60 results about "Antibiotic free" patented technology

The invention relates to the field of biotechnology and female health care, in particular to a stachyose composition for promoting micro-ecological regulation of lactobacillus and application of the stachyose composition, and the composition comprises stachyose and pharmaceutically acceptable auxiliary materials. The stachyose is used as prebiotics to selectively promote proliferation and colonization of the lactobacillus crispatus, the combination of 1%-5% of the stachyose and 0.1%-0.5% of the lactic acid can promote proliferation of the lactobacillus crispatus, the lactobacillus crispatus is one or more of the lactobacillus crispatus, the lactobacillus plantarum and the lactobacillus acidophilus, and the combination of 1%-5% of the stachyose and 0.1%-0.5% of the lactic acid can inhibit gardnerella and candida albicans. The stachyose composition disclosed by the invention is in the form of slow-release gel and bath foam; the composition can be used for treating or preventing bacterial vaginitis, colpitis mycotica and related symptoms, has the advantages of no antibiotics, no drug resistance, high safety and the like, and is suitable for the fields of vagina local nursing and micro-ecological regulation.

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 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 present invention discloses a preparation method of CFS nanozyme. This nanozyme has broad-spectrum bactericidal property, has low toxicity to normal cells, and there is no problem of bacterial drug resistance, and is expected to be used in the field of antibiotic-free sterilization. The CFS-ion-MN wound repair microneedle patch provided by the present invention, as a wound dressing, can transdermally deliver the antibacterial CFS nanozyme to the deep part of the wound, effectively kill bacteria. The CFS nanozyme can also inhibit inflammation in the body through its superoxide dismutase-like activity, promote wound healing, and promote the antibacterial and healing of the wounds of mice infected with Staphylococcus aureus. Among them, Co 2+ and Fe 3+ can inhibit the IL-17 and NF-κB signaling pathways, thereby accelerating wound closure.

The invention belongs to the technical field of genetic engineering, and discloses a plasmid-free genetically engineered bacterium for efficiently synthesizing gamma-aminobutyric acid from beginning by taking a cheap carbon source as a substrate, a method and application of the genetically engineered bacterium. The genetically engineered bacterium is obtained by overexpressing a T7RNA polymerase gene on the basis of a wild type E.coli MG1655; the gene gabT and the gene puuE are deleted; performing overexpression on the genes of gadC, gadbm, gdh, gltA, pyc and ppc; and the expression of sucA and argA genes in a GABA channel produced by escherichia coli is dynamically regulated and controlled by using a growth coupled promoter. The genetic manipulation of the engineering bacterium is carried out on a genome, plasmid residues, antibiotics and inducers are not used, the production performance is stable, and the fermentation process is simple. The engineering bacteria are used for fermenting for 38 hours under a staged pH value control process, the yield of gamma-aminobutyric acid can reach 35.4 g / L, and the engineering bacteria have a relatively good industrial application value.

The invention discloses a mycobacteriophage and application thereof, and relates to the technical field of bacteriophages. The mycobacteriophage is named as Mycobacter phage BZNK001, is preserved in Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 67907-B1, does not contain antibiotic drug-resistant genes and integrase genes, has excellent biological safety and good environmental tolerance, can maintain high biological activity in a temperature range of 37-50 DEG C under an acid-base condition of pH 5-9, and can be used for preparing the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage. The bacillus subtilis has broad-spectrum and high-efficiency non-tuberculous mycobacterium cracking activity, and can effectively crack four clinical key mycobacteria such as mycobacterium abscessus, mycobacterium avium, mycobacterium intracellularis and mycobacterium smegmatis. According to the invention, a mycobacteriophage resource library is enriched, a novel and stable biological prevention and control means is provided for efficient prevention and control of mycobacteria, and the mycobacteriophage has good clinical application and industrialization values.

The invention relates to the technical field of synthesis of natural dyes by a biological method, in particular to an antibiotic-demand-free ornamental blue high-yield strain as well as construction and application of the antibiotic-demand-free ornamental blue high-yield strain. Corynebacterium glutamicum is used as an initial strain, a tricarboxylic acid circulating gene is regulated and interfered, and the copy number of exogenous blue pigment synthetase BpsA in a genome is adjusted, so that the ornamental blue high-yield strain is obtained. The strain does not need to use antibiotics in the fermentation process, the yield is stable, the product production capacity equivalent to the level when antibiotics are used can still be achieved through fermentation under the antibiotic-free condition, and the growth performance of the strain is kept. The method for producing the ornamental blue by using the strain provided by the invention ensures that the ornamental blue product is natural and green, also improves the efficient production capacity of the strain, solves the contradiction between green environmental protection and stable high yield of the ornamental blue dye, is green and environment-friendly, and has a remarkable industrial application prospect.

