1221 results about "Plasmid" patented technology

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 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 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 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 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 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 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.

The invention belongs to the technical field of synthetic biology, and particularly relates to recombinant escherichia coli for efficiently producing O-acetyl-L-homoserine as well as a construction method and application of the recombinant escherichia coli. According to the invention, CRISPR-Cas9 gene editing is combined with metabolic modification, so as to precisely integrate OAH biosynthesis key nodes: introducing and overexpressing a mutant metX gene at a genome pseudogene site to enhance the expression of homoserine acetyltransferase, knocking out byproducts such as poxB and mgsA to synthesize genes, and carrying out high-yield synthesis of OAH. Corynebacterium glutamicum thrA (anti-feedback inhibition), aspB and bacillus subtilis acsA-acuA genes (construction of an acetic acid switch) are introduced, gene expression of gltA, tpiA and the like is optimized to increase supply of precursors such as acetyl CoA and the like, and finally a stable strain without plasmids, antibiotics and inducers is obtained. The production cost is effectively reduced, and the method has the advantage of high genetic stability and shows a good application prospect in OAH industrial production.

The invention relates to the technical field of biology, in particular to a recombinant fusion protein, polynucleotide for coding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell and a method for preparing target polypeptide by using the recombinant fusion protein. The recombinant fusion protein comprises tag polypeptide-target polypeptide-(linker 1-linker x-linker 2-target polypeptide) n from N terminal to C terminal, x is the number of linkers, x is 0 or 1, n is a positive integer not less than 1, the tag polypeptide is SEQ ID NO.1 or SEQ ID NO.2, the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site, and the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site. And the connecting peptide 2 contains a WELQ protease enzyme cutting site. The recombinant fusion protein disclosed by the invention is expressed in an inclusion body form, the synthesis process is simplified, the target polypeptide can be prepared with high yield and high purity by using the recombinant fusion protein disclosed by the invention, and the production cost can be reduced from multiple dimensions.

The invention discloses a method for improving the solubility and thermal stability of sweet protein, and belongs to the technical field of biosynthesis, the method comprises the following steps: constructing a gene tandem recombinant plasmid containing a monellin x-3C-sfGFP-3C-monellin y expression cassette, x is greater than or equal to 1, y is greater than or equal to 1, 5 is greater than or equal to x + y is greater than or equal to 3, a 3C protease recognition sequence is also inserted between adjacent copies of monellin, and each monellin is connected with a purification tag; the gene tandem recombinant plasmid is transformed into escherichia coli and inducible expression is carried out, thalli are split and purified to obtain fusion protein, 3C protease is used for enzyme digestion, and the target protein is obtained after re-purification. The total protein yield and the solubility proportion are remarkably improved by adopting a series construction mode of plasmids, meanwhile, the stability of monellin is effectively improved, the purification step is simpler and more convenient, and the method is suitable for industrial production.

The invention discloses a method for producing mRNA (messenger ribonucleic acid) template plasmids by fermenting escherichia coli. When the recombinant escherichia coli is fermented and cultured, a seed solution of the recombinant escherichia coli is inoculated into a basic culture medium to be fermented and cultured until the OD600 value of fermentation liquor is 18-22, then a fed-batch culture medium is continuously added at the feeding rate of 5-15 mL / L / h to be subjected to continuous fed-batch fermentation culture, the basic culture medium contains 0.05-0.2 mmoL / L of ferric salt compound and 0.05-0.2 mmoL / L of copper chloride dihydrate, and the basic culture medium contains 0.05-0.2 mmoL / L of ferric salt compound and 0.05-0.2 mmoL / L of copper chloride dihydrate. The basal culture medium contains 10-15 g / L of copper chloride dihydrate and 100-300 mg / L of p-hydroxybenzoic acid, the carbon source of the basal culture medium contains 0.1-0.3 mmoL / L of a ferric salt compound, 0.1-0.3 mmoL / L of copper chloride dihydrate and 100-300 mg / L of p-hydroxybenzoic acid, the carbon source of the fed-batch culture medium contains 360-480 g / L of glycerol, and the ferric salt compound is one or more of ferrous citrate, ferric ammonium citrate or ferrous sulfate heptahydrate. According to the method, the yield and superhelix content of the mRNA template plasmids are improved by optimizing the culture medium formula and culture conditions, and large-scale production of the high-quality target mRNA template plasmids is facilitated.

