420 results about "Codon optimization" patented technology

The invention is related to polynucleotide-based cytomegalovirus vaccines. In particular, the invention is plasmids operably encoding HCMV antigens, in which the naturally-occurring coding regions for the HCMV antigens have been modified for improved translation in human or other mammalian cells through codon optimization. HCMV antigens which are useful in the invention include, but are not limited to pp65, glycoprotein B (gB), IE1, and fragments, variants or derivatives of either of these antigens. In certain embodiments, sequences have been deleted, e.g., the Arg435-Lys438 putative kinase in pp65 and the membrane anchor and endocellular domains in gB. The invention is further directed to methods to induce an immune response to HCMV in a mammal, for example, a human, comprising delivering a plasmid encoding a codon-optimized HCMV antigen as described above. The invention is also directed to pharmaceutical compositions comprising plasmids encoding a codon-optimized HCMV antigen as described above, and further comprising adjuvants, excipients, or immune modulators.

The present invention relates to the construction and engineering of cells, more particularly microorganisms for producing PUFAs with four or more double bonds from non-fatty acid substrates through heterologous expression of an oxygen requiring pathway. The invention especially involves improvement of the PUFA content in the host organism through fermentation optimization, e.g. decreasing the temperature and / or designing an optimal medium, or through improving the flux towards fatty acids by metabolic engineering, e.g. through over-expression of fatty acid synthases, over-expression of other enzymes involved in biosynthesis of the precursors for PUFAs, or codon optimization of the heterologous genes, or expression of heterologous enzymes involved in the biosynthesis of the precursor for PUFAs.

A heterologous expression in a host Pseudomonas bacteria of an optimized polynucleotide sequence encoding a protein.

The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

The invention is related to polynucleotide-based cytomegalovirus vaccines. In particular, the invention is plasmids operably encoding HCMV antigens, in which the naturally-occurring coding regions for the HCMV antigens have been modified for improved translation in human or other mammalian cells through codon optimization. HCMV antigens which are useful in the invention include, but are not limited to pp65, glycoprotein B (gB), IE1, and fragments, variants or derivatives of either of these antigens. In certain embodiments, sequences have been deleted, e.g., the Arg435-Lys438 putative kinase in pp65 and the membrane anchor and endocellular domains in gB. The invention is further directed to methods to induce an immune response to HCMV in a mammal, for example, a human, comprising delivering a plasmid encoding a codon-optimized HCMV antigen as described above. The invention is also directed to pharmaceutical compositions comprising plasmids encoding a codon-optimized HCMV antigen as described above, and further comprising adjuvants, excipients, or immune modulators.

The present invention relates to codon optimization utilizing DNA shuffling. A method of producing gene sequences optimized for a desired functional property is described involving synthesizing a library of parental codon variant genes encoding some or all codon choices at some or all amino acid positions of a gene, reassorting the variant codons among the parental codon variant genes using DNA shuffling thereby forming progeny codon variant genes, expressing the progeny codon variant genes in a host; and screening or selecting for progeny codon variant genes encoding a desired functional property.

The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

The invention relates to novel variants of the enzymatic peptide pullulanase, the gene sequences encoding said novel peptides, expression vectors comprising those gene sequences as well as organisms expressing the novel pullulanase variants. The novel pullulanase variants of the present invention were made empirically by the use of codon-optimization procedures, selective truncation of “wild-type” molecules and through the replacement of selected amino acid residues. Furthermore, the invention relates to the use of these novel pullulanase peptides in the textile, fermentation, food and other industries.

The invention provides methods for producing a protein in a cell free protein synthesis system such that the protein does not contain an asparagine (Asn or N) residue at serine (Ser or S) positions. Also provided are compositions and nucleic acid templates for use in the methods described herein.

