193 results about "Recombineering" patented technology

The invention provides a recombinant engineering bacterium for producing lumbrukinase and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, when the lumbrukinase F238 is expressed, one or more genes in TDA3 and KRE9 genes are knocked out in a targeted manner by utilizing a CRISPR-Cas9 gene editing technology, and / or an SGA1 gene is overexpressed and integrated into a strain, so that the expression quantity of the lumbrukinase F238 is increased. According to the recombinant engineering bacterium, the yield of the lumbrukinase can be obviously increased and can reach 235% at most, meanwhile, the activity of the lumbrukinase is obviously enhanced and can reach 699.6 U / mL at most, and the recombinant engineering bacterium has a wide application prospect.

The invention provides an indoxacarb degrading enzyme as well as a coding gene, a production strain and application thereof. Wherein the amino acid sequence of the indoxacarb degrading enzyme is as shown in SEQ ID NO. 1, and the sequence of the coding gene is as shown in SEQ ID NO. 2. The production strain is a recombinant engineering strain comprising the coding gene. The application refers to the application of the indoxacarb degrading enzyme in degrading indoxacarb and repairing an indoxacarb polluted environment, and a corresponding method for degrading indoxacarb or repairing the indoxacarb polluted environment. The indoxacarb degrading enzyme has the advantages that the screened indoxacarb degrading enzyme can efficiently and rapidly degrade indoxacarb, can also be used for repairing environments such as water bodies and soil polluted by indoxacarb, and has important application value.

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

The invention discloses a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, and relates to the technical field of gene engineering, in particular to a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, which comprises the following steps: gene synthesis and vector construction: synthesizing an optimized sequence SEQ ID No.1 of human source XVII type collagen COL17A1; adding proper restriction enzyme cutting sites at two ends of the sequence; the synthesized gene sequence is cloned to a pGAPZ A-alpha A vector, and the expression of the gene under the control of a GAP promoter is ensured. According to the preparation method of the recombinant engineering bacterium for expressing the XVII type collagen COL17A1, by using a GAP promoter, the constructed recombinant engineering bacterium can keep a stable expression level under different environmental conditions, the GAP promoter serving as a constitutive promoter can continuously and stably express recombinant protein in pichia pastoris, an inducer does not need to be added, the production process is simplified, and the production cost is reduced. The production cost is reduced, and the production efficiency is improved.

Provided is a recombinant human serum albumin-collagen binding domain (CBD) fusion protein for tumor-specific targeting matrix and its use in specific targeted treatment of a solid tumor. HSA and the CBD generate gene fusion in the form of different molecular structures, and can be expressed in a constructed recombinant engineered host. A pharmaceutical recombinant fusion protein is obtained through large-scale fermentation. The recombinant fusion protein shows tumor-specific targeting of solid tumors and can be used as a starting material of a broad-spectrum matrix carrier. The recombinant fusion protein is coupled with any tumor treatment drug, small-molecule compound, or cytotoxic protein to form an innovative pharmaceutical composition. The pharmaceutical composition of the recombinant fusion protein is also long-acting, can prolong a half-life of the conjugate drug in vivo, and has a significantly better clinical curative efficacy on anti-tumor than that of a monomer drug.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

The present invention discloses a genetically engineered bacterium producing furfural amine, and a preparation method and application thereof, and the present invention belongs to the technical field of fermentation engineering. The present invention is to solve the problems of large amount of amino donor, high price, metal ion cofactor, large amount of co-substrate, etc. in the existing preparation of furfural amine by bio-transamination catalysis. The method of the present invention comprises the following steps: constructing a recombinant Escherichia coli cell; adding the whole cell of the recombinant engineered bacillus induced for expression into the reaction system; recovering the whole cell catalyst to prepare the product. Furfural amine is prepared by using a cheap inorganic amino donor and glucose as a co-substrate; the amount and cost of the amino donor are effectively reduced, and the amount of glucose is greatly reduced, thereby realizing the green and efficient synthesis of bio-based furfural amine.