The invention relates to an intestinal probiotic genetically engineered bacterium as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. According to the invention, Escherichia coli is used as an expression host, endogenous recessive plasmid pMUT1 gene and pMUT2 gene are knocked out, an exo-algin lyase gene and an open-loop uronic acid monomer dehydrogenase gene are subjected to heterologous expression, and a 2-keto-3-deoxy-gluconate kinase gene and a 2-keto-3-deoxy-6-phosphate-gluconate aldolase gene are subjected to overexpression. According to the invention, an endogenous recessive plasmid pMUT2 of Escherichia coli Nissle 1917 is utilized, metabolic pathways of algin and degradation products thereof are introduced, and a stable passage engineered strain EcNc-pMUT2 '-4 which can be proliferated by utilizing algin and degradation products thereof and is free of antibiotic markers is obtained, and the engineered strain has good intestinal tract probiotic performance.

The present application belongs to the field of synthetic biology and functional food technology, and provides a swallowing safety-oriented xanthan gum directional biosynthesis method. In view of the problem that existing xanthan gum is difficult to reduce the risk of swallowing residue and aspiration by relying on viscosity regulation alone, a multi-dimensional evaluation system combining interfacial tribology and extensional rheology is established to determine the side chain modification parameters suitable for swallowing disorder food. By chromosome scarless reconstruction of xanthan gum synthesis gene cluster, the gumF 、 gumG gene is knocked out and the gumL promoter is replaced, an engineering strain without antibiotic resistance gene residue and with genetic stability is constructed, and directional synthesis of xanthan gum with specific side chain structure is realized. The obtained structure customized xanthan gum has a lower mucosal interfacial friction coefficient and a more optimal extensional rheological property, and can synergistically realize interfacial lubrication and bolus cohesion, significantly reducing the wall residue in the pharynx, and keeping the consistency stable in complex food matrix. The present application can provide a safe, stable and industrialized functional xanthan gum raw material for swallowing disorder food.

The present invention discloses a traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and its preparation process, which relates to the related field of biotechnology. The traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation of the present invention combines the synergistic effects of probiotics and traditional Chinese medicine, and has multiple advantages such as regulating oral flora, antibacterial and anti-inflammatory, antioxidant, and relieving fatigue. Lactobacillus acidophilus NM can tolerate acid and bile salts, effectively inhibit pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella, reduce the occurrence of dental caries, gingivitis, and halitosis. It has no antibiotic resistance and is safer for long-term use. The extracts of Scutellaria baicalensis, honeysuckle, and green tea are rich in flavonoids and tea polyphenols, which can further antibacterial and anti-inflammatory and improve oral health. At the same time, this probiotic can reduce blood lactic acid, blood urea nitrogen, and blood ammonia, enhance the activities of SOD, CAT, and GSH-Px, and inhibit lipid peroxidation, thereby relieving body fatigue and enhancing immunity. The preparation forms are diverse.

The application discloses application of eugenol in preparation of a bacteriostatic agent for enhancing symbiotic nitrogen fixation efficiency of legume-rhizobium, and relates to the field of agricultural microorganisms. ‑1 The application is safe and non-inhibiting to seed germination of Medicago polymorpha and target rhizobium LL2 in a concentration range of 500-1000 muL / L ‑1 of eugenol, can significantly reshape rhizosphere microbial community structure by selectively inhibiting competitive microorganisms such as bacillus and streptomyces in soil, and is environment-friendly and free of antibiotic residue risk, thereby providing a green and efficient new approach for improving symbiotic nitrogen fixation efficiency of legume-rhizobium.

