30 results about "Sialyltransferase" patented technology

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 3-sialyltransferase mutant and application thereof. The a-2, 3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K and A315H are obtained by performing site-specific mutagenesis on six amino acids in an a-2, 3-sialyltransferase amino acid sequence coded by a gene derived from Campylobacter jejuni (OH4384), the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3'-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the P198A, the G173D, the D174Q, the F196K and the A315H, the optimal recombinant microbial strain 3 '-SL-09 (V175N / P198A / D174Q) is obtained through combination of multiple mutation sites, the yield can reach 5.91 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 3'-SL.

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 6-sialyltransferase mutant and application thereof. The a-2, 6-sialyltransferase mutants M133L, E140D, D228H, N289D and G322Q are obtained by carrying out site-specific mutagenesis on five amino acids in an a-2, 6-sialyltransferase amino acid sequence coded by a gene derived from Photobacterium sp.JT-ISH-224, and compared with wild type alpha-2, 6-sialyltransferase, the yield of the single-point mutant 6 '-SL of the E140D and the yield of the single-point mutant 6'-SL of the G322Q are increased by 87.80% and 78.01% respectively. The yield of the optimal recombinant microbial strain 6 '-SL-7 (E140D / G322Q) obtained by mutation site combination can reach 1.471 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 6'-SL.

This invention provides a method for constructing and applying MDCK cells stably integrated with disialotyltransferase, relating to the field of biotechnology. The construction method includes: infecting MDCK cells with lentiviral particles, followed by culture and screening to obtain MDCK cells stably expressing porcine ST3GAL1 and porcine ST6GAL1 genes; the lentiviral particles carry porcine ST3GAL1 and porcine ST6GAL1 genes; the nucleic acid sequence of the porcine ST3GAL1 gene is shown in SEQ ID NO.1; the nucleic acid sequence of the porcine ST6GAL1 gene is shown in SEQ ID NO.2. This construction method is simple and convenient, and the constructed MDCK cells can stably express porcine ST3GAL1 and porcine ST6GAL1 genes, effectively improving the culture efficiency of swine influenza virus, and thus can be used in the preparation of swine influenza virus.

This invention belongs to the field of microbial genetic engineering technology, specifically relating to an α-2,6-sialic acid transferase mutant and its application. This invention utilizes a mutant derived from *Lithocarpus lucida* (…). Photobacterium Five amino acids in the amino acid sequence of α-2,6-sialyl transferase encoded by the gene of sp. JT-ISH-224 were mutated at specific sites to obtain α-2,6-sialyl transferase mutants M133L, E140D, D228H, N289D, and G322Q. Among them, the E140D and G322Q single-site mutants of α-2,6-sialyl transferase showed increases in 6'-SL yield of 87.80% and 78.01%, respectively, compared with wild-type α-2,6-sialyl transferase. The optimal recombinant microbial strain 6'-SL-7 (E140D / G322Q) obtained by combining the mutation sites achieved a yield of 1.471 g / L, effectively solving the bottleneck problem of low catalytic efficiency of sialyl transferase and providing a high-performance enzyme element for the efficient and low-cost biomanufacturing of 6'-SL.

The invention provides a preparation method of an alpha-2, 3 sialic acid glycosyl transferase mediated sugar chain fixed-point coupling antibody drug conjugate, which comprises the following steps: S1, dissolving an antibody, adding UDP-Gal, Mn < 2 + > and beta-1, 4 galactosyl transferase for reaction, and purifying after reaction to obtain an antibody I with galactose at the tail end of a sugar chain; s2, dissolving the antibody I, adding an azide-modified CMP sialic acid derivative, alpha-2, 3 sialic acid glycosyltransferase and alkaline phosphatase for reaction, and purifying after reaction to obtain an antibody II of which the tail end of a carbohydrate chain is connected with the sialic acid derivative; s3, dissolving toxin molecules in DMSO, adding the dissolved toxin molecules into the antibody II for reaction, and performing ultrafiltration after the reaction to obtain ADC molecules; or the ADC molecule is directly prepared by adopting a one-pot enzyme method. The screened ST3Gal3 is used for preparing the carbohydrate chain fixed-point coupling ADC, the DAR value is controllable, batch-to-batch stability is achieved, uniformity is high, repeatability is high, quality control and CMC are better, and then the drug effect of the ADC can be better improved.

