35 results about "Plectasin" patented technology

The invention discloses a preparation method of multi-copy high expressed recombined plectasin by pichia pastoris. The method comprises the following steps: a plectasin expressing gene sequence is designed according to preference performance to codon translated by pichia pastoris; the optimized plectasin gene is fused on an alpha-factor signal peptide C terminus of an expression vector pPICZalphaA to construct a single-copy expression vector, the vector comprises a plectasin expression cassette containing a start signal element alcohol oxygen dehydrogenase strong promoter (AOX), alpha-factor signal peptide gene and a plectasin gene fused in C terminus, a stop signal element AOX (TT) and the like. A complementation principle of restriction endonuclease Bg1II and BamHI cohesive end is used to obtain plectasin gene-containing recombinant plasmid of different copy cascade expression cassettes, pichia pastoris is electrotransformed and secreted and expressed plectasin with high efficiency under the methanol induction. The expression level and plectasin gene copy number exist a linear relation. The constructed multi-copy high expressed yeast cells can be used for raising the output and reducing the cost, and is adapted to large scale production of plectasin.

The invention discloses a recombinant antibacterial peptide which has an amino acid sequence shown in SEQ ID NO.1. The invention also discloses a nucleotide sequence for encoding the recombinant antibacterial peptide, a recombinant vector and recombinant bacteria comprising the nucleotide sequence, and a preparation method and antibacterial application of the recombinant antibacterial peptide. The recombinant antibacterial peptide has high antibacterial activity, and the method for preparing the recombinant antibacterial peptide is simple and high in yield and has good application prospect.

The invention discloses recombinant bacteria for expressing plectasin and an application thereof. The classification name of the recombinant bacteria is lactococcus lactis NZ9700-pGMN3, the preservation serial number of the recombinant bacteria is CCTCC NO: M2014611, the recombinant bacteria can achieve upper-high expression of plectasin protein, the expression quantity of the recombinant bacteria far exceeds that of other anode recombinant bacteria, and the recombinant bacteria can be directly used for preparing anti-bacteria medicament, have no hidden safety risks and hardly have any side effects. The recombinant bacteria have the functions of inhibition of methicillin-resistant staphylococcus aureus, streptococcus agalactiae, streptococcus pyogenes, enterococcus faecalis and the like. Bacterial strains of the recombinant bacteria can achieve efficient and continuous expression under self-induction, the recombinant bacteria can express and secrete fusion protein containing plectasin in the fermentation process, the total yield of protein is more than 1.41g/L, plectasin accounts for more than 70%, and the recombinant bacteria have the potential of treating mammitis and infectious diseases of a genital tract.

The invention discloses an application of plectasin in inhibiting streptococcus agalactiae. The plectasin is found to be good at inhibiting the streptococcus agalactiae through bacteriostasis experiments; the plectasin is further prepared into corresponding agentia, such as plectasin gel and plectasin emulsion, the agentia achieve good curative effects for mastitis, wherein the antibacterial anti-inflammatory effect of the gel is better, the action time lasts longer, the moisturizing effect is better, action sites can be effectively isolated from making contact with air, and a wound is prevented from infection and inflammation. The plectasin can be used for preparing bacteriostat of the streptococcus agalactiae and further used for curing the mastitis, and especially for the mastitis caused by the streptococcus agalactiae which is difficult to cure at present, and a good curative effect is achieved.

The invention discloses a piglet compound feed containing antimicrobial peptide for the later stage of nursery and a preparation method of the piglet compound feed. The compound feed is prepared from the following components in parts by weight: 58 to 61 parts of maize, 14 to 16 parts of bean pulp, 8 to 12 parts of imported DDGS (Distillers Dried Grains with Soluble), 5 to 8 parts of fermented soybean meal, 2 to 4 parts of glucose, 1.8 to 5 parts of imported fish meal, 0.4 to 0.8 part of calcium hydrophosphate, 0.15 to 0.2 part of lysine, 0.1 to 0.4 parts of table salt, 0.8 to 1.0 part of stone powder, 0.15 to 0.2 part of composite trace element premix, 0.10 to 0.15 part of rice bran, 0.08 to 0.10 part of methionine, 0.05 to 0.10 part of threonine, 0.03 to 0.10 part of choline, 0.02 to 0.05 part of composite multivitamin premix, 0.02 to 0.05 part of antioxidant, 0.01 to 0.03 part of plectasin antibacterial peptide, 0.01 to 0.03 part of cecropin and 0.02 to 0.05 part of plant essential oil complex. Proved by experiments, the antibacterial peptide feed disclosed by the invention can obtain body weight which is slightly higher than that of an antibiotic group and better feed rewards and has no influence on metabolism of piglets, body tissue proteinosis and an immunologic function. The piglet compound feed has important economic value and social value for ensuring piglet growth, intestinal health and food safety of human beings.