The invention discloses an industrial aquaculture management system for antibiotic-free bullfrogs, and belongs to the technical field of aquaculture. The system comprises circulating water antibiotic-free treatment, antibiotic-free seedling cultivation, antibiotic-free cultivation management and a novel drainage vertical pipe system. The circulating water non-antibiotic treatment adopts a closed-loop flow, and antibiotics and ammonia nitrogen are efficiently removed through the working procedures of sewage tank microfiltration, ozone oxidation disinfection, constructed wetland biological photodegradation, aeration tank ammonia nitrogen removal, clean water tank plant bacterium adding purification reflux and the like; a staged plant nutrition and probiotic feeding mode is adopted for antibiotic-free seedling cultivation; the antibiotic-free cultivation management realizes antibiotic-free cultivation through density control, water quality and water temperature regulation and control, scientific feeding and animal health care; the novel drainage vertical pipe adopts a circular hole and vertical seam structure to prevent blocking. According to the method, whole-course antibiotic-free breeding is achieved, the survival rate in the seedling stage reaches 80% or above, the adult frog breeding period is about 120 days, the product is free of antibiotic residues, the national environmental protection and food safety specifications are met, and an efficient solution is provided for factory-like antibiotic-free bullfrog breeding.

The invention belongs to the technical field of biology, and particularly relates to a recombinant strain of high-yield riemerella anatipestifer bacteriophage lyase, a mutant strain, a construction method and application. The invention provides a coding gene of riemerella anatipestifer bacteriophage lyase, and further constructs recombinant lactococcus lactis of high-yield riemerella anatipestifer bacteriophage lyase. The mutant strain provided by the invention is obtained by taking the recombinant lactococcus lactis as a starting strain through a mutation breeding technology and a specific high-throughput screening strategy, has higher secretory expression quantity of riemerella anatipestifer bacteriophage lyase, is good in hereditary stability in an antibiotic-free culture medium, and has a good application prospect. The method is suitable for industrial efficient production of active lyase. The riemerella anatipestifer bacteriophage lyase expressed by the mutant strain has an obvious splitting effect on riemerella anatipestifer in vitro, has obvious prevention and treatment effects on riemerella anatipestifer infection in vivo, and can effectively improve the survival rate of infected chicks and remove pathogenic bacteria in vivo.

The invention discloses an imidazolyl lipid assembly as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: mixing diphenyl phosphate, alcohol and pyridine, and heating for reaction to obtain alkyl phosphate; mixing N, N-diethylethylenediamine, 5-imidazole formaldehyde and a solvent, carrying out a heating reaction in a protective atmosphere, then adding the alkyl phosphate, and continuing the heating reaction to obtain an imidazolyl lipid molecule; dissolving the imidazolyl lipid molecules in an organic solvent to obtain a mother solution; the imidazolyl lipid assembly is obtained from the mother liquor through a film hydration method. The imidazole-based lipid assembly constructed by the invention can utilize the imidazole group to destroy the normal structure of bacteria under the condition of no antibiotic load, and utilizes the catalytic action of the imidazole group to complete the destroy of a biological membrane matrix and an inflammation activation medium, so that the synergism of antibiosis and anti-inflammation is realized, and the imidazole-based lipid assembly has the effect of buffering local acidic pH.

The invention relates to a quick starting method of a biological filter bed based on fungus-bacterium coordinated regulation. The quick starting method adopts a two-step method and comprises the following steps: firstly, quickly starting a bacterium filter bed by taking benzyl alcohol as a carbon source; adjusting the pH value to 5.5-6.2, adding wheat bran, and implementing intermittent hypoxia stimulation (replacing antibiotics) until the absolute copy number ratio of fungi / bacteria is (F / B) gt; and the starting is finished when the temperature is 0.35. The whole process is free of antibiotics, the starting period is smaller than or equal to 14 days, the methylbenzene removal rate is larger than or equal to 95%, the mineralization rate is larger than or equal to 80%, and the method is remarkably superior to the prior art and suitable for large-air-volume and low-concentration hydrophobic VOCs treatment.

The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting the infection rate of lentivirus on target cells. Comprising the following steps: infecting target cells with a lentivirus-containing sample to be detected; preparing a single-cell suspension from the target cells integrated with the lentivirus, uniformly dividing the single-cell suspension into two parallel samples which are identical in number and state, and inoculating the parallel samples to obtain antibiotic treated cells and antibiotic-free treated cells; adding antibiotics into the medicine sieve group for screening, counting to obtain positive infected cells Ne, and counting the control group without adding the medicines at three time points, namely when the dichotomy operation is executed, when the antibiotics start to screen and after the antibiotics are screened, so as to obtain the total cell numbers No.2, No.1 and No.3; and calculating the infection rate result of the lentivirus on the target cells based on the total cell number. According to the method, the sampling time node of the control group is optimized, the cell number of the control group is selected as the denominator of the infection rate calculation when the medicine sieve group samples start to be added, and the accuracy of the experimental result is further improved.