The invention provides application of protein in construction of engineering bacteria for producing 6 '-sialic acid lactose, and relates to the technical field of microbial genetic engineering. According to the invention, new alpha2, 6-sialyltransferase with the potential of producing 6 '-sialyllactose is obtained through screening, and genes for coding precursor CMP-Neu5Ac synthetic pathway enzyme and alpha2, 6-sialyltransferase are introduced into escherichia coli to construct a new biosynthesis pathway of 6'-sialyllactose so as to construct engineering bacteria for efficiently producing 6 '-sialyllactose. Besides, a precursor CMP-Neu5Ac synthetic pathway enzyme and alpha2, 6-sialyltransferase are integrated and expressed in the engineering bacteria, so that the instability problem caused by plasmids is avoided, meanwhile, the dependence on antibiotics in the production process is reduced, and the environmental risk in the production process is reduced.

The present invention provides recombinant Corynebacterium glutamicum genetically engineered to introduce the N-acetylmannosamine (ManNAc) synthesis pathway, and genetically engineered to express an exogenous N-acetylneuraminic acid synthetase, an exogenous CMP-N-acetylneuraminic acid synthetase, an exogenous lactose transporter, and an exogenous sialyltransferase. The invention also provides a method for producing sialic acid lactose by using the recombinant corynebacterium glutamicum.

The invention discloses recombinant lactobacillus casei for expressing sialyltransferase ST6GALNAC1, and belongs to the technical field of genetic engineering and microorganisms. The recombinant bacterium is obtained by cloning an ST6GALNAC1 gene to a pPG612 vector and electrically transforming the ST6GALNAC1 gene into lactobacillus casei ATCC393. Experiments show that the engineering bacterium can significantly enhance glycosylation modification of intestinal mucoprotein MUC2, improve expression of tight junction proteins (ZO-1, Occludin and Claudin-1), inhibit activation of a p38 / MAPK inflammation pathway and regulate an intestinal flora structure, so that intestinal injury and systemic inflammatory response caused by salmonella are effectively relieved. The invention provides a strain basis and technical support for developing a novel oral microecological preparation.

Immunogenic compositions containing a lipo-oligosaccharide sialyltransferase (LST) protein or antigenic fragment thereof and an adjuvant are provided. Further immunogenic compositions contain a lipid nanoparticle and a nucleic acid encoding LST protein or an antigenic fragment thereof enclosed within the lipid nanoparticle. Vaccine compositions and methods of protecting or treating a subject from a bacterial infection are also provided.

The present invention discloses a humanized animal model for human influenza virus infection and a preparation method. The present invention provides a method for constructing a mouse model for human influenza virus infection, comprising the following steps: introducing the endothelial cell-specific promoter K18, the human sialyltransferase ST6GAL1 encoding gene, and the luciferase encoding gene into wild-type mice; subsequently breeding to obtain the F2 generation, which is a mouse model for human influenza virus infection; and each generation is subjected to genotyping and in vivo imaging identification. The present invention inserts the human ST6GAL1 gene into the mouse gene sequence, overexpresses the human sialyltransferase ST6GAL1 that synthesizes the human α2,6-linked sialic acid receptor, and constructs a mouse model for human influenza virus infection. The model can be directly infected with human influenza virus without the need for an in vivo adaptation process in mice, providing a humanized mouse model that conforms to the clinical characteristics of influenza for in-depth elucidation of the pathogenesis of influenza, verification of new drug targets, and development of new drugs and vaccine evaluation.

The present disclosure relates to the production of sialylated Human Milk Oligosaccharides (HMOs), in particular to the production of sialyl-lacto-N-tetraose a (LST-a), from precursor oligosaccharides and the genetic engineering of suitable cells for use in said production, as well as to methods for producing said sialylated HMOs.

The invention relates to a preparation method and application of a sialylated glycan derivative, which comprises the following steps of: uniformly mixing chemically modified sialic acid monosaccharide with a receptor substrate, cytidine triphosphate (CTP), MgCl2, a CMP-sialic acid synthetase preferable mutant and sialyltransferase, transferring a sialic acid derivative to a 3-site or 6-site hydroxyl group on non-reducing end galactose of glycan by a one-pot enzymatic method, and carrying out reaction to obtain the sialylated glycan derivative. And collecting a reaction product to obtain the sialylated glycan derivative. The method is simple and can be used for editing glycans on the surfaces of the immune cells so as to enhance the specificity and cytotoxicity of the immune cells to tumor cells.

