2064 results about "Plasmid" patented technology

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

The invention relates to the field of enzyme engineering, in particular to modified xylanase, a DNA (deoxyribonucleic acid) molecule, a recombinant plasmid and a transfected cell. The modified xylanase protected by the application is formed by replacing a deletion segment of a receptor enzyme with a substitution segment, the receptor enzyme is wild type xylanase, the sequence of the receptor enzyme is SEQ ID NO: 1, the deletion segment comprises a carbohydrate binding module of the receptor enzyme, the substitution segment is a carbohydrate binding module of a donor enzyme, the donor enzyme is a carbohydrate active enzyme, and the donor enzyme is a carbohydrate active enzyme. The sequence of the donor enzyme is one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The carbohydrate binding module of the carbohydrate active enzyme with relatively high activity is replaced into the receptor enzyme, so that the binding capacity of the receptor enzyme and the carbohydrate is improved, and the activity of the receptor enzyme is improved.

The invention relates to the technical field of microbial genetic engineering, in particular to a method for improving cordycepin synthesized by saccharomyces cerevisiae and saccharomyces cerevisiae engineering bacteria. According to the invention, codon-optimized 2 '-carbonyl-3'-deoxyadenosine reductase gene and 3 '-adenosine monophosphate phosphohydrolase gene from cordyceps militaris are expressed by using plasmids, and then the plasmids are transformed into host cells to obtain the saccharomyces cerevisiae engineering bacteria. And inoculating a seed solution obtained by seed culture of the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium containing a metabolic effector, and carrying out fermentation culture to synthesize cordycepin. Metabolic effectors (Cu < 2 + >, Fe < 2 + >, Mg < 2 + >, Zn < 2 + >, citric acid, cysteine, aspartic acid, glycine, VB1 and tea polyphenol) are added into a fermentation culture medium strain, and the variety and concentration of the metabolic effectors are optimized, so that the yield and synthesis efficiency of cordycepin are remarkably improved. By using the combination of the strain obtained by the invention and the optimal effector, the yield of cordycepin in a fermentation tank reaches 2.9 g / L.

The invention relates to a gene editing vector of agrobacterium, a gene editing method of the gene editing vector and an agrobacterium editing strain. The agrobacterium gene editing vector comprises the following gene elements: a plasmid skeleton, a Pvan promoter, a spCas9 gene, a green fluorescent protein gene GFP, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. According to the agrobacterium gene editing vector, a CRISPR / spCas9-GFP gene editing system is adopted, an efficient, simple, convenient and traceless genome editing system is constructed in agrobacterium, and gene knockout and insertion can be achieved.

A stable transformant, in which a gene of interest is incorporated into the genome, is simply and efficiently produced.A method for producing a transformant, comprising a step of introducing into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into the predetermined position in the host genome and the gene of interest is expressed therein.

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

The invention discloses construction and application of an escherichia coli L-tryptophan biosensor, and belongs to the technical field of bioengineering. The L-tryptophan biosensor provided by the invention comprises a mutated leader peptide fragment tnaC-tnaA, a promoter of the mutated leader peptide fragment tnaC-tnaA, a green fluorescent protein coding gene eGFP and a pTrc99a plasmid skeleton, a recombinant strain containing the L-tryptophan biosensor is cultured, the expression of green fluorescent protein in a culture solution is gradually enhanced along with the increase of the concentration of the L-tryptophan, and the fluorescence value of a unit cell shows better correlation when the concentration of the L-tryptophan is 0-1.5 g / L; therefore, when the biosensor is used for detecting the L-tryptophan in the fermentation liquor, the biosensor has the characteristics of simplicity in operation, sensitive response and high threshold value.