The invention provides a resveratrol production strain and a construction method and application thereof, according to the strain, tyrAfbr, tyrB, aroE, pntAB, ompF, aroGfbr and accA genes are up-regulated, a carbon storage regulation factor csrA gene is knocked out, the transcriptional level of a fabF gene is down-regulated through a BBaJ23113 promoter, a TAL gene from Rhodotorula glutinis, a 4CL gene from Arabidopsis thaliana and an STS gene from Vitis vinifera are heterologously expressed on a genome, and the strain carries a high-copy plasmid PET-28a-WAL at the same time; the strain takes glucose as a carbon source, resveratrol is efficiently and stably synthesized from the beginning by adopting a fermentation method, the production cost is low, and efficient production of resveratrol is realized.

The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-PR recombinant rabbit monoclonal antibody and application thereof.The anti-PR recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5. The invention also relates to a nucleotide sequence for coding the anti-PR recombinant rabbit monoclonal antibody, a recombinant plasmid or an expression vector, a preparation method, application of the anti-PR recombinant rabbit monoclonal antibody in a PR protein detection method or device, and the like. The PR recombinant rabbit monoclonal antibody disclosed by the invention has the characteristics of good specificity, strong positive signal and the like, so that scoring is easier in IHC staining, and cancer detection and distinguishing are more accurate.

Provided are a preparation method for a yeast-expressed type I recombinant human collagen with a tri-helical structure and use thereof. The preparation method comprises the steps of performing fermentation using an engineered bacterium and performing purification to obtain the type I recombinant human collagen with a tri-helical structure. A method for constructing the engineered bacterium comprises the step of transforming a host bacterium with plasmids pMChZ-α1(I), pMCrN-α2(I), pPIC9K-P4H(DP)-1, and pMCeH-[α1(I)] 2α2(I) to construct the engineered bacterium. The type I recombinant human collagen with a tri-helical structure prepared using the preparation method comprises hydroxyproline, and hydroxyproline accounts for 30% or more of total proline.

The invention relates to the field of microbial metabolism engineering, and discloses a method for increasing the yield of 2 '-fucosyllactose (2'-FL) through the synergistic effect of various alpha-1, 2-fucosyltransferases. According to the method, two or more alpha-1, 2-fucosyltransferases with different sources are co-expressed in microbial cells, and the complementary enzymatic characteristics of the alpha-1, 2-fucosyltransferases are utilized, so that the synthesis efficiency of the 2 '-FL is remarkably improved, and the yield of the 2'-FL is effectively improved. The microbial cell is escherichia coli C43 (DE3) (E. coli C43 lacZ waaF with lacZ and waaF genes knocked out, the serial number is SL), a pETDuet-1 plasmid is constructed, and genes manB, manC, gmd, wcaG and zwf are overexpressed on a pETDuet-1 vector, so that the microbial cell can be used for preparing the microbial cell. In a 5L fermentation tank, after the final strain SL07 is used for final fermentation for 68 hours, the yield of 2 '-FL reaches 148g / L, and the production intensity reaches 2.18 g / L / h. The embodiment shows that the engineering strain for co-expressing the alpha-1, 2-fucosyltransferase from different sources can obviously improve the yield of the 2 '-FL.

The invention discloses a recombinant pichia pastoris strain as well as a construction method and application thereof, and belongs to the field of microbial fermentation. The construction method specifically comprises the following steps that pGAPZB is used as a plasmid skeleton, a recombinant expression plasmid pGAPZB-ACSec containing an ACSec gene is constructed, and the sequence of the ACSec gene is shown as SEQ ID No.1; linearizing the recombinant expression plasmid, and transforming the linearized recombinant expression plasmid into a pichia pastoris host; and screening a pichia pastoris strain capable of expressing the ACSec gene. According to the invention, the ability of pichia pastoris to grow and metabolize by utilizing acetic acid is improved, and the application potential of pichia pastoris in fermentation production and synthesis of chemical products taking acetic acid as a precursor by utilizing a cheap carbon source is shown.

The invention relates to a gene, a protein and a plasmid of a trachinotus ovatus apoptosis regulatory factor Bok and application of the gene, the protein and the plasmid, and belongs to the field of molecular biology, the sequence of cDNA nucleotide of the trachinotus ovatus apoptosis regulatory factor Bok is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2. The invention further provides a construction method of the eukaryotic expression plasmid of the trachinotus ovatus apoptosis regulatory factor Bok. The regulation effect of the constructed eukaryotic expression vector on other apoptosis-related factors of the trachinotus ovatus is verified, and after the eukaryotic expression plasmid for the apoptosis regulation factor Bok of the trachinotus ovatus is injected into a fish body, the disease resistance of the fish body can be remarkably improved, and a remarkable immune protection effect is achieved.

