344 results about "Gene synthesis" patented technology

The present invention relates generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms. Thus, the present invention outlines a novel approach to utilizing the results of genomic sequence information by computer directed gene synthesis based on computing on the human genome database. Specifically, the present invention contemplates and describes the chemical synthesis and resynthesis of genes defined by the genome sequence in a host vector and transfer and expression of these sequences into suitable hosts.

In embodiments of the present invention, methods are provided for removing double-stranded oligonucleotide (e.g., DNA) molecules containing one or more sequence errors, generated during nucleic acid synthesis, from a population of correct oligonucleotide duplexes. In one embodiment, the oligonucleotides are generated enzymatically. Heteroduplex (containing mismatched bases) oligonucleotides may be created by denaturing and reannealing the population of duplexes. The reannealed oligonucleotide duplexes are contacted with a mismatch recognition protein that interacts with (e.g., binds and/or cleaves) the duplexes containing a base pair mismatch. The oligonucleotide heteroduplexes that have interacted with such a protein are separated, simultaneously with contacting or sequentially in a separate step, from homoduplexes. These methods are also used in another embodiment to remove heteroduplex oligonucleotides (e.g., DNA) that are formed directly from chemical nucleic acid synthesis. In other embodiments of the present invention, kits and compositions useful for the methods are provided.

The invention provides recombinant human collagen and a method for preparing the recombinant human collagen. The method comprises the following steps: 1)preparing plasmid: performing artificial whole gene synthesis of a sequence of SEQ ID No:3 in a sequence table, then performing Xho I and EcoR I double enzyme digestion on a pPIC9K carrier and human collagen in SEQ ID No:3 through artificial whole gene synthesis, then extracting plasmid; 2) converting: mixing the prepared plasmid and pichia yeast for conversion to obtained converted bacterium colony; 3) screening of multicopy additive recombinants; 4) obtaining a broth through fermentation; and 5) purifying to obtain the product. The invention also provides a gene sequence for coding the recombinant human collagen. The recombinant human collagen has good biological compatibility, and the purity can reach more than 95%.

The invention belongs to the field of gene engineering, and discloses a method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation. Recombinant escherichia coli is constructed by the following method: according to a published proline-4-hydroxylase gene and tryptophan promoter sequence, first optimally designing a tryptophan tandem promoter and a proline-4-hydroxylase structure gene, then after the total gene synthesis of the tryptophan tandem gene promoter and the proline-4-hydroxylase structure gene, connecting the promoter and the structure gene to pAMP plasmid to construct recombinant plasmid pAMP-P2trp-Hyp for overexpression of proline-4-hydroxylase. The invention also discloses the application of the escherichia coli in the production of the 4-hydroxyproline, the shake flask fermentation results show that the 4-hydroxyproline prepared by the recombinant escherichia coli has a yield reached 0.31g / L, and suggest that the recombinant escherichia coli has good industrial development prospects.

Devices and methods integrate nanopore and microfluidic technologies for recording molecular characteristics of individual molecules such as, for example, biomolecules. Devices comprise a first substrate comprising a microchannel, a second substrate comprising a microchannel, the second substrate positioned below the first substrate, and a membrane having a thickness of about 0.3 nm to about 1 nm and comprising at least one nanopore, the membrane positioned between the first substrate and the second substrate, wherein a single nanopore of the membrane is constructed and arranged for electrical and fluid communication between the microchannel of the first substrate and the microchannel of the second substrate. To mitigate the effect of errors that occur during de novo DNA synthesis, longer DNA molecules are typically synthesized from shorter oligonucleotides by polymerase construction and amplification (PCA), or by other methods.

The invention provides aminopeptidase for catalytic synthesis of carnosine, and a preparation method and application thereof. The aminopeptidase has the amino acid sequence shown by SEQ ID NO:2. For the preparation of biological enzymes, firstly, a gene engineering strain of the biological enzymes is built; the biological enzyme gene fragment obtained through full gene synthesis is subjected to linearized plasmid recombination by using restriction enzymes SalI. The built gene-engineered strain is suitable for high-density culture; the exoenzyme is biologically synthesized; solid-liquid separation is performed; ultrafiltration concentration is performed to obtain enzyme liquid; histidine and beta-alanine can be catalyzed in normal-temperature and normal-pressure water solution to efficiently synthesize carnosine; the reaction environment is friendly; the production cost is low; the conversion time is short; the process operation is simple; the reaction system impurity content is low; the post treatment is easy; the wide prospects of large-scale industrial application are realized.

