293 results about "Competent cell" patented technology

The invention discloses a recombinant humanized XVII type collagen as well as an expression vector and a genetically engineered bacterium thereof. Four functional fragments with high activity are obtained through screening, one of the fragments is optimized and then combined according to a certain sequence to obtain recombinant humanized XVII type collagen JYC1703, or one of the fragments is optimized and then combined according to a certain sequence, and GPP sequences are fused at an amino terminal and a carboxyl terminal respectively to obtain recombinant humanized XVII type collagen JYC1701 and JYC1702, and the recombinant humanized XVII type collagen JYC1701 and JYC1702 are combined according to a certain sequence to obtain the recombinant humanized XVII type collagen JYC1701 and JYC1702. After codon optimization, amplifying and cloning into an expression vector, transforming into pichia pastoris competent cells after enzyme digestion linearization, and screening high-copy recombinants through G418 resistance gradient, so as to obtain pichia pastoris genetically engineered bacteria for expressing recombinant humanized XVII type collagen. The recombinant humanized XVII type collagen JYC1701, JYC1702 and JYC1703 disclosed by the invention show excellent functions of promoting cell adhesion, cell proliferation and cell migration, and are suitable for preparing products such as skin care products or medical instruments and the like.

The invention belongs to the field of molecular biology and immunology, and particularly relates to an anti-CD3 nano antibody capable of activating the activity of T cells as well as a preparation method and application of the anti-CD3 nano antibody. The invention provides a preparation method of the anti-CD3 nano antibody. The preparation method comprises the following steps: constructing a CD3 plasmid and an overexpression cell line, and immunizing alpaca to obtain peripheral blood mononuclear cells (PBMC) of the alpaca; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription to obtain cDNA; carrying out PCR amplification on the antibody gene segment and cloning the antibody gene segment to a phage display vector; and electrically transforming the vector into SS320 competent cells to obtain the nano antibody library. After two rounds of panning, detecting and screening an antibody clone capable of being combined with the CD3 protein through ELISA (Enzyme-Linked Immunosorbent Assay); an antibody sequence is selected through sequencing, and an antibody protein is expressed by using a mammalian cell high-throughput expression system; and then detecting and screening the antibody which is only specifically combined with the CD3. The method disclosed by the invention is simple, convenient and rapid to operate, and can be used for preparing active antibodies for treating potential tumors and proliferative diseases.

The invention relates to the field of animal breeding, and discloses a pig beta defensin compound preparation which comprises pig beta defensin PBD1, PBD2 and PBD114, and the weight ratio of the PBD1 to the PBD2 to the PBD114 is 1: 1: 1. The porcine beta defensin PBD1, the porcine beta defensin PBD2 and the porcine beta defensin PBD114 are recombinant proteins, and are respectively expressed in escherichia coli BL21 (DE3) PLysS competent cells by a pET-32a expression vector. Through the synergistic effect of the defensins PBD1, PBD2 and PBD114, the minimum inhibitory concentration (MIC) of the EPEC is reduced to 18.75 mu g / mL from 75 mu g / mL of single defensins, and is reduced by 75%; within 24 hours, the bacteriostasis rate of the compound preparation reaches 90%, and the inhibition effect on EPEC is remarkably improved.

The invention provides an SlTAGL1 gene for regulating and controlling the lutein content of tomato fruits, a method and application, a coding sequence of the SlTAGL1 gene is obtained from a tomato genome, a target spot is connected to CRISPR / Cas9 by designing the target spot, the tomato is successfully infected by using agrobacterium competent cells, a CRISPR / Cas9 gene editing plant of the tomato is obtained, the gene editing plant is analyzed, and the content of lutein in the tomato fruits is regulated and controlled. The result shows that compared with the wild tomato, the lutein content in the tomato fruits after the SlTAGL1 is knocked out is obviously increased by 1.6-2.2 times. According to the invention, an efficient and stable genetic method is adopted to improve the content of xanthophyll in tomato fruits, so that the quality of the tomato fruits is improved, and gene resources and strategies are provided for culturing high-xanthophyll tomato varieties.

The invention discloses preparation of a human CD56 immune phage display antibody library, which comprises the following steps: S1, animal immunization: expressing recombinant hCD56 protein in escherichia coli, then immunizing a Balb / c mouse, and detecting immune titer; s2, splenocyte preparation: extracting RNA (Ribonucleic Acid) of immune mouse spleen cells, and performing reverse transcription to obtain cDNA (Complementary Deoxyribonucleic Acid); s3, preparation of an initial bacteriophage display library: amplifying a heavy chain variable region and a light chain variable region by using a PCR technology, and then assembling to form scFv; constructing scFv phagocytids, electrically transforming the scFv phagocytids into TG1 competent cells, and constructing a bacterial library; s4, constructing a phage single-chain library of the hCD56: infecting the bacterial library by using a helper phage M13K07, and constructing a phage single-chain antibody library; and S5, elutriation and screening of a phage library: specifically enriching a phage single-chain antibody library, screening positive clones from the phage single-chain antibody library, and determining a sequence. According to the invention, the human hCD56 antibody scFv sequence fragment can be screened out, and the research and development period is shortened.