The invention discloses a method for increasing expression amount of pichia pastoris for secreting and expressing plectasin. The method is characterized in that a pichia pastoris genome is used as a template, a PDI (protein disulfide isomerase) gene is amplified, a PDI segment is treated by BstBI and EcoRI double enzyme digestion, and is connected to a pPIC6aphlaA carrier segment which is also treated by the BstBI and EcoRI double enzyme digestion, and an expression carrier pPIC-PDI is formed; the PDI is expressed in a plectasin expression strain, three pairs of disulfide bonds of the plectasin are promoted to form a correct space structure, the plectasin is smoothly secreted and expressed out of cells, and the purpose of increasing the expression amount of plectasin is realized. The method has the advantage that the expression amount of plectasin is increased by 30%.

The invention discloses a plectasin mutant, a gene for coding the plectasin mutant, and a preparation method and use of the plectasin mutant. The plectasin mutant has an amino acid sequence shown in the formula of SEQ ID No.2 in the sequence table. A nucleotide sequence of the gene for coding the plectasin mutant is shown in the formula of SEQ ID No.3. The invention also discloses a fusion protein of the plectasin mutant and a gene for coding the fusion protein. Compared with the existing plectasin, the prepared plectasin mutant has higher bacteriostatic activity, less plectasin use amount, better bacteriostatic effects and a wide medical application prospect.

The invention discloses a recombinant plectasin as well as an expression and purification method and application thereof. The amino acid sequence of the recombinant plectasin is as shown in SEQIDNO.2. The recombinant plectasin is prepared by sequentially carrying out enrichment culture, inductive expression culture and centrifugalization on a recombinant strain containing a gene for encoding an amino acid; sequentially taking, cracking and centrifugalizing thalli, so that a cracked supernatant is obtained; and purifying the cracked supernatant, so that the recombinant plectasin is obtained. The obtained recombinant plectasin is subjected to enzyme digestion by enzyme SUMOpro, and then the obtained product is dialyzed, so that plectasin can be obtained. According to the invention, through the optimization on the expression and purification method of recombinant plectasin, a large amount of high-purity and high-activity plectasin can be obtained, and the obtained plectasin is good in antibacterial effect, wide in antibacterial range, and large in antibacterial spectrum.

The invention discloses a method for preparing plectasins by saccharomyces cerevisiae. The method comprises the following steps: a, transforming and amplifying saccharomyces cerevisiae genes; b, building recombinant plasmids pYES2-CPS-Plec of the saccharomyces cerevisiae genes in yeast cells; c, transforming INVSC1 cells of saccharomyces cerevisiae genes by the recombinant plasmids pYES2-CPS-Plec; d, screening positive bacteria strains, and performing digestion and identification; e, cultivating transformed bacteria to obtain antimicrobial peptide saccharomyces cerevisiae genes; and f, identifying the activity of the antimicrobial peptide saccharomyces cerevisiae genes. The plectasins provided by the invention has an obvious bacteriostatic action on a saccharomyces cerevisiae expression system, and the antibacterial effect is stronger than that of the antibacterial effect before modification. The saccharomyces cerevisiae expression system provided by the invention has the following advantages of being better in antibacterial effect, fast in growth rate and high in yield, is likely to realize high-density culture and does not need adopt methanol induction; furthermore, a protein product for expressing secretion is likely to be separated and purified; moreover, the saccharomyces cerevisiae expression system has a plurality of posttranslational modification features, such as polypeptide folding, glycosylation, methylation, acetylation and the like; in addition, due to a clear genetic background, the saccharomyces cerevisiae expression system is likely to operate an expression vector.