The invention discloses a construction method of nicotinamide engineering bacteria for cosmetics, which comprises the following steps: amplifying target DNA, recovering the target DNA, carrying out seamless cloning or CPEC connection on the recovered DNA fragment and a vector, carrying out seamless cloning by using a seamless cloning kit of an all-type gold company, carrying out connection by using a sterile ultrapure water complementing system, and carrying out plasmid construction. The method comprises the following steps: inoculating a liquid culture medium with a strain which is verified to be correct, carrying out plasmid extraction on the obtained bacterial liquid by using an AxyPrep plasmid small extraction kit, carrying out shake flask small test on enzyme activity, thallus propagation and protein expression after chassis strain gene editing, adding a substrate 3-cyanopyridine solution into the crushed thallus for reaction, and carrying out HPLC (High Performance Liquid Chromatography) detection on nicotinamide. According to the method, the nitrile hydratase is expressed through metabolism of microorganisms, conversion from 3-cyanopyridine to nicotinamide is achieved through one-step reaction after cell disruption, the enzyme activity of the nitrile hydratase is improved, and meanwhile the generation amount of the by-product nicotinic acid is reduced.

The invention provides a valine production strain as well as a construction method and application thereof, the strain is constructed by utilizing a CRIPSR / Cas9 system gene editing technology based on allele exchange, an artificial operon is designed and constructed, the artificial operon is constructed at a ygaY site, a strong promoter Ptrc is used for controlling ilvIH (G14N S17F)-pykF to carry out synchronous multi-copy expression, and the valine production strain can be used for producing valine. On the basis of the strain, an artificial operon is constructed at an ycgH site, a strong promoter Ptrc is used for controlling ilvC-ilvD to carry out synchronous multi-copy expression, meanwhile, feedback inhibition of valine on ilvIH is relieved, and an ilvIH natural promoter is replaced by the strong promoter Ptrc, so that more pyruvic acid flows to valine; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for efficiently synthesizing valine from the beginning by taking glucose as a substrate.

The invention discloses preparation and application of grouper enterospora sporowall protein SWP26 and a polyclonal antibody of the grouper enterospora sporowall protein SWP26, and belongs to the field of animal quarantine, the grouper enterospora sporowall protein SWP26 is obtained by amplifying grouper enterospora genes by using a PCR (Polymerase Chain Reaction) method, shearing a target band and transferring the target band into a pET-32a vector to construct a recombinant plasmid, and then transforming escherichia coli BL21 (DE3) for induced expression. A Ni-NTA affinity chromatography method is used for protein purification, animal immunization and polyclonal antibody purification are carried out on the purified SWP26 recombinant protein, western blot detection is carried out on the purified antibody, an obvious signal appears at about 43kDa, and it is proved that the anti-SWP26 polyclonal antibody can be subjected to a specific reaction with the SWP26 purified protein. The invention clones and identifies the high-abundance spore wall protein SWP26 positioned on the surface of the enterosporidium of the grouper for the first time, belongs to a specific protein of the enterosporidium of the grouper, and can be used as a drug target for treating enterocytozoonosis of the grouper.

The invention provides a chimeric envelope glycoprotein, a preparation method thereof and an envelope plasmid, a packaging method and a kit of the chimeric envelope glycoprotein, and particularly relates to a preparation method of the chimeric envelope glycoprotein, the chimeric envelope glycoprotein and a lentivirus envelope plasmid. The invention discloses a pseudotyped packaging method of lentivirus and a kit. The amino acid sequence of the wild type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of other envelope glycoproteins, and compared with the initial wild type envelope glycoprotein, the obtained chimeric envelope glycoprotein can effectively improve the lentivirus packaging efficiency, biological activity and other properties.

The application discloses an Escherichia coli probiotic with high beta-alanine yield and application thereof, and the recombinant genetically engineered bacterium takes a wild-type Escherichia coli probiotic as a starting strain and is subjected to one or more gene edits, including Trc promoter strengthening of a panD gene, an aspC gene, an sdhC gene and a ppc gene, overexpression of a panD gene from Bacillus subtilis, an aspB gene from Corynebacterium glutamicum, an aspA gene and a pyc gene from Corynebacterium glutamicum on a plasmid pTrc99a, and knockout of a cycA gene. 600 Compared with an existing reported Escherichia coli probiotic beta-alanine production strain, the OD of the recombinant genetically engineered bacterium is 4.4 times that of the existing reported strain, and the beta-alanine yield is 6 times that of the existing reported strain, so that the recombinant genetically engineered bacterium has great industrial application potential.