The invention belongs to the technical field of biology, and particularly relates to a method for detecting and enriching glycosylated RNA (Ribonucleic Acid) based on in-vitro enzyme chemical labeling of alpha-2, 3, alpha-2, 6 type sialyltransferase. According to the application, sialidase is firstly used for digesting sialic acid at the tail end of a naturally generated glycoRNA sugar chain, and sialidase digestion is not needed for an RNA sample which does not have sialic acid at the tail end of the glycoRNA sugar chain but has potential sialic acid modification sites; sialic acid with a labeled group is transferred to a potential sialic acid modification site at the tail end of a glycoRNA carbohydrate chain by using alpha-2, 3, alpha-2, 6 type sialyltransferase, such as PmST1, PmST3, Ph2, 3siat and Pd26ST, so that glycoRNA with a detectable group can be directly obtained, or the detectable group is further introduced through subsequent reaction, so that subsequent signal detection or enrichment can be carried out; the RNA part can be further sequenced, and the glycosyl part can be further subjected to mass spectrum identification. The application provides a very efficient, simple and convenient detection means for detection and enrichment of sialylated glycoRNA, has the characteristics of high specificity and high sensitivity, and has a wide application prospect.

Provided is a recombinant Corynebacterium glutamicum, which is genetically engineered to introduce an N-acetyl-mannosamine (ManNAc) synthetic pathway, and is genetically engineered to express a heterologous N-acetylneuraminic acid synthase, a heterologous CMP-N-acetylneuraminic acid synthase, a heterologous lactose transporter, and a heterologous sialyltransferase. Provided is a method for using the recombinant Corynebacterium glutamicum to produce sialyllactose.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an immobilized enzyme. Compared with free enzyme, the immobilized enzyme prepared from the selected specific resin can be reused and is high in stability; the immobilized enzyme is easily separated from a reaction system, so that a product purification process can be simplified, and the product yield and quality are improved. Moreover, by immobilizing a sugar nucleotide producing enzyme and four sialyltransferases, a new strategy suitable for large-scale synthesis of functional sugar chains such as sialylated sugar chains is developed, the stability of related enzymes is improved, long-term storage and reutilization of the enzymes are realized, and the production cost of the functional sugar chains is reduced.

A method for producing sialyllactose by co-fermentation of two strains is provided, belonging to the technical fields of synthetic biology and fermentation engineering. An engineered Escherichia coli strain was constructed for the co-expression of α-2,6-sialyltransferase (ST6) or α-2,3-sialyltransferase (ST3) and NeuA enzyme. A two-step fermentation process was adopted: in a first stage, the engineered strain was cultured; in a second stage, said strain was co-fermented with yeast of two strains, using lactose and sialic acid as substrates to achieve the synthesis of sialyllactose. Under a co-fermentation mode of the engineered strain and yeast, shake flask horizontal fermentation is performed for 30 hours, the concentration of 6'-sialyllactose being 35.58 g / L, and the conversion rate of sialic acid being 93.69%; shake flask horizontal fermentation is performed for 24 hours, an optimal concentration of 3'-sialyllactose production being 21.8 g / L. In a 7L fermenter, horizontal fermentation is performed for 16, the highest yield of 6'-sialyllactose reaching 52.29 g / L, and the conversion rate being 97.99%. These results lay a technical foundation for the large-scale production of sialyllactose.

The present application relates to the technical field of bioengineering, and particularly relates to a preparation method and application of immobilized enzyme. Compared with free enzyme, the immobilized enzyme prepared by selecting specific resin can be reused and has high stability; the immobilized enzyme is easy to separate from the reaction system, and can simplify the product purification process, improve the product yield and quality. Moreover, the present application develops a new strategy suitable for the large-scale synthesis of functional sugar chains such as sialylated sugar chains by immobilizing one kind of sugar nucleotide synthase and four kinds of sialyltransferase, improves the stability of related enzymes, realizes long-term preservation and reuse of the enzymes, and reduces the production cost of functional sugar chains.

The application provides a method for preparing sialylated pullulan, which comprises the following steps: adding 1.0-30.0 g / L of sialic acid to a fermentation liquor of a strain of Aureobasidium pullulans EP1001 and / or a genetically engineered strain of Aureobasidium pullulans EP1001 after 24.0-60.0 hours of fermentation; catalyzing, by sialyltransferase, the transfer of sialic acid from an activated glycosyl donor to the end of a saccharide chain of a receptor pullulan to form a sialylated pullulan complex, i.e., sialylated pullulan; precipitating the supernatant of the centrifuged fermentation liquor by using an organic solvent; and finally obtaining the sialylated pullulan. The content of the pullulan in the sialylated pullulan is 55.05 g / L, the content of the sialylated pullulan in the sialylated pullulan is 9.58 g / L, the content of free sialic acid in the culture and / or the culture liquor is 0.42 g / L, and the sialic acid conversion rate is up to 95.8%.