The invention relates to a method for producing R-3-aminobutanol. The method comprises the following steps: artificially designing a sequence shown in SEQID NO: 1, carrying out full gene synthesis, cloning the synthesized gene fragments into an expression vector pET22b to prepare a recombinant expression vector, transferring the recombinant expression vector into Escherichia coli to prepare a genetically engineered bacterium of a recombinant D-amino acid dehydrogenase, culturing the genetically engineered bacterium to prepare the recombinant D-amino acid dehydrogenase, sequentially adding a substrate of which the final concentration is 10-300mmol/L, a 2-15wt% cosolvent, a cofactor of which the final concentration is 0.1-1mmol/L, an amino donor of which the final concentration is 0.02-1mmol/L and the 0.01-1wt% D-amino acid dehydrogenase into a reaction solution to constitute a reaction system, reacting, after the reaction is completed, and extracting R-3-aminobutanol in the reaction solution. The method disclosed by the invention has the advantages that the cost is low, the conversion rate is greater than 95%, the product yield is greater than 85%, no by-product is generated and method is more suitable for industrial applications.

The invention provides a system and method for synthesizing polynucleotides by solid phase assembly oligonucleotide precursors, in accordance with the method, a polynucleotide is partitioned into an ordered set of subunits, wherein each subunit is assembled in a single reaction from a subset of oligonucleotide precursors that uniquely anneal together to produce the subunit. The subunits are then assembled to form the desired polynucleotide. An important feature of the invention is the selection of subunits that are free of undesired sequence elements, such as palindromes, repetitive sequences, and the like, which would result in more than one subunit product alter ligating a pool of oligonucleotide precursors.

The invention provides a biological preparation method of R-3-aminobutanol. The method includes: conducting whole-gene synthesis according to an artificially designed sequence shown as SEQ ID NO:1, and performing cloning into pET24a so as to obtain a recombinant expression vector; transferring the recombinant expression vector into E.coli to obtain a recombinant D-transaminase genetic engineering bacterium; culturing the bacterium to prepare recombinant D-transaminase; adding 3-carbonyl butanol with a final concentration of 10-300mmol/L, dimethyl sulfoxide or acetonitrile with a mass percentage concentration of 2-15%, pyridoxal phosphate with a final concentration of 0.1-1mmol/L, isopropylamine or D-alanine with a final concentration of 0.02-1mol/L, and recombinant D-transaminase with a mass percentage concentration of 0.01-1% are added into the reaction solution to compose a reaction system for reaction; and at the end of the reaction, extracting R-3-aminobutanol from the reaction solution. With a low cost, the method provided by the invention can achieve a conversion rate of more than 97% and a product yield of greater than 87%, and does not generate by-product, thus being more suitable for industrial application.

The invention belongs to the technical field of antigen preparation processes, and particularly relates to a coronavirus fusion protein, and a preparation method and application thereof. According tothe coronavirus fusion protein provided by the invention, the nucleotide sequence of the RBD structural domain of a 2019-nCoV S protein and the nucleotide sequence of a 2019-nCoV N protein are subjected to complete gene synthesis and then are constructed into an insect cell expression vector to prepare a recombinant plasmid containing the RBD structural domain sequence of the 2019-nCoV N protein and the RBD structural domain sequence of the 2019-nCoV S protein, escherichia coli is transformed by the recombinant plasmid to obtain a recombinant plasmid containing the gene sequence of the fusionprotein, and host cells are transfected by the recombinant plasmid to complete then fusion expression of the RBD structural domains of the 2019-nCoV N protein and the 2019-nCoV S protein. The expression process of the fusion protein is simple, meanwhile, and when the fusion protein is applied to a 2019-nCoV antibody detection kit, the sensitivity of the detection kit is remarkably improved, the specificity is also improved, the detection rate is efficiently increased, and the overall use performance is more optimized.