The invention discloses a bacterial strain for producing farnesene and an application of the bacterial strain, belonging to the field of synthetic biology. The bacterial strain for producing the farnesene contains related genes for synthesizing the farnesene through a mevalonic acid pathway and codon optimization; and the sequence of a farnesene synthetic gene afs optimized by codons is as shown by SEQ ID NO.1. The bacterial strain can be used for producing the farnesene, and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression to obtain the farnesene. The gene for synthesizing the farnesene is subjected to codon optimization or the farnesene is synthesized by using the mevalonic acid pathway to ensure that each protein is closer to a ratio of AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi:IspA:AFS=1:10:2:5:5:2:5:2:2, so that the production of the farnesene can be further promoted. By adopting the bacterial strain, the output of the farnesene is greatly improved to be more than 1g / L.

The invention discloses a glucose oxidase mutant gene, expression and application thereof. In the invention, 272 basic groups are changed through codon optimization and GC content change and the content of GC is reduced to 48.44% from 55.54% so as to obtain the glucose oxidase mutant gene, wherein the basic group is represented by SEQ ID NO.2. The glucose oxidase mutant gene is transferred into pichia yeast to express; the experimental result shows that the secretory expression level of the glucose oxidase mutant gene in the pichia yeast is significantly improved by comparing with the same before mutation; compared with partial research at home and abroad, the final secretory expression of the glucose oxidase mutant gene achieves high expression level, thereby building the foundation for further expansion of industrial production. The determination of enzymatic properties of the glucose oxidase mutant gene shows that the recombinant glucose oxidase protein expressed by the glucose oxidase mutant gene has good thermal stability and high enzyme activity.

The invention belongs to the biotechnical field, and concretely relates to a recombinant humanized anti-PD-1 and c-MET bispecific antibody, and a preparation method and an application thereof. Humanized antibody database comparison, computer molecular structure simulation, molecular docking simulation and sequence analysis are carried out, and recombinant humanized anti-PD-1 and c-MET bispecific antibody molecule designing comprising affinity maturation, structure optimization, humanization and codon optimization is carried out, and an expression plasmid of the recombinant humanized anti-PD-1 and c-MET bispecific antibody is constructed on the basis of an optimized signal peptide sequence and a plasmid expression vector replicon. The bispecific antibody has the characteristics of low immunogenicity, simple and safe preparation process, high yield, high purity and high activity, and can be used to further prepare new specific drugs for treating non-small cell lung cancer.

The invention provides a DNA sequence encoding African swine fever virus antigen, a composition of the antigen encoded thereby and use thereof in immunological detection. and also provides an application of the composition of the antigen encoded by the above DNA sequence, and the above antigen composition for an immunological detection method for detecting the African swine fever virus. A codon optimization method capable of eliminating rare codons is used for optimizing the DNA sequences of CP204L, PK205R, PB602L, CP530R, E183L, and PB646L in the major membrane proteins and capsid proteins encoding the African swine fever virus, the above-described codon-optimized nucleotide sequence can efficiently express the above-mentioned main membrane proteins and capsid proteins in a suitable expression system, and an established immunological detection method can detect Africa swine fever virus quickly and simply on a large scale.

The invention provides a recombinant DNA sequence, hansenula polymorpha, a preparation method for hepatitis B surface antigen, and a hepatitis B vaccine. The recombinant DNA sequence is obtained by codon optimization of coding genes of the hepatitis B virus surface antigen according to codon usage frequency of the hansenula polymorpha. The invention also provides the hansenula polymorpha comprising the recombinant DNA sequence, a method for preparing adr sub-type hepatitis B surface antigen by using the recombinant DNA sequence, and the hepatitis B vaccine. The adr sub-type hepatitis B surface antigen has high expression level of the recombinant DNA sequence. The recombinant hansenula polymorpha is fast in growth speed, has high HBsAg yield, can be fermented in high cell density by using a cheap chemically synthetic medium, has low fermentation contamination rate and is beneficial to large-scale production; and HBsAg adr vaccine provided by the invention has high trend of Th1 and Th2 type cellullar immunologic response.