This invention belongs to the field of genetic engineering, specifically relating to a 6'-sialyltransferase mutant and its applications. The 6'-sialyltransferase mutant described in this invention is derived from a mutation of the 6'-sialyltransferase Pd2,6ST gene of *Bacillus damselae*. Compared to the wild-type 6'-sialyltransferase, the 6'-sialyltransferase mutant constructed in this invention exhibits higher substrate CTP tolerance, making it more suitable for the production of sialyl lactose and also adaptable to ectopic regeneration systems to achieve CTP regeneration.

The invention relates to recombinant escherichia coli for synthesizing sialic acid lactose-N-tetrasaccharide c as well as a construction method and application of the recombinant escherichia coli. The supply of a precursor CMP-Neu5Ac of the recombinant escherichia coli is optimized by adding a new synthesis route; alpha 2, 6-sialyltransferase with high efficiency and high specificity is screened and integrated into an escherichia coli BL21 (DE3) genome, so that the yield of sialyllactose-N-tetrasaccharide c is further increased, and accumulation of precursors LNnT and LNTII and a byproduct 6 '-SL is reduced; by adding the sugar efflux transporter gene, the inhibition effect caused by intracellular accumulation of high-concentration sugar products is reduced. The recombinant escherichia coli is used for culture and fermentation, high-yield sialic acid lactose-N-tetrasaccharide c can be synthesized, and the proportion of the obtained product sialic acid lactose-N-tetrasaccharide c in total HMO is further increased.

The application discloses a recombinant lactobacillus casei expressing sialyltransferase ST6GALNAC1, and belongs to the field of genetic engineering and microbial technology. The recombinant bacterium is obtained by cloning the ST6GALNAC1 gene into a pPG612 vector and electrotransforming the ST6GALNAC1 gene into lactobacillus casei ATCC393. Experiments show that the engineering bacterium can significantly enhance glycosylation modification of intestinal mucus protein MUC2, improve expression of tight junction proteins (ZO-1, Occludin and Claudin-1), inhibit activation of a p38 / MAPK inflammatory pathway, and regulate intestinal flora structure, thereby effectively relieving intestinal injury and systemic inflammatory response caused by salmonella. The application provides a strain basis and technical support for development of a new type of oral microecological preparation.

The present application relates to a kind of recombinant escherichia coli of synthetic sialyllactose-N-tetrasaccharide c and its construction method and application, by adding new synthetic pathway, the supply of its precursor CMP-Neu5Ac is optimized;By screening high-efficiency, specific strong alpha2,6-sialyltransferase, and it is integrated into escherichia coli BL21 (DE3) genome, further improve the yield of sialyllactose-N-tetrasaccharide c, reduce the accumulation of precursor LNnT, LNTII and by-product 6'-SL;By adding sugar efflux transporter gene, reduce the inhibitory effect brought by intracellular accumulation of high-concentration sugar product.Synthetic sialyllactose-N-tetrasaccharide c of high yield can be obtained by culturing and fermenting recombinant escherichia coli, and further improve the proportion of the obtained product sialyllactose-N-tetrasaccharide c in total HMO.

This invention relates to a polymeric probe for assessing sialylation levels in vivo. It uses a dendritic polymer with alkyne-terminated branches as a carrier, and its surface is simultaneously modified with galactose, cyanidin 5 fluorescent dye, and folic acid. The detection protocol involves subcutaneously injecting the prepared polymeric probe into the tumor region of tumor-bearing mice. Folic acid-mediated targeted endocytosis allows the polymeric probe to enter the tumor cells. In vivo sialylates bind sialic acid to the galactose terminal of the polymeric probe. Sodium periodate is used to oxidize the sialic acid terminal in mouse tumor tissue sections to form an aldehyde group, which is then further coupled with an acylhydrazide fluorescein molecule. Fluorescence signals from cyanidin 5 and fluorescein are simultaneously collected using fluorescence microscopy, and the fluorescence intensity of fluorescein in the superimposed region of the two fluorescence signals is obtained using software, which can then be used to assess the sialylation level of the tumor tissue.