The invention discloses construction and application of a plasmid-free and auxotrophy-free L-tryptophan production strain, and belongs to the technical field of bioengineering. According to the invention, a plurality of key anti-feedback inhibition enzymes in a tryptophan synthesis route are optimized and combined, and the optimal combination is integrated into an escherichia coli genome by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats)-ASPR (clustered regularly interspaced short palindromic repeats)-trans-spansses (clustered regularly interspaced short palindromic repeats) system. According to the invention, the plasmid-free and nutritional deficiency-free engineering strain ZH-3 for producing the L-tryptophan is successfully constructed, and under the conditions of no use of antibiotics, use of inducers and no addition of additional substances, the total yield of the L-tryptophan reaches 43g / L and the sugar-acid conversion rate is increased to 0.180 g / g after the strain is cultured for 35h in a 3L fermentation tank. The strategy used by the invention is not only suitable for construction of tryptophan genome integration expression strains, but also provides reference for construction of other plasmid-free amino acid production strains.

The invention provides a genetically engineered bacterium for producing O-succinyl-L-homoserine as well as a construction method and application of the genetically engineered bacterium. In a chassis bacterium genome, the expression of a 2-ketoglutaric acid decarboxylase encoding gene sucA is enhanced, and the expression of a succinyl-coenzyme A synthetase encoding gene sucD is weakened, so that the supply of succinyl-coenzyme A is increased; the method comprises the following steps: increasing the NADPH (Nicotinamide Adenine Dinucleotide Phosphate) reducing capacity and ATP (Adenosine Triphosphate) energy supply of a chassis bacterium, increasing DNA (Deoxyribose Nucleic Acid) in combination with a transcription dual regulatory factor ompR to improve the stress resistance of escherichia coli under high osmotic pressure, and introducing an overexpression plasmid containing a homoserine transsuccinylase coding gene metA to construct the genetically engineered bacterium for producing O-succinyl-L-homoserine. The engineering strain obtained through a systematic metabolic engineering modification strategy can realize effective accumulation of OSH, the shake flask yield of OSH reaches 19.8 g / L, the fed-batch fermentation yield of a 5L fermentation tank reaches 110.5 g / L, the sugar-acid conversion rate reaches 52.6%, and a foundation is laid for subsequent construction of high-yield OSH engineering bacteria.

The invention belongs to the field of biosynthesis, and in particular relates to LBD (Levandanenediol Pyrophosphate) synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LSLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained by performing mutation and optimization on LSLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 26.11 g / L and is greatly improved compared with the prior art.

The invention provides a subunit fusion protein RBD-Fc on the surface of a porcine D-type coronavirus and a preparation method and application thereof, the preparation method comprises the following steps: 1) cloning a gene sequence as shown in SEQ ID NO.5 into a eukaryotic expression vector to obtain a recombinant plasmid containing a fusion protein RBD-Fc coding gene; (2) transfecting a recombinant plasmid containing the fusion protein RBD-Fc coding gene into an expression cell; 3) culturing, screening and domesticating the expression cells in the step 2) to obtain a highly expressed cell strain; and 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain the subunit fusion protein RBD-Fc. According to the present invention, the RBD-Fc can be subjected to mass soluble expression, the protein is stable, the problems that the porcine D-coronavirus surface S protein cannot be subjected to large-scale expression and the like in the prior art are solved, the preparation method is simple, and the cost is low.

The invention belongs to the field of biosynthesis, and particularly relates to sclareol synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, sclareol biosynthesis yield is generally low, sclareol synthase is mutated and optimized, an SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 21.13 g / L and is greatly improved compared with the prior art.

The invention relates to an Escherichia coli engineering strain for producing 2, 3-pentanediol as well as a construction method and application of the Escherichia coli engineering strain, and the method comprises the following steps: constructing recombinant plasmids pACYC-IlvA *-ThrA * BC for expressing an IlvA * gene of threonine deaminase, a ThrA gene of asparaginic acid kinase I, a Thr B gene of homoserine kinase and a ThrC gene of threonine synthetase; the method comprises the following steps: constructing a recombinant plasmid pRSF-Ilv2C-Aro10 for expressing an Ilv2C gene of acetohydroxy acid synthetase and an Aro10 gene of propiophenone dehydrogenase; and chemically converting the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10, and transferring the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10 into escherichia coli so as to obtain the escherichia coli engineering strain for producing the 2, 3-pentanediol. According to the escherichia coli engineering strain, the biosynthesis of 1.61 g / L of 2, 3-pentanediol is realized by using L-threonine and 40 g / L of glucose.