The present invention relates to a resistance-free plasmid production system. The present invention combines toxic transcripts / anti-toxic transcripts, operon-related elements and small RNAs to construct a plasmid comprising a nucleic acid fragment 3 encoding a small RNA, and host cell expression cassettes comprising a toxic gene expression cassette and an anti-toxic gene expression cassette, wherein the toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing the toxic transcript, and the anti-toxic gene expression cassette comprises a promoter, an operon and a nucleic acid fragment 2 for transcribing the anti-toxic transcript. The reverse complementation of the nucleic acid fragment 3 and the nucleic acid fragment 1 produces a steric effect to inhibit the function of the toxic transcript, so that the entire plasmid production process does not need to be added with antibiotics or other inhibitors to realize the screening and production of the plasmid, thereby the harm of antibiotics and inhibitors to the plasmid in cell gene therapy is solved.

Embodiments relate to a composition comprising a bacteriophage or a mutant thereof and a storage solution, wherein the bacteriophage or the mutant thereof is capable of killing a Gram-positive bacterium comprising Staphylococcus aureus, wherein the composition is stable for at least about a year at 39.2-46.4° F. (4-8° C.).

The invention discloses a stable and antibiotic-free plasmid expression system and application thereof in preparation of antitumor drugs. The plasmid expression system comprises a replicon, a regulatable promoter, a ribosome binding site RBS and an axe / txe toxin-antitoxin stabilization module, and the plasmid expression system can realize long-term stable expression of exogenous genes under the antibiotic-free condition. The replicon is selected from one or a combination of more of pUC, pMB1, p15A or pSC101; the promoter is selected from one or a combination of more of J23100, J23150 or J23112, and the RBS is selected from one or a combination of more of B0032, B0033 or B0034. According to the invention, an antibiotic-free and lasting plasmid platform is established for salmonella-based therapy, and a framework, synthetic biology and cancer treatment opportunity are provided for reliable gene delivery in vivo.

The application discloses a traditional Chinese medicine feed additive for preventing and treating Simmental respiratory disease syndrome and a preparation method and application thereof, and belongs to the technical field of livestock feed additives. The additive is prepared from traditional Chinese medicine raw materials, such as ephedra, honeysuckle, forsythia, radix scrophulariae, radix houttuyniae, radix isatidis, platycodon, bitter apricot kernel, radix glycyrrhizae, radix astragali, rhizoma atractylodis, mint, loquat leaf and radix codonopsis, through decocting, extracting, low-temperature extracting after mint, concentrating and spray drying. The application adopts a three-step prevention and treatment strategy of 'dispelling lung-evil, clearing heat and resolving toxin and supporting healthy body', can effectively prevent and adjuvantly treat Simmental respiratory disease syndrome caused by mixed infection of multiple pathogens, significantly reduces the morbidity of calf BRDC, enhances the immune function of the body, promotes growth and weight gain, has the characteristics of no antibiotic residue, no drug resistance, good palatability and the like, and meets the requirements of green and healthy breeding.

The present invention provides for the production of improved viral and non-viral nanoplasmid vectors. Specifically, the invention provides an antibiotic-label-free covalent closed circular recombinant DNA molecule comprising: a. An antibiotic-label-free insert comprising a structured DNA sequence selected from the group consisting of a reverse repeat, a forward repeat, a homopolymerized repeat and an eukaryotic origin of replication; b. A Pol III-dependent origin of replication comprising an R6K [gamma] origin of replication having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 18; and c. An RNA-OUT RNA selection marker, which comprises an RNA-IN modulating RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6, and a method for detecting the RNA-OUT RNA.