The invention provides a recombined porcine interferon Gamma, a coding gene thereof, and an expressing, purifying and inclusion body refolding method, and belongs to the field of biological gene engineering. The recombined porcine interferon Gamma serves as a non-specific broad-spectrum anti-viral biological preparation, and has a wide medicinal prospect in the field of veterinary medicines; but as most of gene engineering veterinary medicines, the porcine interferon Gamma has the problems of underproduction, high prices, non-uniform quality and the like. According to the recombined porcine interferon Gamma, the coding gene thereof and the expressing method, an escherichia coli expression system is used for performing heterologous expression for the recombined porcine interferon Gamma subjected to codon optimization. Besides, aiming at the problem that the porcine interferon Gamma in a prokaryotic expression system is usually expressed in a manner of an inclusion body, the invention further provides the purifying and refolding method for the inclusion body of the recombined porcine interferon Gamma, so that the prepared recombined porcine interferon Gamma has high activity and meets a standard for industrialized production.

The invention relates to a recombinant human III-type collagen and a prokaryotic expression method thereof. The amino acid sequence of the humanized III-type collagen is shown as SEQ ID NO.1, and thenucleotide sequence of an encoding gene is shown as SEQ ID NO.2. The method comprises the following steps: carrying out codon optimization and design to obtain the gene sequences; inserting the gene sequences into a position between enzyme cutting sites NdeI and XhoI of an expression vector PET30a(+) to construct a recombinant expression vector pET30-1230; transforming Escherichia coli BL211(DE3)competent cells; selecting positive clones; and performing culture induction for efficient expression.

The invention relates to the field of enzyme engineering, and in particular discloses acid-resistant zearalenone detoxifying enzyme and an encoding gene thereof, and application of the enzyme and the gene. The amino acid sequence of the zearalenone detoxifying enzyme is optimized, 8 amino acid residues (with 8 amino acid difference from zlhy-6 detoxifying enzyme) of the detoxifying enzyme are changed, the optimal reaction pH of recombinant expression zearalenone detoxifying enzyme is reduced from 8.5 to 6.5 and is very identical with the pH of pig and chicken intestinal canals, and the enzyme activity of the detoxifying enzyme still maintains 40 percent or more when the pH is reduced to 5.0, so the detoxifying efficiency of the zearalenone detoxifying enzyme serving as a feed additive to actual application is greatly improved. The zearalenone detoxifying enzyme gene is subjected to codon optimization, and the copy number of the gene in a recombinant yeast genome is increased to be 4, so the expression quantity of the zearalenone detoxifying enzyme is greatly increased.

The invention relates to the technical field of biological medicine, in particular to nucleic acid for encoding SARSCoV2 virus spike protein and application of the nucleic acid. Through codon optimization, the invention provides the Spike high-expression novel coronavirus mRNA vaccine based on the TC83Venezuelan Equine Encephalosis Virus (VEEV) in the alpha virus family, and the vaccine can detecta spike antibody after 14 days of inoculation in an animal experiment.

The invention discloses recombinant escherichia coli for producing tyrosol and a construction method and application thereof and belongs to the technical field of bioengineering. The escherichia coliheterologously expresses a saccharomyces cerevisiae pyruvate decarboxylase gene ARO10* after codon optimization. According to the recombinant escherichia coli, the ARO10* gene is integrated while a lacI locus, a trpE locus, a pabB locus, a pabA locus and a pykF locus of an escherichia coli genome are deleted, and a strain containing multiple copies of the ARO10* gene is obtained. On the basis of the recombinant strain, the ARO10* gene is randomly integrated at the multiple loci, and the ARO10* gene is inserted at a yccX locus so that the strain with high tyrosol yield can be obtained. No inducer nor antibiotic is required for fermentation by using this strain. After 48 hours of fermentation, the yield of tyrosol can reach 28 mM.