The invention provides a recombinant engineering bacterium for preparing recombinant human albumin based on a transport secretion way and application, and relates to the technical field of gene engineering. One or more of endoplasmic reticulum related genes SRP54 and ERJ5, vesicle transport related genes YOS1 and SLY1 and Golgi apparatus related genes GOS1 and YIP1 are integrated into a recombinant engineering bacterium genome through a gene recombination technology, so that efficient secretory expression of recombinant human albumin is realized. According to the method provided by the invention, the expression quantity of the recombinant human albumin is remarkably improved, and the method has a wide application prospect.

The invention relates to a diacylglycerol acyltransferase capable of synthesizing triglyceride as well as a coding gene and application of the diacylglycerol acyltransferase, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the diacylglycerol acyltransferase is as shown in SEQ ID NO.1, and the amino acid sequence of the diacylglycerol acyltransferase is as shown in SEQ ID NO.2. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The invention relates to the technical field of collagen, and discloses a preparation method of recombinant engineering bacteria for expressing III-type collagen COL17A1, which comprises the following steps: synthesizing an optimized sequence of human III-type collagen, and adding proper restriction enzyme cutting sites at two ends of the sequence. According to the preparation method of the recombinant engineering bacterium for expressing the III-type collagen COL17A1, codon optimization is carried out on a target sequence by adopting a yeast expression system, OptimGeneTM codon optimization is provided according to codon preference of yeast strains such as pichia pastoris, and the yield of recombinant protein is further improved. And a proper vector is selected to realize high-efficiency expression in pichia pastoris. For example, a pGAPZ A-alpha A vector stably expresses recombinant protein in pichia pastoris by using a GAP promoter, is a constitutive gene expression vector, and can maintain a stable expression rate in cells no matter how the environment changes. An inducer does not need to be added, the production process is simplified, the production cost is reduced, and the production efficiency is improved.

The invention belongs to the field of biotechnology and environmental governance, and particularly relates to a manganese peroxidase mutant based on high-throughput screening modification and application of the manganese peroxidase mutant to straw degradation. Aiming at the problems of low natural secretion amount, insufficient specific activity, complex preparation process and the like of the existing manganese peroxidase, the method comprises the following steps: firstly, constructing engineering bacteria based on an escherichia coli exocrine system, constructing a mutant library through error-prone PCR in combination with a directed evolution technology, and performing high-throughput screening by utilizing an ABTS colorimetric method; the recombinant manganese peroxidase mutant with high catalytic activity is obtained, and the enzyme activity is improved by 1.29 times compared with that of a wild type. Furthermore, recombinant engineering bacteria are constructed on the basis of a bacillus subtilis exocrine system, and efficient secretion of the mutant enzyme is achieved. The mutant enzyme preparation is used for wheat straw degradation, and the lignin degradation rate reaches 41.37% (improved by 30.09% compared with that of a wild type). The method is simple and convenient in process, low in cost and remarkable in effect, and has a wide industrial application prospect.

The invention provides a recombinant engineering bacterium for preparing recombinant equine albumin and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, one or more of genes SED1 and TIP1 related to cell wall composition is / are knocked out in a targeted manner while the recombinant equine albumin is expressed, and / or one or more of genes PAC10 and YDJ1 related to protein folding promotion is / are overexpressed and integrated into a strain, so that the expression quantity of the recombinant equine albumin is improved. According to the recombinant engineering bacterium disclosed by the invention, the yield of the recombinant equine albumin can be obviously improved, the highest yield can reach 51.27 g / L, and compared with an unmodified recombinant engineering bacterium, the yield is improved by 5.38 times, and the recombinant engineering bacterium has a wide application prospect.

The present invention provides a recombinant engineered bacterium for preparing recombinant albumin based on transcription factors and its application, and relates to the field of genetic engineering technology. The present invention increases the expression of recombinant albumin by simultaneously overexpressing SUS1 gene and SUA7 gene into the strain while expressing recombinant albumin, and / or using CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1. The recombinant engineered bacterium of the present invention can significantly increase the yield of recombinant albumin and has great application value.