Disclosed herein is a method of making an antibiotic-resistant bacteria-free fermented food product to treat gut microbiota dysbiosis. Also disclosed is a fermented food product comprising a combination of unique starter strains of bacteria of Leuconostoc sp., Lactobacillus sp., Pediococcus sp., and / or other genera of lactic acid bacteria, such as Leuconostoc K6-Leu13 (WL-1) Leuconostoc mesenteroides Pediococcus K6-Pedio (WL-2), Pediococcus inopinatus, Lacticaseibacillus paracasei, Leuconostoc citreum, Lactobacillus SH16 (WL-3), Lactobacillus brevis, Pediococcus acidilactici, Latilactobacillus sakei, etc., for successful fermented products, with or without microbial succession.

The invention discloses a method for treating antibiotic-containing aquaculture tail water by utilizing bacteria-algae granular sludge. The method comprises the following steps: (1) domesticating the bacteria-algae granular sludge; (2) an antibiotic-free exposure test stage; (3) a low-concentration antibiotic exposure experiment stage; (4) a high-concentration antibiotic exposure experiment stage; and (5) an antibiotic exposure recovery experiment stage. According to the method, a symbiotic system is formed by inoculating scenedesmus and aerobic granular sludge, oxygen is supplied by utilizing photosynthesis of algae, a carbon source is produced by metabolism of bacteria, low-energy-consumption operation is realized, an antibiotic degradation path is strengthened, and synchronous removal of nitrite nitrogen and antibiotics is promoted by combining a reflux device. The problems that the concentration of nutrient substances in traditional aquaculture tail water is low, antibiotic residues inhibit microbial activity, the concentration of nitrite nitrogen in effluent is high, and the antibiotic removal efficiency is low are solved, and the method is suitable for efficient treatment of the antibiotic-containing aquaculture tail water.

The invention provides an antibiotic-free fermented feed for sows and a preparation method thereof, and relates to the technical field of feeds for sows, the antibiotic-free fermented feed for sows and the preparation method thereof comprise 30-35% of corn, 26-28% of wheat, 10-15% of bran and 12-15% of soybean meal. The feed is prepared from the following raw materials in percentage by weight: 0.5-1% of calcium phosphate, 0.5-1% of calcium hydrophosphate, 0.2-0.5% of sodium chloride, 1-2% of soybean oil, 0.2-0.5% of lysine, 0.1-0.3% of threonine, 1-2% of premix and 0.6-1.2% of compound Chinese herbal medicine through a fermentation process. The fermented feed is free of antibiotics, avoids the situation that the balance of animal intestinal microorganisms is damaged by abuse of antibiotics, is good in palatability and high in nutrient substance utilization rate, can improve the intestinal microflora, can increase the feed intake of lactating sows and piglets, reduces the number of escherichia coli in fecal microorganisms, increases the number of probiotics, and further has the effects of improving the yield of the lactating sows and the piglets. And the added composite Chinese herbal medicine agent can enhance the immunity of the sows and improve the defense resistance of the body when the sows eat the feed, which means that the sows have better disease resistance.

The invention discloses an antibiotic-free screening gene expression system, plasmid, engineering bacteria and application thereof, and relates to the technical field of bioengineering. The expression system provided by the invention comprises a first strain, a second strain, a third strain and plasmids of antibiotic-free screening genes, the first strain, the second strain and the third strain are escherichia coli strains with a thyA gene knocked out, the first strain is used for molecular cloning, the second strain is used for expression of target protein, and the third strain is used for biosynthesis of natural products such as polyketone and non-cellulose peptide; a thyA complementary gene expression cassette is inserted into the plasmid of the antibiotic-free screening gene, and a resistance gene is knocked out; the thyA complementary gene expression box contains a thyA gene.

The application relates to a traditional Chinese medicine and additive compound preparation for treating gizzarditis of laying hens and application, and relates to the technical field of veterinary drugs. The compound preparation is prepared by mixing a traditional Chinese medicine extract and a functional feed additive. The raw material composition of the traditional Chinese medicine extract comprises torrefied malt, torrefied medicated leaven, radix codonopsis, rhizoma atractylodis macrocephalae, rhizoma coptidis, andrographis, dried tangerine or orange peel, white arillus, and liquorice. The additive comprises neutral protease, a mould inhibitor and probiotics. The mould inhibitor is selected from aspergillus flavus B1 degrading enzyme, corn strobukenone degrading enzyme, fumonisin degrading enzyme and vomitoxin degrading enzyme. The probiotics are selected from lactobacillus plantarum, bacillus subtilis and fructo-oligosaccharide. The application is based on the traditional Chinese veterinary theory of food digestion and spleen health, dampness expelling and qi regulation, heat clearing and toxin resolving and mucosa repairing. In combination with the effects of the additive on digestion, mould expelling and intestinal micro-ecological regulation, the application has a remarkable treatment effect on gizzarditis of laying hens, can improve the feed intake and weight gain, reduce the gizzard index, promote the repair of gastric mucosa, and has no antibiotic residue and no toxic side effect.