The invention belongs to the technical field of bioengineering, and particularly relates to a production method for a yeast recombinant collagen, which includes construction of recombinant pichia pastoris engineered strain and production of constitutively and secretively expressed recombinant collagen without methanol induction. The invention first utilizes conventional codons of pichia pastoris at the genetic level to optimize a collagen gene sequence and performs artificial total gene synthesis, the optimized collagen gene sequence is then integrated into yeast chromosome, and thereby a constitutively and secretively expressed recombinant pichia pastoris engineered strain is constructed. The fermentation process is easy to operate, yield is high, and the separation and purification of the product are simple; methanol induction is not needed, the fire-proof and explosion-proof indexes of production equipment and factory buildings are low, and the production environment is environmentally friendly and pollution-free; and no toxic and harmful substances are use, so the product is safer. A specific affinity purification label is carried, the product with higher purity can be easily obtained, and the product can be applied to biomedical materials.

The invention discloses a biological preparation method for R-3-aminopiperidine. The biological preparation method comprises the following steps: carrying out gene synthesis according to a sequence as shown in SEQ ID NO:1, cloning the gene into an expression vector pET22a to prepare a recombinant expression vector, and transferring the vector into escherichia coli to prepare a genetically engineered bacterium of recombinant D-transaminase; culturing the bacterium to prepare recombinant D-transaminase genetically engineered bacterium; adding 3-ketopiperidine with final concentration of 10-300mmol/L, 2-15wt% dimethyl sulfoxide or acetonitrile, phosphopyridoxal with final concentration of 0.1-1mmol/L, isopropylamine or D-alanine with final concentration of 0.02-1mol/L and 0.2-0.4wt% recombinant D-transaminase to form a reaction system to carry out reaction; and extracting the R-3-aminopiperidine in the reaction liquor after the reaction is ended. According to the biological preparation method, the cost is low, the conversion ratio is over 97%, the product yield is greater than 85% and byproducts are not produced, so that the biological preparation method is more suitable for industrial application.

The present invention relates to a nucleic acid splicing method based on bidirectional isothermal extension. The method is as follows: an extension system is set up, the extension system comprises a starting double strand, a group of oligonucleotides which are mutually different and orderly spliced, mixed enzyme with connection, polymerization and restriction endonuclease activity, and a reaction buffer matching with the mixed enzyme; in the cooperation of multiple kinds of enzymes, and starting from the starting double strand, the oligonucleotides can be isothermally spliced for synthesis of a target DNA long chain. The nucleic acid splicing method has the characteristics of high success rate, simple design and simple operation, high automation, and the like, so that the nucleic acid splicing method has a potential low cost advantage, and has potential values of application in promotion of gene synthesis, and development of biological engineering, biomedical and bioinformatics fields.

The invention provides a helicobacter pylori multiepitope vaccine. The active ingredient of the helicobacter pylori multiepitope vaccine is a piece of polypeptide; and the helicobacter pylori multiepitope vaccine mainly comprises a multi-copy body of Th and B cell antigen epitopes of helicobacter pylori urease A and B bi-subunit and a mucosal immune adjuvant of a cholera toxin B subunit. The preparation method mainly comprises the following steps of: synthesizing an artificial gene by using a gene synthesis technology, wherein the artificial gene comprises a gene sequence of the multi-copy body of the Th and B cell antigen epitopes of the helicobacter pylori urease A and B bi-subunit; coupling the artificial gene with the gene sequence of the cholera toxin B subunit to form a fusion gene;and expressing the fusion gene by using an escherichia coli prokaryotic expression system, and performing protein purification to obtain the helicobacter pylori multiepitope vaccine. The helicobacterpylori multiepitope vaccine can induce an organism to generate T cell immune response and high-titred specific antibody humoral immune response aiming at the urease A and B bi-subunit, and can be used for preventing and treating helicobacter pylori infection related diseases.