The invention discloses a recombinant engineering bacterium for expressing fetuB and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the recombinant engineering bacteria, pichia pastoris serves as host bacteria, after fetuB genes are introduced, efficient expression of the fetuB in the pichia pastoris is achieved by optimizing codons and improving fermentation conditions, and the expression quantities of CBS7435 under the shake-flask culture condition are 320 + / -25 mg / L, SMD116 under the shake-flask culture condition is 280 + / -30 mg / L, and SuperMan5 under the shake-flask culture condition is 580 + / -45 mg / L. After fermentation conditions are optimized, the culture yield of a fermentation tank can reach 27.35 g / L. The fetuin B disclosed by the invention is good in biological activity, high in expression, simple in operation process and suitable for large-scale industrial production.

The invention discloses a preparation method and application of CHO cell expression triple helix structure recombinant humanized collagen, and relates to the technical field of biology. The preparation method comprises the following steps: fermenting and purifying recombinant engineering cells to obtain the recombinant humanized collagen with the triple-helix structure; the construction method of the recombinant engineering cell comprises the following steps: transforming a recombinant plasmid for expressing alpha1 (III) T1 and a recombinant plasmid for expressing hP4H alpha1 and hP4HB into a host cell, and constructing to obtain the recombinant engineering cell. The recombinant humanized collagen with the triple helix structure and high proline hydroxylation rate can be prepared by utilizing the preparation method, can be widely applied to the fields of cosmetics, medical cosmetology, medical instruments, biomedical materials and the like, and has important market application value.

The invention discloses an application of silkworm fatty acid dehydrogenase Bmdesat13 in enzymatic catalytic generation of trans-palmitoleic acid and a method thereof, the silkworm fatty acid dehydrogenase Bmdesat13 gene ORF sequence is cloned from silkworms, and a brewer's yeast eukaryotic expression system is used for heterologous expression of Bmdesat13, so that the trans-palmitoleic acid is obtained. An enzyme activity product which can be synthesized by the gene in a yeast positive recombinant engineering strain is identified to be trans-9-hexadecenoic acid (trans-palmitoleic acid) through GC-MS (gas chromatography-mass spectrometry), so that theoretical and technical supports are provided for mass production of trans-palmitoleic acid.

The present disclosure provides recombineering-editing systems using CRISPR and recombination enzymes as well as methods, vectors, nucleic acid compositions, and kits thereof. The methods and systems provide means for altering target DNA, including genomic DNA in a host cell.

The invention relates to a combined bacterium system for efficiently synthesizing resveratrol and a method for synthesizing resveratrol, and belongs to the technical field of bioengineering and biosynthesis. The preparation method comprises the following steps: respectively connecting 4CL, STS and FCS with scaffold proteins, constructing recombinant expression vectors for expressing 4CL-SpyTag, STS-SpyCatcher and FCS-SpyTag, respectively introducing the recombinant expression vectors into escherichia coli, carrying out expression to obtain a compound enzyme connected with the scaffold proteins, and then carrying out self-assembly to obtain a multienzyme complex 4CL-SpyTag-STS-SpyCatcher and FCS-SpyTag-STS-SpyCatcher; the constructed recombinant engineering bacterium or enzyme complex is added into p-coumaric acid synthesized from agricultural wastes for further synthesis of resveratrol; the multi-enzyme complex constructed by the invention and the new method for synthesizing the resveratrol not only provide a new high-valued utilization approach for the lignin, but also realize efficient and high-yield synthesis of the resveratrol.

The invention discloses a PD1 gene engineering exosome preparation for cancer immunotherapy and a preparation method of the PD1 gene engineering exosome preparation. The invention provides a method for preparing an exosome for expressing PD1, and further provides any one of the following applications of the exosome as a drug carrier, delivery of drugs, preparation of tumor treatment products, preparation of postoperative tumor recurrence resistant products, preparation of postoperative tumor metastasis resistant products and the like. Genetically recombinantly engineered exosomes can be used to enhance the response of cancer immune checkpoint blocking therapies. The research also provides a new strategy with a prospect, and is used for enhancing the PD1 / PDL1 treatment effect, and postoperative tumor recurrence or metastasis is resisted by reconstructing the immunity of a patient, so that the overall prognosis is consolidated.