The invention provides a resistance-free selection marker plasmid, a preparation method, recombinant bacteria and application, and belongs to the technical field of gene engineering. The plasmid comprises an EM7 promoter, a ccdA41 antitoxin gene and an R6K gamma replication origin, the length of the plasmid molecule is less than 600bp, and the plasmid molecule does not contain antibiotic resistance genes. The plasmid is small in molecular weight and free of antibiotic resistance genes, effectively avoids safety risks caused by use of antibiotics, strictly depends on designated host replication of chromosome integration ccdB, meets biological safety requirements and is suitable for safe production of biological products such as vaccines and recombinant proteins. According to the invention, the recombinant bacterium is used for producing the plasmid without the resistance selection marker simply, conveniently and quickly, the success rate is high, and the recombinant bacterium utilizes the GT115-ccdb strain and the plasmid containing the ccdA41 antitoxin gene to realize stable maintenance and expression of the plasmid in a host through a toxin-antitoxin composite regulation mechanism.

The invention discloses canned highland barley milk and a production method thereof, and relates to the technical field of dairy product production. Comprising the following steps: S1, pretreatment and stirring; preparing raw materials: selecting fresh raw milk, and ensuring no antibiotic residue and pathogen pollution; highland barley is subjected to baking, grinding and enzymolysis treatment in advance; heating: heating the raw milk to 55-60 DEG C, and keeping a stable temperature by adopting a temperature control system; adding the cow milk, the enzymolysis highland barley powder and the crystalline fructose according to the proportion; and stirring and fixing the volume: stirring for 25-30 minutes at a preset temperature to ensure full mixing, and then fixing the volume to a preset volume. Through baking, shelling, grinding and enzymolysis treatment, active ingredients of the highland barley are fully released, and the solubility of the highland barley and the absorptivity of the highland barley in milk are improved; the process not only optimizes the nutritional value of the product, but also significantly improves the taste.

The invention discloses a functional nutritional feed for promoting muscle gaining and fat reducing of cattle and sheep and a preparation method thereof, and belongs to the technical field of enzyme engineering feed processing. The feed is suitable for the fattening stage of cattle and sheep, and can effectively improve the lean meat percentage, reduce visceral fat deposition and improve the meat quality through scientifically proportioned nutritional ingredients. The feed comprises the following main components: soybean meal, oligosaccharides, nattokinase, conjugated linoleic acid, a compound enzyme preparation, dry yeast powder and the like, and can promote the growth of bones and muscles and reduce the deposition of fat at the same time. Experimental results show that the nutritional feed has remarkable effects on improving daily gain, carcass weight, dressing percentage and lean meat percentage of cattle and sheep, is free of antibiotic and hormone residues, and is safe and green. By using the fattening feed, the problem of excessive fat accumulation caused by a traditional fattening method can be effectively solved, the breeding benefits are improved, and the requirements of the market for high-quality beef and mutton are met.

The invention relates to the field of construction of gene deletion strains, in particular to a Brucella M5bvfA gene deletion strain as well as a construction method and application thereof. The specific construction method comprises the following steps: connecting and synthesizing upstream and downstream homologous recombination sequences of a bvfA gene to obtain a targeting fragment, and cloning the targeting fragment into a suicide plasmid containing a gentamicin resistance gene to obtain a targeting plasmid; the targeting plasmids are electrically transformed into escherichia coli, and donor bacteria are obtained; the method comprises the following steps: jointing donor bacteria with brucella M5 recipient bacteria, and screening to obtain gentamicin-resistant brucella clones; the method comprises the following steps: inoculating gentamicin-resistant brucella cloned bacterial liquid into a culture medium containing cane sugar, culturing until monoclone is formed, and identifying and screening to obtain a traceless brucella M5bvfA gene deleted strain. The deletion strain can distinguish natural infection from vaccine immunity, has no antibiotic gene residue, and has the advantages of low toxicity and traceless deletion.