The invention relates to a construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris, in particular to cellulase protein gene codon optimization and construction of a cellulase protein and vitreoscilla hemoglobin protein (VHb)-co-expressed pichia pastoris system. According to the construction method, codon bias optimization is performed on the nucleotide sequence of EG II (GenBank Accession No.DQ178347.1) through Gene Designer (DNA2.0, Menlo Park, CA, USA) software, a pPIC9K-eg2 expression vector is constructed, and a recombined pichia pastoris strain is obtained by taking Pichiapastoris GS115 as a host through electrotransformation. In addition, the nucleotide sequence of the VHb (GenBank Accession No.M30794.1) is obtained from NCBI and artificially synthesized into a gene, then a pPICZalphaA-vgb expression vector is constructed, and the co-expressed pichia pastoris strain is obtained by taking the recombined pichia pastoris strain containing the EG II gene as a host through electrotransformation. Detection shows that the co-expressed strain is improved on the aspects of bacterial concentration growth and enzyme activity, wherein the OD600 value is increased by 7.2%, and the enzyme activity is improved by 2.2%.

The invention discloses a production process of fusion expression recombinant chicken interferon alpha. The process includes the steps of: S1, according to the preference of Escherichia coli codon, conducting codon optimization on a chicken interferon alpha gene sequence published in Genebank, and artificially synthesizing the chicken interferon alpha gene; S2, according to the codon optimized chicken interferon alpha gene, designing three specific primers; S3, constructing recombinant chicken interferon alpha plasmid containing ProS2 dissolution promoting label; S4, transforming and identifying the recombinant expression plasmid; S5, conducting inducible expression of recombinant chicken interferon alpha; S6, extracting an expression product and conducting protein renaturation purification: S61, inclusion body extraction and treatment; S62, inclusion body denaturation; S63, denaturation solution renaturation; and S64, nickel column affinity purification. By means of cell cytopathic inhibition, the invention detects that the interferon has the activity of inhibiting vesicular stomatitis virus proliferation, and the activity unit reaches 7.32*10<7>UI / mg.

The invention relates to a method for preparing alpha-aminobutyric acid by utilizing series connection amino acid dehydrogenase and codon optimization formate dehydrogenase recombinant escherichia coli. First, codon optimization is performed on a Candida boidinii formate dehydrogenase gene sequence according to the codon preference of the escherichia coli. After codon optimization on formate dehydrogenase, the expression quantity of formate dehydrogenase in the escherichia coli can be improved remarkably, the yield of alpha-aminobutyric acid is increased, and in the escherichia coli, co-expression formate dehydrogenase and L-amino acid dehydrogenase can promote circulation of cofactors in mycetome without needing the adding of any exogenous cofactors. In addition, the process of utilizing whole-cell transformation bulk chemical L-threonine to produce alpha-aminobutyric acid is simple and quick, and the cost is low. In a 5 L fermentation tank, the yield of alpha-aminobutyric acid obtained through the method can be 81.5 g / L, and an actually effective strategy is provided for industrial production of alpha-aminobutyric acid.

The invention provides a method for producing beta-amyrin with a saccharomyces cerevisiae engineering bacterium, and belongs to the field of biochemical engineering. According to the method, a 2,3-oxidized squalene monooxygenase gene is cloned from candida albicans, a glycyrrhiza glabra beta-amyrin synthase gene subjected to codon optimization is chemically synthesized, a gene expression cassette is constructed by utilizing a saccharomyces cerevisiae constitutive promoter, the gene expression cassette and a plasmid after double digestion jointly invert saccharomyces cerevisiae, and assembly of the gene expression cassette and the plasmid is achieved by a homologous recombination function of the saccharomyces cerevisiae, so that 2,3-oxidized squalene monooxygenase is reinforced, and beta-amyrin synthase is led in. Saccharomyces cerevisiae engineering bacterium metabolizable glucose directly synthesizes beta-amyrin, and synthesis of beta-amyrin is coupled with growth of the saccharomyces cerevisiae, so that artificial synthesis of the saccharomyces cerevisiae of a plant secondary metabolism product, namely beta-amyrin is achieved. The method requires no effect agent induction, is simple in technology, and can be used for fermenting and producing beta-amyrin.

The invention discloses a preparation method of an EV71 vaccine and the vaccine prepared by the preparation method, relating to the preparation method of taking a Pichia yeast as an expression system for co-expression of P1 and 3CD proteins of the EV71 after codon optimization by utilizing different promoters, and obtaining the viral like particle vaccine with immunogenicity, and also relating to the vaccine prepared by the preparation method.