The invention relates to recombinant escherichia coli of 3 '-sialic acid lactose as well as a construction method and application of the recombinant escherichia coli, and belongs to the field of gene engineering. According to the present invention, plasmids for expressing a precursor CMP-Neu5Ac synthesis pathway gene NeuBCA and an alpha 2, 3-sialyltransferase gene 406nst are introduced into a BL21 (DE3) host bacterium of lacZ, nano A and nano K genes to preliminarily construct the 3 '-sialyllactose biosynthesis pathway; and introducing plasmids expressing cofactors ppk, ndk, cmk and pyrG for promoting the regeneration of the CTP to obtain the high-yield strain E3. Subsequently, neuBCA, 406nst and ndk are integrated on chromosomes of the engineering bacteria independently or in a combined manner, and gene expression is realized by adopting a strong promoter PJ23119, so that a plasmid type high-yield 3 '-sialic acid lactose strain EN7 (the extracellular yield of 3'-sialic acid lactose is 3.080 g / L) and a high-yield whole genome strain ENB2 (the extracellular yield of 3 '-sialic acid lactose is 1.359 g / L) are obtained. Certain reference significance is provided for large-scale industrial application.

The present application relates to the field of biotechnology, and more particularly to a reagent and method for high-throughput plate screening of 3-sialyltransferase mutants. The present application realizes efficient screening of 3SL high-yield mutants in large-scale mutant libraries by adopting solid plate color screening technology combined with pH indicator. The method quickly identifies the target mutant by directly observing the color change on the plate, thereby omitting the cumbersome steps of strain construction, sequencing, transfer and shake flask culture in the traditional screening process, significantly improving the screening speed and efficiency. Compared with the prior art, the present application not only greatly shortens the experimental time and simplifies the operation process, but also reduces the requirements for human resources and equipment, providing great convenience for screening of enzyme mutant libraries. The technical advantage of the present application is that it is fast, efficient and low-cost, and is expected to be widely applied in the fields of biomedicine and food science.

The present application relates to the technical field of biotechnology, and particularly relates to a sialyltransferase mutant and application thereof in 3'-sialyllactose synthesis.The present application provides a sialyltransferase mutant SL54-M10, which has high selectivity, single product and simple purification; and has high catalytic activity, high thermal stability and high productivity, and can be applied in industrialization to produce 3'-sialyllactose pure product.

Provided are an engineered bacterium producing 3'-sialyllactose, a construction method and application thereof. The engineered bacterium has the ability to produce 3'-sialyllactose, and has an introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidyltransferase gene (css) and an alpha-2,3-sialyltransferase gene (ST) in its genome or a recombinant plasmid carried thereby. The engineered bacterium has a higher ability to produce 3'-sialyllactose without the need for antibiotics.

The invention discloses a synthesis method of a uniform sialylated AFP-L3 antigen, serum polyclonal antibody preparation and application thereof, and belongs to the field of immunology, the uniform sialylated AFP-L3 antigen comprises a sialylated AFP-L3 glycopeptide antigen or a sialylated AFP-L3 glycoprotein antigen, and the synthesis method comprises the following steps: transferring a carbohydrate chain to a starting substrate by using Endo-M N175Q transglycosylation reaction to obtain an intermediate I; wherein the starting substrate comprises a glycopeptide Fmoc-ATKVN (GlcNAc) FTEAQKAALDV-NH2 or an AFP (Alpha Fetal Protein) protein which is expressed in HEK293T and is subjected to hydrolysis treatment; modifying the intermediate I through fucose transferase FuT8 to obtain an intermediate II; the intermediate II is modified and extended through galactose transferase GalT and sialyltransferase SiaT, and an intermediate III or a sialylated AFP-L3 glycoprotein antigen is obtained; the sialylated AFP-L3 glycopeptide antigen is obtained through the steps that the sialylated AFP-L3 glycopeptide antigen is obtained, the intermediate III is subjected to Fmoc removal treatment and then conjugated with bis-succinimide glutaric acid ester and hemocyanin, and the serum polyclonal antibody generated based on the antigen has high titer and specificity.

The invention relates to the technical field of biology, in particular to a sialyltransferase mutant and application of the sialyltransferase mutant in synthesis of 3 '-sialyllactose. The invention provides the sialyltransferase mutant SL54-M10, on one hand, the sialyltransferase mutant SL54-M10 is high in selectivity, single in product and simple in purification; on the other hand, the 3 '-sialyllactose has high catalytic activity, high thermal stability and high productivity, can be industrially applied, and is used for producing a pure product of 3'-sialyllactose.

The present invention relates to a method for producing an Fc fusion protein comprising an EPO-hybrid Fc fusion protein (i.e., epoetin alpha) having a C-terminal lysine content of about 1–10% and a sialic acid content of about 17–27 mol / mol. The method comprises culturing host cells transformed with DNA encoding the Fc fusion protein and DNA encoding human α-2,3-sialyltransferase under optimized culture conditions, and purifying the Fc fusion protein using an optimized purification process including anion exchange chromatography.