The invention discloses a phytoene dehydrogenase mutant gene and application thereof in carotenoid synthesis. The mutant gene is obtained by performing multiple rounds of mutation on a phytoene dehydrogenase coding gene by means of iterative orthogenesis and through a high-throughput screening method based on bacterial colony color change. When the phytoene dehydrogenase mutant gene is used to synthesize lycopene, A178S mutant is obtained in the first round of orthogenesis, and lycopene yield is increased by 27.3%; A178S/N280T mutant and A178S/M234I mutant are obtained by using the A178S mutant as a base for orthogenesis, and lycopene yield is further increased by 51.4% and 18.3% while the lycopene yield is not further increased by combining A178S, N280T and M234I; A178S/N280T/E239P mutantis obtained by performing orthogenesis on the A178S/N280T mutant, and A178S/N280T/E239P mutant is finally obtained through three rounds of orthogenesis and screening.

The invention discloses bacillus circulans chitoanase as well as a preparation method and application thereof. According to the preference of pichia pastoris codons, a chitoanase coding gene sequencein bacillus circulans is obtained by utilizing a whole gene synthesis method; an optimized nucleic acid sequence is shown as SEQ ID NO. 2. Furthermore, efficient secreting expression is carried out onan optimized chitoanase coding gene by utilizing a pichia pastoris expression system to obtain the bacillus circulans chitoanase; an amino acid sequence of the bacillus circulans chitoanase is shownas SEQ ID NO. 1. The chitoanase disclosed by the invention has relatively high hydrolysis activity on a chitosan substrate and a crude enzyme solution produced by shake-flask fermentation has a hydrolysis capability of degrading 5g of chitosan by utilizing 1mL of the crude enzyme solution (with the protein content of about 0.77mg); when the chitosan with the same amount is degraded, about 150mg ofneutral proteinase obtained from bacillus subtilis is needed; the efficiency is theoretically improved by about 200 times; the bacillus circulans chitoanase has a very good industrial application prospect.

The invention provides a mycoplasma pneumonia mosaic antigen amino acid sequence containing an amino acid sequence as shown in SEQ ID NO:1 as well as a full-gene synthesized mycoplasma pneumonia mosaic antigen full-gene sequence containing an amino acid sequence as shown in SEQ ID NO:2. The invention also provides a method for constructing the two gene sequences. The invention also provides a preparation method of the mycoplasma pneumonia mosaic antigen containing full-gene synthesis, a mycoplasma pneumonia detection kit and a preparation method thereof. Mp recombinant mosaic antigen is selected as a mark material and is applied to a gold immunochromatography system, and the detection system is directly marked and captured, so the sensitivity is greatly improved, the specificity of the antigen is high, the antigen is easy in cultivation and purification, and cost is reduced; and a new method for detecting mycoplasma pneumoniae IgG rapidly and accurately is provided for clinical use, and a good market prospect is achieved.

The invention belongs to the field of bio-genetic engineering and provides a DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and an expression method thereof. The method comprises the following steps: directly obtaining a gene of egg white lysozyme by gene synthesis means; carrying out operations such as digestion and connection, and then introducing the gene into an eukaryotic expression vector pPIC9K; transforming into pichia pastoris GS115, screening high-copy recons, and then realizing efficient expression of the recombinant egg white lysozyme in vitro through methanol induction. The method is not limited by a raw material source; the copy number of the lysozyme can be increased; the expression amount and the activity of the enzyme are improved; the later separation and purification technologies are simplified; large-scale industrialized production of the egg white lysozyme is realized; and the DNA segment has the characteristics of being stable, pure, and high in biological activity, and can be widely applied to many fields such as pharmacy and feeds.

Belonging to the field of genetic engineering, the invention discloses a recombinant pichia pastoris for enhanced expression of xylanase from Streptomyces sp.FA1. According to the invention, a target gene with a nucleotide sequence shown as SEQ ID NO:1 is acquired by amplification, the target gene is connected to the expression vector pGAPZ alpha A-PARS, and then the obtained recombinant vector is introduced into pichia pastoris KM71/pPIC9K-XynA so as to obtain the recombinant pichia pastoris. The invention also discloses a production method for the recombinant engineered pichia pastoris. The method includes the steps of: (1) synthesizing an autonomously replicating sequence PARS by whole gene synthesis technology; (2) constructing the expression vector pGAPZ alpha A-PARS; (3) constructing the recombinant expression vector pGAPZ alpha A-PARS-XynA; (4) converting a pichia pastoris host and screening a positive transformant; (5) conducting flask shaking induction culture; and (6) inducing recombinant engineered pichia pastoris in a fermentation tank to produce xylanase.