The invention belongs to the technical field of microbial engineering, and discloses a method for producing eicosapentaenoic acid (EPA) with high yield by schizochytrium limacinum based on random mutation and low-temperature screening and application, and the method comprises the following steps: driving overexpression of a G418 resistance gene through a P2520 promoter, constructing a random insertion mutant library, and screening to obtain a schizochytrium limacinum mutant strain with a randomly mutated genome; coating in a culture medium containing G418 resistance for culturing, and screening out strains which are resistant to low temperature and good in growth state at 15 DEG C; and inoculating into a fermentation culture medium, adding MgSO4 with the final mass concentration of 0.1% at the initial stage of fermentation, supplementing MnCl2 with the final mass concentration of 0.05% when fermentation is performed for 72 hours, and culturing to obtain a mutant strain. According to the invention, the G418 resistance gene carried by the plasmid is randomly inserted into the schizochytrium limacinum genome, and low-temperature induced screening is combined, so that the target limitation of traditional homologous recombination is broken through, and a stable strain with multiple mutation superposition can be obtained through enrichment.

The invention discloses a carbapenemase OXA-58-like positive plasmid and a host bacterium propagation risk assessment method thereof, which are used for carrying out integrated assessment on the plasmid and the host bacterium in three dimensions of drug resistance, pathogenic potential and propagation potential, and carrying out comprehensive risk grading and classification by taking the OXA-58-like positive plasmid and the host bacterium thereof as a plasmid-host complex. The invention develops a set of OXA-58-like gene screening and systematic analysis process based on a public plasmid database aiming at the defects that the existing research of the OXA-58-like gene is mostly limited to a single species or a local sample and a standardized automatic analysis method is lacked. According to the method, all OXA-58-like gene positive plasmids in a database can be subjected to unified and efficient drug-resistant gene mining, the association rule between the drug-resistant genes and plasmid pedigree, host strains and geographical distribution is systematically disclosed, and important technical support is provided for drug resistance propagation monitoring and risk assessment.

The invention discloses a primer and a method for quantitatively monitoring the biomass of sargassum hemiphyllum based on an environmental DNA technology, and belongs to the technical field of molecular ecology. The primer probe group comprises an upstream primer, a downstream primer and a fluorescent probe which are specifically targeted to the sargassum hemiphyllum mitochondria COX1 gene, and the sequence is shown as SEQ ID NO.1-3. The kit comprises the primer probe group, a qPCR (quantitative polymerase chain reaction) premixed solution, nuclease-free water and a sargassum hemiphyllum plasmid positive control. The method comprises the following steps: collecting a water sample, enriching eDNA, performing qPCR detection by using the primer probe group after extraction and purification, and realizing qualitative detection and quantitative evaluation of sargassum hemiphyllum through a Cq value or a standard curve. The method disclosed by the invention has the advantages of high sensitivity, strong specificity, no damage to the environment and target organisms, capability of realizing large-scale rapid general survey and the like, and is suitable for early warning of gulfweed blooms, investigation of population distribution and evaluation of ecological influence.

The invention relates to the technical field of bioengineering, in particular to a penicillin G acylase mutant, polynucleotide, an expression vector and application. The penicillin G acylase mutant is obtained by carrying out site-directed mutagenesis on a wild type penicillin G acylase gene of parent Escherichia coli, and carrying out site-directed mutagenesis on the wild type penicillin G acylase gene to obtain the penicillin G acylase mutant. Phenylalanine (F) at the 24th site of an alpha chain, proline (P) at the 383rd site of a beta chain, threonine (T) at the 384th site of the beta chain, glutamic acid (G) at the 385th site of the beta chain and serine (S) at the 386th site of the beta chain of the penicillin G acylase are introduced and mutated by a whole plasmid PCR (Polymerase Chain Reaction) technology to obtain mutants. When the penicillin G acylase mutant obtained by the invention is used for catalyzing methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid to synthesize cefamandole, the synthesis activity is improved, and meanwhile, the side reaction rate is greatly reduced.

The invention relates to the field of biological medicine preparation, and particularly provides liraglutide precursor peptide tandem recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered host cell and a method for preparing target polypeptide. The recombinant fusion protein is fusion peptide-target polypeptide-(linker peptide-target polypeptide) n from the N terminal to the C terminal, n is a positive integer from 4 to 6, the fusion peptide is SEQ ID NO.1, the target polypeptide is liraglutide precursor peptide, the linker peptide comprises a spacer peptide and a protease restriction enzyme cutting site, the spacer peptide is SEQ ID NO.2, the isoelectric point of the recombinant fusion protein is 4.6-4.7, and the average hydrophilic value is-0.780--0.765. The proportion of the target polypeptide in the recombinant fusion protein is high, use of solvents under extreme conditions is avoided in the production process, the enzyme digestion efficiency is high, and the product purity and yield are high.