The invention provides a genetically engineered bacterium and a construction method of the genetically engineered bacterium, and the genetically engineered bacterium is characterized in that a green fluorescent protein sfGFP is used for mediating protein glutaminase, PG (protein glutaminase), PGF (protein glutaminase), PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF and PGF; eC 3.5. 1.44) is efficiently secreted and expressed in engineering bacteria, and belongs to the technical field of biological engineering. The method comprises the following steps: an obtained sfGFP gene is derived from an NCBI database (GenBank numbering: CP035486.1), an obtained PG zymogen gene PP (Propeptide-Protein glutaminase) is derived from a Chryseobacterium prion prgA gene (GenBank numbering: AB046594.1) in the NCBI database, a recombinant plasmid pHT01 / sfGFP-PP is introduced into bacillus subtilis WB800N to obtain an engineering bacterium, extracellular secretion expression of PP is realized, the sfGFP-PP is activated by trypsin to obtain PG, and the PG is subjected to enzyme activation to obtain the recombinant bacillus subtilis. The expression quantity and the secretion efficiency of the PG in the bacillus subtilis are greatly improved. The recombinant PG produced by the method can generate 23.5 U / mL enzymatic activity under activation of trypsin, can be used for improving protein functional characteristics, and has relatively high industrial production and application values.

The invention belongs to the technical field of biological detection, and discloses a kit and a method for quantitatively detecting residual quantity of host cell protein of pichia pastoris in order to solve the problems of limit of quantitation and low detection sensitivity of an existing detection method. According to the present invention, the empty plasmid without the recombinant human serum albumin gene is introduced into the pichia pastoris, and purification is performed by combining the Protein A chromatographic column and the host cell protein coupling affinity column, such that the prepared polyclonal antibody can be effectively used for the quantitative detection of the residual amount of the pichia pastoris host cell protein in the biological product; the kit formed by the polyclonal antibody has good accuracy and repeatability on the quantitative detection of the host cell protein residues in the pichia pastoris source biological products, the quantification limit is 0.2 ng / mL, and the kit provided by the invention can meet the quantitative detection of the host cell protein residues in all the pichia pastoris source biological products.

The invention discloses an SaRNA vaccine for echinococcosis as well as a preparation method and application of the SaRNA vaccine. The preparation method of the SaRNA vaccine comprises the following steps: carrying out codon optimization on a modified target antigen protein through a genetic engineering technology, then assembling the modified target antigen protein with a self-replicating protein sequence, 5 'UTR, 3' UTR and Poly (A) tail, carrying out gene synthesis, then cloning the synthesized gene into a plasmid, and carrying out purification to obtain the SaRNA vaccine. The preparation method comprises the following steps: constructing recombinant plasmids, sequentially carrying out plasmid linearization, in-vitro transcription and purification on the constructed recombinant plasmids to prepare SaRNA molecules, and finally wrapping the SaRNA molecules in lipid nanoparticles to form the SaRNA vaccine for the echinococcosis. Experiments prove that the SaRNA vaccine can activate humoral immunity and cellular immunity of mice at the same time, high-level EG95 specific antibodies and cytokines can be generated through low-dose immunity, and the SaRNA vaccine has wide application prospects in the aspect of preventing and / or treating the echinococcosis.

The invention provides an intratumoral bacterium detection and analysis method based on circular DNA enrichment sequencing and related model construction, and belongs to the field of molecular biology. The intratumoral bacterium detection method provided by the invention comprises the following steps: extracting total DNA of cancer tissue or para-carcinoma tissue, carrying out linear digestion, purifying a linear digestion product, and enriching to obtain circular DNA; the method comprises the following steps: amplifying circular DNA, and breaking a DNA amplification product to 300-500bp; purifying DNA fragments, constructing a library, and sequencing; and performing genome comparison according to a sequencing result to obtain information of the intracellular bacteria. According to the method, complete bacterial genome and plasmid genome information can be captured, and the detection sensitivity of the intracellular bacteria and the richness of the bacterial genome information are improved. The invention further provides an intratumoral bacterium analysis method which can be used for screening generic cancer bacterial markers, and provides a cancer risk assessment machine learning model based on the intratumoral bacterium, so that the clinical application possibility of the intratumoral bacterium is improved.