The invention provides a process for synthesizing genes and other long double stranded polynucleotides by assembling very short oligonucleotides into partly double stranded polynucleotides, and then connecting these partly double stranded polynucleotide subassemblies with linkers comprised of very short oligonucleotides. In one embodiment, the correct order of the polynucleotide subassemblies is coded in overhangs present at each end of the partly double stranded polynucleotide subassemblies. Linkers having a sequence complimentary to the combined overhangs connect adjacent subassemblies, which are then ligated together. In one preferred embodiment the oligos are six bases long, for which there are only 4096 different possible sequence permutations. A complete library of oligos of this size and scale can be cost-effectively synthesized and quality controlled, avoiding the typical errors and yield issues associated with phosphoramidite synthesis of longer oligos. Furthermore, the limited oligo library size supports development of a laboratory-scale gene synthesis machine.

The invention provides nicotinamide nucleoside kinase whole yeast cells and a process for biocatalytically synthesizing NMN by the nicotinamide nucleoside kinase whole yeast cells, and relates to the technical field of enzyme engineering. The process for biocatalytically synthesizing the NMN comprise the following steps: S1, constructing a nicotinamide nucleoside kinase recombinant display expression vector of saccharomyces cerevisiae: designing an encoding DNA sequence of NrK1 according to an amino acid sequence (SEQ ID NO: 1) of an NrK1 mutant, adopting preferred codons of the saccharomyces cerevisiae as codons, and synthesizing encoding DNA (SEQ ID NO: 2) of NrK1 in a whole-gene synthesis manner; and according to the nicotinamide nucleoside kinase whole yeast cell and the NMN biocatalytic synthesis process thereof, the nicotinamide nucleoside kinase whole yeast cell and the NMN biocatalytic synthesis process thereof can anchor and express high-activity enzymes capable of catalyzing NMN synthesis from different biological sources on the cell wall surface of saccharomyces cerevisiae through a genetic engineering technology; and the whole-yeast engineering cell enzyme capable of efficiently synthesizing the NMN is formed.

The invention discloses a gene synthesis of Alpha-interferon of wild boars and vector construction thereof as well as a production method of a product. A number of problems of low expression capacity, products expressed in a fusion state, products with purification tags or no fermentation technology with high density exist in the domestic recombinant strains. The invention includes a method that a codon and codon pairs, preferred by Escherichia coli, are used for synthesizing an Alpha-interferon gene of the wild boar, establishing a high-efficiency expression vector and transforming a high-efficiency expression strain as well as methods of high-density fermentation of engineering bacteria, separation and purification of inclusion bodies, the modification, the renaturation and the purification of target protein and the determination of biological activity of the expressed product. The gene synthesis, the vector construction and the production method pertain to the technical field of the production of polypeptide drugs by genetic engineering in biopharmaceuticals.

The invention relates to a pseudo-virus standard substance for nucleic acid diagnosis of novel coronavirus 2019-nCov and application thereof. The pseudo-virus standard substance is prepared through the steps of gene synthesis, plasmid extraction, pseudo-virus packaging and the like. Wherein the synthesized gene comprises a novel coronavirus 2019-nCov gene RdRp, a Gene E and a Gene N; the 2019-nCovpseudo-virus standard substance is provided for the first time, is different from RNA synthesized in vitro and clinical positive living virus in the past, perfectly overcomes the defects of RNA and clinical positive living virus, is really suitable for performance evaluation of test kit, comprises limit of detection, specificity, repeatability and the like, and is further applied to clinical diagnosis of 2019-nCov; the pseudo-virus standard substance has the advantages of no pathogenicity, reproducibility, reliable quality control method and stable batch-to-batch, can be stably prepared and supplied for a long time, requires the biological safety level of P2 in a laboratory, and meets the safety requirements of many units.