The invention discloses a SARS-CoV-2 coronavirus vaccine and a preparation method thereof. An S gene of a SARS-CoV-2 coronavirus is subjected to codon optimization, truncated body and mutant sequences of the S gene are introduced into a secretory defective adenovirus vector, and packaging is conducted to obtain a corresponding recombinant adenovirus; and the recombinant adenovirus can express SARS-CoV-2 virus related protein in vivo, processing, folding, glycosylation and other modifications are completed, the native conformation of S protein is basically maintained, and the characteristics of high biological activity, long half-life period, lasting immunogenicity and the like are achieved. According to the vaccine and the method, by adopting the defective adenovirus vector carrying secretory peptide, a recombinant adenovirus vaccine can be secreted out of cells after being expressed in vivo, so that the humoral immunity is activated.

The invention provides an I-group 4-type fowl adenovirus DNA vaccine and application thereof. According to the technical scheme, based on experimental means, research finds and shows that fiber protrusions have good immunity prototypes, in this way, with 4-type fowl adenovirus fibrous protein C-terminal genes being antigen substances, codon optimization is carried out on the basis of a natural sequence, an eukaryotic expression vector pCAGGS is cloned, and the DNA vaccine pCAGoptiFAV4C is constructed. According to the researched fowl adenovirus NDA vaccine, a method of gene engineering fermentation is adopted for preparing antigens, and therefore the I-group 4-type fowl adenovirus DNA vaccine is low in cost, pure in antigen and safe to use. By the utilization of a serology method and an immunity virus attack method, the immunity effect of the vaccine is evaluated. The result shows that the vaccine can provide effective immune protection for fowl, and has good commercial development prospects. Compared with a traditional vaccine, the DNA vaccine achieves the safety of subunit vaccines and inactivated vaccines, and also has the features of simultaneous induction of humoral immunity and cellular immune response, wherein the features only belong to attenuated vaccines or recombinant vaccines.

The present invention provides a recombinant yeast system for expressing the glycoprotein E2 of classical swine fever virus (CSFV), in which the expression level of yE2 is improved by codon optimization and shortening coding region of E2 gene. The truncated E2 subunits are used as major active ingredient in anti-CSFV vaccines and useful diagnostic blocking ELISA kits for CSFV infection with easy manipulation and low cost.

The invention relates to the technical field of biology, in particular to a fusion protein of porcine pseudorabies virus and a preparation method, application and vaccine of the fusion protein. The fusion protein of porcine pseudorabies virus comprises a gB section and a gD section, wherein the gB section is expressed by the nucleotide sequence shown in SEQ ID NO.1; and the gD section is expressedby the nucleotide sequence shown in SEQ ID NO.2. The sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are the sequences obtained through contrast and analysis by selecting genes of classical strains and current epidemic strains as research objects, and codon optimization and modification are preformed on the sequences, so that the broad spectrum of fusion protein antigen is further improved and theantigen expression amount is increased. The invention further provides a preparation method and application of the fusion protein, and the vaccine for preparation.

The invention discloses a method of preparing tauroursodeoxycholic acid by biotransformation and application of the method. Biotransformation includes genetic codon optimization, engineered bacteria construction, engineered bacteria cultivation, substrate transformation and product preparation. Tauroursodeoxycholic acid is prepared by transforming a substrate through direct fermentation of engineered bacteria; the substrate is taurochenodeoxycholic acid. The substrate may reach 250 g / L in concentration; the reaction time is short; substrate transformation rate reaches 98% and above; the obtained product reaches 99% and above in purity; cyclic regeneration of NAD+ (nicotinamide adenine dinucleotide +) in the reaction system helps greatly reduce the usage of the coenzyme NAD+; the cost of enzymic catalytic reaction is reduced; industrial amplification is benefited. hydroxysteroid dehydrogenase and the regenerated coenzyme are connected via a flexible polypeptide sequence to form a protein fusion polymer; binding distances to the substrate and coenzyme are shorter; transformation progress is more facilitated; the number of fermenting times in industrial production is decreased; the process is simplified; time cost and material cost are saved.