The invention discloses a fusion expression plectasin, a preparation method and application thereof. The amino acid sequence of the recombinant plectasin is SEQ ID NO:1; and a nucleotide sequence designed according to an escherichia coli preferred codon is SEQ ID NO:2. According to the invention, a prokaryotic expression vector is utilized to construct the escherichia coli BL21 (DE3) host strain capable of expressing plectasin-thioredoxin fusion protein. The strain is subjected to multiplication culture, isopropyl-beta-D-isopropylthiogalactoside induction expression is carried out, centrifugation is performed to harvest thallus, the thallus is cracked, then centrifugation is conducted to acquire the supernatant, and affinity chromatography purification is carried out to obtain plectasin fused protein, the growth of gram positive staphylococcus aureus, streptococcus pneumoniae and the like can be significantly inhibited without removing thioredoxin by enzyme digestion. According to the invention, plectasin fused protein has obvious bacteriostatic effect without removing fusion protein by enzyme digestion, and the production cost is greatly reduced.

The invention relates to a plectasin mature polypeptide dimer fusion protein, genes for coding the protein and an expression vector containing a DNA sequence for coding the protein. The invention also relates to a method for expressing the plectasin mature polypeptide dimer fusion protein in recombinant Pichia pastoris. The method comprises the following steps: using the expression vector containing the DNA sequence for coding the plectasin mature polypeptide dimer fusion protein to convert Pichia pastoris and obtain recombinant Pichia pastoris, and performing fermentation culture to secrete the plectasin mature polypeptide dimer fusion protein. The obtained plectasin mature polypeptide dimer fusion protein has bioactivity and can be widely used in the fields such as medical treatments, foods, feeds and scientific researches.

The invention discloses a monoclonal antibody against plectasin and a detection kit for plectasin. The monoclonal antibody is secreted by an anti-plectasin monoclonal antibody hybridoma 5D11 which ispreserved in China General Microbiological Culture Collection Center on May 30, 2018, with an accession number of CGMCC No. 15790. The kit comprises a 96-well plate coated by a plectasin antigen, theanti-plectasin monoclonal antibody, a plectasin standard substance, horseradish peroxidase-labeled goat anti-mouse IgG, a sample diluent, a sample washing solution, a TMB developing solution and a stopping solution. The anti-plectasin monoclonal antibody secreted by the hybridoma has high specificity and high titer; and the kit can detect trace amounts of plectasin in various samples and has the advantages of simple operation, high sensitivity, good repeatability and the like.

The invention discloses a method for producing plectasin through sweet wormwood herbs. The method includes the following steps of firstly, refitting and obtaining a carrier T202; secondly, optimizing and amplifying a plectasin gene; thirdly, establishing recombinant plasmid T202-Plec for expressing the plectasin gene in sweet wormwood herb cells; fourthly, converting agrobacterium EHA105 cells through the recombinant plasmid T202-Plec; fifthly, screening out positive strains, and conducting enzymatic detection; sixthly, transforming the sweet wormwood herbs to obtain sweet wormwood herb transformation plants with positive antimicrobial peptide plectasin; seventhly, quantitatively authenticating the expression amount of plectasin through PCR(qRT-PCR) in real time. An sweet wormwood herb expression system adopted for the method has the advantages that according to the codon preference of the sweet wormwood herbs, the nucleotide sequence of the plectasin gene is optimized, and the efficient and stable mRNA transcription, rapid and accurate translation and high external protein accumulation are ensured; plant expression plectasin is low in cost, safe and high in economic benefit; the expressed protein product can be directly used for being added without being purified.

The invention discloses a recombinant engineering bacterium for efficiently expressing plectasin, the genome of the recombinant engineering bacterium contains a plectasin quadruple concatemer, the plectasin quadruple concatemer is formed by connecting four gene segments for encoding plectasin in series through gene segments for encoding T2A peptide, and the nucleotide sequence of the plectasin quadruple concatemer is shown as SEQ ID NO.5. The construction method comprises the following steps: adding a 6 * His tag at the N end of plectasin for purification, adding a T2A peptide sequence at the C end of plectasin for connection with a next group of gene expression cassettes, and constructing a quadruple concatemer of plectasin in this way; and transforming the recombinant expression vector into a host competent cell to obtain the recombinant engineering bacterium. The recombinant engineering bacterium capable of efficiently expressing plectasin is applied to preparation of plectasin, inhibition of staphylococcus aureus and preparation of a preparation for inhibiting staphylococcus aureus. The recombinant engineering bacterium can efficiently express plectasin and has a good application prospect.