid positioning signal, and the human c-myc pyrenoid positioning signal can effectively guide aggregation of the transposase in a nucleus. The transposase is loaded in a transposon integration system, can mediate efficient integration of the exogenous gene in host cells, and can efficiently and stably express.

The invention provides a liver cancer tissue specific RNA (Ribose Nucleic Acid) interference system, as well as a construction method and an application method thereof. The liver cancer tissue specific RNA interference system comprises an AFP (Alpha Fetal Protein)-Cre vector and an LoxP-shRNA vector which are both constructed by lentiviral vectors, wherein the AFP-Cre vector contains an AFP promoter and a Cre recombinase gene placed at the downstream of the AFP promoter; the LoxP-shRNA vector contains a reconstructive H6 promoter and an shRNA sequence placed at the downstream of the U6 promoter; the reconstructive U6 promoter contained in the LoxP-shRNA vector is the U6 promoter in which a structure, capable of indicating whether the AFP-Cre / LoxP-shRNA system is in work, is inserted inside; and the structure, capable for indicating whether the AFP-Cre / LoxP-shRNA system is in work, is shown as LoxP-CMV-eGFP-LoxP. The RNA interference system, provided by the invention, has the characteristics of being high in liver cancer tissue specificity, long-lasting in effect, high in efficiency, excellent in indication performance, and simple and convenient in operation; and the liver cancer tissue specific RNA interference system enables the liver cancer RNA interferential targeted treatment of liver cancer, provides high convenience to relative research and analysis, and can be widely applied to the treatment of the liver cancer and the basic research.

The invention provides a recombinant expression of shrimp antibacterial peptide in probiotic bacteria and an application thereof, which comprises the clone of a shrimp antibacterial peptide gene, the construction of the recombinant carrier of the shrimp antibacterial peptide gene, the conversion and the screening of the recombinant carrier of the shrimp antibacterial peptide gene, the engineering bacteria of the fermentation culture expressed antibacterial peptide, and the verification and the purification of an expression product. The invention also provides the application of recombining probiotic bacteria with the shrimp antibacterial peptide gene. The probiotic bacteria which is used for aquaculture feed, in particular to feed chiefly used for shrimps. The recombinant probiotic bacteria can continually propagate in an organism body, is expressed continually within a certain period, is provided for the antibacterial peptide of the organism body, and has good specificity and validity.

The invention relates to a high-efficiency stable mammal cell expression system. The high-efficiency stable mammal cell expression system efficiently expresses a target gene in an appropriate mammal host cell strain by applying a novel high-efficiency expression vector. The high-efficiency stable mammal cell expression system disclosed by the invention can outstandingly enhance the expression level of the target gene in a host cell, greatly reduce the screening time and cost of a high-expression cell strain and realize the high-yield stable expression of a target protein in a mammal cell.

The invention discloses a recombinant human interleukin (IL) 15 long peptide fragment and a production method thereof. The production method comprises the following steps: obtaining a human IL-15 long peptide fragment gene; establishing an eukaryotic expression vector and transforming the vector into Pichia pastoris X-33; performing resistance screening on high-level secretory-expressed yeast transformants; performing induced expression, purifying the supernate through a nickel column and an ion exchange column to obtain rhIL-15L. The production method is characterized in that the amino acid residue Ala of the site Kex2P1' of the vector pPICZAlphaA is mutated into Pro and the Pichia pastoris X-33 is taken as host bacteria, and therefore, mass high-efficiency stable expression of the rhIL-15L is realized; the recombinant protein rhIL-15L produced by the method is marked with HIS and C-MYC tags and prone to protein purification and detection; besides, the recombinant protein rhIL-15L is a glycoprotein which is modified through glycosylation to a certain extent, and therefore, the recombinant protein rhIL-15L has excellent bioactivity and is capable of effectively maintaining the proliferation and the bioactivity of human NK (Natural killer) cells in vitro and of a mouse in vivo.

The invention discloses a nonresistant bifunctional DNA vaccine vector, and a construction method and an application thereof, and belongs to the technical field of animal gene engineering. A nutrition selection marker is used as the screening marker of the nonresistant bifunctional DNA vaccine vector to substitute antibiotic resistance gene in order to construct a eukaryotic expression plasmid pcD-asd and fundamentally block the diffusion propagation of the antibiotic resistance gene, the safety is good, and the expression efficiency and the copy number are high. The DNA vaccine vector can complement with asd gene defect Gram-negative bacteria to construct a chromosome-plasmid balanced lethal system, and the system can highly and stably express exogenous gene in the eukaryotic cell, and can be used as a nucleic acid vaccine expression vector, for example, attenuated Salmonella typhimunum is used as a vector to construct bivalent and multivalent live bacterial vaccines.

The invention discloses a high-throughput expression method of alkaline bacterial laccase in yeast. The high-throughput expression method comprises the following steps: (A) a pichia pastoris expression vector carrying with alkaline bacterial laccase genes is constructed; (B) positive transformants are screened; (C) the yeast expression vector is linearized; (D) the yeast galvanic competence is prepared; (E) recombinants are expressed and identified; (F) fusion proteins are purified; (G) enzyme kinetic parameters are determined. The high-throughput expression method helps modelers to finish the compositional modeling of models more efficiently and reasonably through the model discovery based on the semantics, so that the problems of easy formation of inclusion bodies by the alkaline bacterial laccase in the prokaryotic expression system, low yield and low enzymatic activity are solved.