The invention provides a mutant of xylose reductase and application of the mutant in synthesis of D-tagatose. According to the present invention, the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification on the basis of the sequence and structure-oriented semi-rational modification strategy so as to obtain the high activity xylose reductase mutant, and the xylose reductase mutant has the following characteristics that: the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification so as to obtain the xylose reductase mutant, according to the invention, the mutant of the xylose reductase is integrated into a redox pathway for synthesizing the D-tagatose by virtue of a genetic engineering means, and is expressed in bacillus subtilis 168, so that a recombinant engineering bacterium for synthesizing the D-tagatose by fermenting D-galactose is successfully constructed, the efficient synthesis of the D-tagatose is realized, and the yield of the D-tagatose is improved. And the method has obvious practical application value and economic benefit.

The invention relates to the field of protein, in particular to a preparation method of recombinant human albumin and a product thereof. The preparation method comprises the following steps: constructing recombinant engineering bacteria which can silence or weaken the expression of one or more genes of PEP4, YPS1, MKC7, PRC1 and PRB1 through a gene editing technology while expressing recombinant human albumin, and silence or weaken the expression of one or more genes of PMT1, PMT2 and PMT4 at the same time; and then, carrying out low O-glycosylation fermentation culture on the obtained engineering bacteria, and carrying out separation and purification to obtain a high-purity recombinant human albumin product. According to the present invention, the protein degradation and the O-glycosylation pathway are cooperatively regulated, and the specific fermentation process is combined, such that the yield of the obtained recombinant human albumin is significantly improved, the characteristics of low immunogenicity and low glycosylation level are provided, and the performance optimization and the clinical application expansion of the recombinant human albumin product can be significantly promoted.

The invention belongs to the technical field of gene cloning and assembling, and particularly relates to a plasmid library construction method based on Red / ET recombination engineering and application of the plasmid library construction method. According to the invention, an appropriate linear expression vector is constructed, and 50bp short sequences at two ends of the vector are used as homologous arms. The two homologous arms are introduced to the two ends of all DNA sequences to be studied, so that the two homologous arms can be subjected to homologous recombination with the vector, the cotransformation linearization vector and the target DNA sequence are integrated into escherichia coli with sufficient RecE and RecT expression, and plasmid libraries carrying different target DNAs can be constructed through one-step line-line recombination. On the basis, homologous arms of A-B or B-A versions are introduced to the two ends of a plurality of genes with a synergistic effect relationship, and co-expression plasmids in different combination modes can be constructed by one-step line-line recombination, so that the synergistic effect between the genes is conveniently explored or interaction proteins are determined, and the method has a good practical application value.

The invention relates to a nucleic acid molecule for preparing indigoidine, an expression vector and a recombinant engineering strain. The nucleic acid molecule comprises a sequence as shown in SEQ ID NO.2 or a nucleotide sequence which has at least 98% sequence identity with the SEQ ID NO.2. According to the present invention, the Wlpl protein encoded by the nucleic acid molecule is derived from Williamsiapylosphaerae bacteria, the amino acid sequence of the Wlpl protein is represented by SEQ ID NO.1, the protein has excellent 4 '-phosphoric acid pantetheine transferase catalytic activity, and when the Wlpl protein is used for catalyzing the synthesis process of glutamine to synthesize the indigoidine, the yield of the indigoidine can achieve 14.26 g / L, such that the indigoidine yield is substantially improved, and the good industrialization prospect is provided;

The invention relates to a recombinant engineering bacterium for expressing pure iron nitrogenase as well as a construction method and application of the recombinant engineering bacterium, and belongs to the field of biotechnology and bioengineering. Key gene clusters of rhodopseudomonas and Vilnelande nitrogen-fixing bacteria are amplified and cloned into an expression vector, escherichia coli is converted to obtain recombinant engineering bacteria for expressing pure iron nitrogenase, and the pure iron nitrogenase expressed by the recombinant engineering bacteria can reduce carbon dioxide (CO2) into methane (CH4); the recombinant engineering bacteria are cultured in an LB culture medium added with ferric chloride, ampicillin and spectinomycin, oxygen exposure is reduced as much as possible under the optimized semi-anaerobic condition, the enzyme activity can be improved to the maximum extent, and compared with a traditional system, the methane yield is remarkably increased; by providing a novel methane production scheme, the technical defects of low efficiency and oxygen sensitivity of the traditional nitrogenase process are improved; the process of the present invention contributes to carbon capture and utilization by converting waste carbon dioxide into valuable methane.