The invention discloses a method for synthesizing a protein-RNA compound in vivo for nucleic acid detection, and relates to the technical field of biology. The technical key points are as follows: a series of recombinant plasmids capable of realizing the simultaneous expression of CRISPR / Cas effect proteins [including all sgRNA-guided Cas proteins such as Cas9, Cas12 (a, b, g, f and the like), Cas13, Cas14 and the like] and crRNA / tracrRNA (sgRNA) thereof in a series of expression strains of escherichia coli and assembling in the escherichia coli body are constructed, wherein the recombinant plasmids can be used for realizing the simultaneous expression of the CRISPR / Cas effect proteins [including all sgRNA-guided Cas proteins such as Cas9, Cas12 (a, b, g, f and the like); according to the present invention, the His tag is added to the 3'end of the compound gene, such that the compounds can be purified by using the nickel column so as to be used for subsequent nucleic acid detection, and the CRISPR / Cas protein nucleic acid compound assembled in vivo has characteristics of high enzyme activity, high sensitivity and high specificity so as to create good conditions for the CRISPR system to establish the nucleic acid detection platform;

The invention relates to the technical field of stem cells, in particular to a lentivirus envelope plasmid combination and application thereof, a lentivirus and a packaging method thereof and a method for transduction of hematopoietic stem cells. The invention provides an envelope plasmid combination for lentivirus packaging, which is composed of lentivirus packaging plasmids containing VSVG glycoprotein and lentivirus packaging plasmids containing BaEV glycoprotein in a ratio of 3: 7. The invention further provides a method for transduction of the hematopoietic stem cells by the lentivirus, the method is simple and convenient to operate, the long-term dryness of the hematopoietic stem cells in vitro can be maintained, efficient and stable transduction of the hematopoietic stem cells can be realized, and the transduction rate is greater than 90%. The lentivirus transduction method hematopoietic stem cells can be used for hematopoietic stem cell gene therapy hematopoietic system genetic diseases, such as severe combined immunodeficiency, beta-thalassemia and sickle cell disease, Wiskott-Aldrich syndrome and the like, and the application prospect is good.

The invention discloses a method for promoting lactobacillus activity enhancement, which comprises the following steps: S1, amplifying a target gene corresponding to an LPxTG structural protein, and amplifying a target gene corresponding to a signal peptide; s2, obtaining a target gene connected with the LPxTG structure and the signal peptide; s3, connecting the target gene obtained in the step S2 with a plasmid gene sequence to construct a recombinant expression vector; s4, introducing the recombinant expression vector obtained in the step S3 into escherichia coli BL-21 so as to increase the number of the recombinant expression vector; s5, introducing the recombinant expression vector obtained in the step S4 into lactobacillus plantarum C8 to obtain a recombinant strain; and step S6, co-culturing lactobacillus plantarum C8 and the recombinant strain obtained in the step S5. The lactobacillus plantarum C8 has the advantages that the whole lactobacillus plantarum C8 and the recombinant strain have better survival condition and adhesiveness in gastric juice and intestinal juice.

The present invention relates to means for recombinant manufacture. In particular, it relates to a recombinant bacterial cell comprising in its genome at least one essential gene the endogenous promoter of which has been replaced by a heterologous expression control sequence being inducible by an inducer molecule, such that the expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule. Further contemplated are methods for generating the recombinant bacterial cell of the invention as well as a method for recombinant manufacture of a compound of interest. The present invention also provides for use the recombinant bacterial cell of the invention for recombinant manufacture of a compound of interest and a kit for manufacture of a compound of interest.

The invention belongs to the technical field of microorganisms, and particularly relates to recombinant escherichia coli containing a PDRN sequence and a preparation method thereof, and the preparation method comprises the following steps: S1, preparing a PDRN solution; s2, complementing the double strands of the PDRN into blunt ends; s3, constructing a recombinant plasmid; s4, carrying out mixed culture on a product obtained in the step S3 and escherichia coli DH5alpha; s5, screening the recombinants; s6, carrying out PCR (Polymerase Chain Reaction) amplification, and recording a strain from a fragment with the length of more than 200bp; s7, preparing a seed solution by adopting the strain prepared in the step S6; s8, strain fermentation; and S9, centrifugally collecting thalli. The method comprises the following steps: constructing a recombinant plasmid containing a PDRN fragment, introducing the recombinant plasmid into Escherichia coli, subsequently culturing the recombinant Escherichia coli to copy DNA in the Escherichia coli, extracting a purified exogenous gene fragment to obtain a strain containing a single fragment PDRN sequence, and getting rid of dependence on raw materials during subsequent extraction.

The invention discloses escherichia coli recombinant bacteria with high glycolic acid yield and a construction method of the escherichia coli recombinant bacteria, and belongs to the technical field of bioengineering. Lactate dehydrogenase ldhA, phosphate transacetylase pta, acetokinase ackA and pyruvate oxidase poxB are knocked out in escherichia coli, key enzymes isocitrate lyase aceA and glyoxylate reductase ghrA in a glycollic acid synthesis path are over-expressed in a genome level, conversion of carbon flow to glycollic acid is enhanced, and the glycolic acid is converted into glycolic acid. On the basis, malic acid synthase AaceB, malic acid synthase GglcB and glycollic acid oxidase glcDEF are knocked out, a branch metabolic pathway is blocked, an efficient strain which is free of plasmids and resistance genes and does not need to induce glycollic acid production is constructed, the accumulation amount of L-valine fermented in a 5L fermentation tank for 48 hours reaches 51.2 g / L, the yield is 0.6 g / g, and the production intensity is 1.06 g / L / h.

The invention relates to the technical field of molecular biology, in particular to a multichannel virus reporting system based on TAT trans-activation. The invention relates to a multichannel virus reporting system based on TAT trans-activation. The multichannel virus reporting system comprises an activated virus and a reporting cell, the activated virus is a human enterovirus A71 for expressing TAT protein; the reporter cells are cells transfected with reporter plasmids; the reporter plasmid comprises an LTR sequence and a plurality of reporter genes which are started to express by the LTR sequence. When the virus is activated to infect the reporter cell, the TAT protein carried by the virus enters the reporter cell to activate the expression of the downstream reporter gene, and the expression level of the reporter gene can reflect the virus replication condition, so that the screening or mechanism research of related antiviral drugs can be realized. According to the technical scheme, the technical problem that the flexibility, stability and high-throughput screening adaptability of a virus reporting system in the prior art are not ideal can be solved, and the virus reporting system has ideal popularization and application prospects.

The invention provides a plasmid, an alpha-ketoglutaric acid engineering strain and a construction method and application of the alpha-ketoglutaric acid engineering strain, the strain is obtained by adopting a directional transformation method, transcriptional levels of katE, katG, sodB and grxA genes are up-regulated, a catR gene and a katA gene are heterologously expressed, a high-copy plasmid pETKG is carried, and the plasmid simultaneously expresses L-glutamate oxidase LGOX genes from Streptomyces sp and Chain A; the strain self-synthesized catalase eliminates H2O2 generated in the process of producing alpha-ketoglutaric acid through enzyme catalysis, engineering bacteria of L-glutamate oxidase LGOX expressed by pETKG plasmids are used for performing whole-cell catalysis on L-sodium glutamate to synthesize alpha-ketoglutaric acid, and the engineering strain is a high-level engineering strain capable of industrially and stably producing alpha-ketoglutaric acid.