The invention relates to an artificial efficient nitrogen fixation system and a preparation method and application thereof. The artificial efficient nitrogen fixation system contains recombinant engineering bacteria constructed by an artificial combination module and nitrogen fixation chassis microorganisms; wherein the artificial combination module sequentially comprises a constitutive promoter element Pc and a coding gene of methyltransferase selected from nitrogen-fixing pseudomonas stutzeri A1501 in the direction from upstream to downstream. According to the recombinant engineering bacteria constructed by introducing the artificial combination module into the nitrogen-fixing chassis microorganism, the nitrogenase activity of the nitrogen-fixing chassis microorganism can be remarkably improved, the nitrogen-fixing capacity of the nitrogen-fixing chassis microorganism is remarkably improved, and the recombinant engineering bacteria have a relatively good application prospect in the aspect of promoting plant growth.

Provided herein are methods and compositions for truncating and, optionally, joining polynucleotides. The methods bypass inefficient cloning methods and the requirement for expensive enzymes by using in vivo homologous recombination in conjunction with specific endonuclease digestion. The methods may further be used to assemble long fragments of DNA from existing DNA constructs or libraries or to generate novel DNA libraries.

The present invention provides a fusion protein KP-Ag1 used as a Klebsiella pneumoniae vaccine antigen, which is composed of KP6 protein, KP21 protein and KP24 protein connected by a flexible amino acid Linker; wherein, the amino acid sequence of the KP6 is SEQ ID NO:4, the amino acid sequence of the KP21 is SEQ ID NO:5, and the amino acid sequence of the KP24 is SEQ ID NO:6. The present invention also provides its expression vector and recombinant engineering diagram, as well as its application as a Klebsiella pneumoniae antigen. The antigen protein prepared by the method described in the present invention can effectively stimulate the body to produce a higher humoral immune response, and can provide obvious protection against lethal-dose Klebsiella pneumoniae infection, and can be used as a candidate antigen for Klebsiella pneumoniae vaccine.

The invention relates to euphausia superba-derived diacylglycerol acyltransferase as well as a coding gene and application thereof, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the enzyme is as shown in SEQ ID NO.1, and the amino acid sequence of the enzyme is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the euphausia superba-derived diacylglycerol acyltransferase. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The present invention provides a Klebsiella pneumonia KPC-Pal recombinant protein, which is composed of a KPC peptide segment extracted from the Klebsiella pneumonia KPC-2 protein and a Pal peptide segment extracted from the Klebsiella pneumonia Pal protein connected by a polypeptide linker, wherein the amino acid sequence of the Klebsiella pneumonia KPC-2 protein is SEQ ID NO: 3, and the amino acid sequence of the Klebsiella pneumonia Pal protein is SEQ ID NO: 4. The present invention also provides its expression vector and recombinant engineered bacteria, as well as its application in the preparation of a preparation for diagnosing, preventing or treating Klebsiella pneumonia infection. The Klebsiella pneumonia KPC-Pal recombinant protein provided by the present invention can effectively stimulate the body to cause a protective immune response, thereby resisting the lethal infection of Klebsiella pneumonia.

The present invention provides a recombinant engineered bacterium for producing recombinant canine albumin and its application, relating to the field of biochemistry, specifically, genetic engineering technology. By integrating overexpression of one or more of the protein-folding-related genes PBN1, KAR2, and ERV2 into the strain while simultaneously expressing recombinant canine albumin, the present invention significantly improves canine albumin expression, with relative protein expression reaching up to 491% and specific yields reaching up to 32.83 g / L, demonstrating broad application prospects.