The invention provides a nicotinamide nucleotide transhydrogenase mutant and application thereof, valine at the 169th site of the nicotinamide nucleotide transhydrogenase mutant is designed to be mutated into isoleucine, the isoleucine is used for constructing a lysine production strain, and the new strain does not contain plasmids, does not have growth defects, does not need induction, and has the advantages of good genetic stability, high fermentation yield and the like. The lysine can be efficiently and stably produced from the beginning by taking glucose as a substrate.

The invention provides a genome editor enDelIscB and a genome editor enDelIscB-T5E based on an IscB system, and further provides a base editor ICBE and a base editor IABE based on the enDelIscBD60A. According to the invention, a genome editor and a base editor which are more compact in size and higher in cleavage activity and insertion / deletion efficiency are realized by optimizing a single mutation site of the activity of a DelIscB protein cleavage plasmid, an optimized variant of the DelIscB protein and an optimized sgRNA variant of the DelIscB. According to the present invention, it is verified that the albinism model is efficiently constructed by using the enDelIscB editor and the enDelIscB-T5E editor through micro-injection of the mouse embryo, such that the enDelIscB editor and the enDelIscB-T5E editor have great potential in the field of genome editing.

The present invention belongs to the field of genetic engineering technology and discloses a plasmid-free genetically engineered bacterium that can efficiently synthesize γ-aminobutyric acid from scratch using a cheap carbon source as a substrate, a method and an application thereof. The engineered bacterium is based on wild-type E. coli MG1655, overexpresses the T7 RNA polymerase gene; deletes the genes gabT and puuE; overexpresses gadC and gad bm , gdh, gltA, pyc, and ppc genes; and a growth-coupled promoter was used to dynamically regulate the expression of the sucA and argA genes in the GABA production pathway in Escherichia coli. All genetic manipulations in this engineered bacterium were performed within the genome, leaving no plasmid residues and requiring no antibiotics or inducers. The resulting strain exhibited stable production performance and a simple fermentation process. Using the engineered bacterium under a staged pH control process for 38 hours, the yield of γ-aminobutyric acid reached 35.4 g / L, demonstrating promising industrial application potential.

The invention relates to a vaccine for preventing and treating sheep echinococcosis infection and a preparation method thereof.The preparation method comprises the steps that echinococcosis Eg95 full-length gene antigen epitopes are screened through bioinformatics software, screened fragments with high antigenicity are optimized and then subjected to double-copy connection through a flexible linker, His tag sequences are added to the two ends of a tandem sequence, the sequence is as shown in SEQ ID NO.2, and the vaccine for preventing and treating sheep echinococcosis infection is obtained; and the pEE12.4-double Eg95 plasmid is connected with a CHO eukaryotic cell expression vector, so that the pEE12.4-double Eg95 plasmid is obtained. The plasmid is integrated into a CHO eukaryotic cell expression system, the expression quantity of the purified Eg95 protein reaches 10g / L, and a stable and high-yield veterinary vaccine is provided for large-scale production.

The invention discloses an echinococcosis mRNA (messenger Ribonucleic Acid) vaccine as well as a preparation method and application thereof. The preparation method of the mRNA vaccine for the echinococcosis comprises the following steps: carrying out codon optimization on a modified target antigen protein, then assembling the modified target antigen protein with 5 'UTR, 3' UTR and Poly (A) tail, carrying out gene synthesis, then cloning the synthesized gene into a pUC57 plasmid, and sequentially carrying out plasmid linearization, in-vitro transcription and purification on the constructed recombinant plasmid to prepare an mRNA molecule, thereby obtaining the mRNA vaccine for the echinococcosis. Finally, mRNA molecules are wrapped in lipid nanoparticles through a microfluidic method to form the mRNA vaccine for the echinococcosis, and immune effect evaluation is carried out on the mRNA vaccine for the echinococcosis. Experiments prove that the prepared mRNA vaccine for the echinococcosis can activate humoral immunity and cellular immunity of mice at the same time, can provide an effective protection effect for the mice attacking insects, and has a wide application prospect in the aspect of preventing and / or treating the echinococcosis.