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention relates to the technical field of ginsenoside, aims to solve the problem of low conversion rate of ginsenoside Rg3 prepared by adopting a microbial conversion method in the prior art, and particularly discloses beta-glucosidase and a preparation method thereof, and application of the beta-glucosidase in production of ginsenoside Rg3. The preparation method of the beta-glucosidase comprises the following steps: S1, inserting a beta-glucosidase gene into a pET vector to obtain a recombinant plasmid; s2, inoculating the recombinant plasmids into competent cells, culturing the competent cells in an antibiotic-free culture medium and a resistant culture medium in sequence to form monoclonal antibodies, performing IPTG (isopropyl-beta-d-thiogalactoside) induction culture to form monoclonal strains, and storing the monoclonal strains in glycerol to obtain glycerol bacteria; s3, glycerol bacteria are placed in an LB culture medium to be cultured and then transferred into a fermentation culture medium, the fermentation culture medium is placed in a fermentation tank to be fermented, then centrifugal separation, thallus collection and re-suspension are conducted, beta-glucosidase is obtained and used for decomposing ginseng total saponins to produce ginsenoside Rg3, and the conversion rate of main products can reach 98%.

The invention provides a method for efficiently expressing amidase and application of the amidase, and belongs to the technical field of enzyme technology and gene engineering. The method comprises the following steps: A1, cloning a fusion tag coding sequence for enhancing amidase expression into an escherichia coli expression vector to obtain a first recombinant expression vector; a2, cloning an amidase coding sequence into a first recombinant expression vector to construct a second recombinant expression vector, wherein amidase and the fusion tag are subjected to fusion expression, and the amidase is located at the downstream of the fusion tag; a3, introducing the second recombinant expression vector into escherichia coli competent cells to construct recombinant genetic engineering bacteria; and A4, fermenting the obtained recombinant genetically engineered bacterium to express amidase. According to the method disclosed by the invention, the expression quantity of amidase is remarkably improved by utilizing the fusion tag, chiral alkylaniline with high optical purity can be generated by utilizing the prepared amidase, and the e.e. Value of the obtained compound is greater than 97%.

The invention relates to pichia pastoris engineering bacteria and a preparation method thereof, and a method for producing cellooligosaccharide / disaccharide by using corn straws or wheat straws, and belongs to the technical field of genetic engineering. A cellulose incision enzyme gene and a cellulose excision enzyme gene are transferred into pichia pastoris engineering bacteria. The preparation method comprises the following steps: respectively inserting a cellulose incision enzyme gene and a cellulose excision enzyme gene into plasmids to construct expression vectors; transferring the expression vector into a competent cell of pichia pastoris GS115; and screening and identifying to obtain successfully transformed pichia pastoris engineering bacteria. According to the present invention, the pichia pastoris heterologous expression cellulose incision enzyme and cellulose excision enzyme are adopted to construct the pichia pastoris engineering strain capable of degrading the plant straw into the cellooligosaccharide / disaccharide, such that the reliable chassis strain is provided for the production of the starch from the cellulose;

The invention discloses a construction method of a nocardia seriolae mycolic acid deficient strain, which belongs to the technical field of aquaculture prevention and control, and comprises the following steps: strain and material preparation, culture and genome extraction, upstream and downstream fragment cloning to construct deletion plasmids, competent cell preparation, electrotransformation and screening, and biological characteristic and toxicity analysis. The nocardia seriola seriola mycolic acid defect type strain is constructed and compared with a wild strain and a back-filling strain for research, the effect of the mycolic acid in the aspects of growth, form, toxicity and the like of thalli can be defined, the relationship between the mycolic acid and the pathogenicity of the thalli can be deeply known, and an important clue is provided for revealing the pathogenic mechanism of the nocardia seriola seriola.

The invention discloses a plant immunomodulatory gene RDP as well as an expression vector and application thereof. The inventor finds a plant immunomodulatory factor gene RDP from potatoes, after a corresponding sequence is obtained through cloning, agrobacterium competent cells are transferred into nicotiana benthamiana, it is verified that the plant immunomodulatory factor gene RDP can be successfully expressed in other plants, and it is verified that the pathogenic effects of various phytophthora are good. The gene is proved to be capable of effectively improving the disease resistance of the nicotiana benthamiana after being expressed. Through objective evaluation of an agrobacterium tumefaciens-mediated transient expression system, it is found that the plant immunomodulatory factor RDP can induce plants to generate resistance, the gene can be directly introduced into the plants through a transgenic technology due to the powerful function of the gene, disease-resistant varieties are developed and cultivated, and the gene has great significance in prevention and treatment of plant diseases.

The invention discloses a method for constructing an antibacterial peptide Apidaecin IB high-expression genetically engineered bacterium, which comprises the following steps of: 1, designing three pairs of primers according to a DNA sequence of an amplification template coding region, carrying out PCR (Polymerase Chain Reaction) amplification, and constructing expression plasmids in escherichia coli; step 2, carrying out multiplication culture on engineering bacteria saccharomyces cerevisiae CENPK2, preparing saccharomyces cerevisiae competent cells, and subpackaging and refrigerating for later use; step 3, carrying out enzyme digestion on the constructed expression plasmid, and carrying out mixed transformation on the expression plasmid and a competent cell of saccharomyces cerevisiae CENPK2, so as to obtain the high-expression genetic engineering bacterium CENPK2-Apidaecin IB-Ty1Cons2 of the antibacterial peptide Apidaecin IB, wherein the high-expression genetic engineering bacterium CENPK2-Apidaecin IB-Ty1Cons2 is obtained; according to the invention, the Ty1Cons2 transposon is integrated into the saccharomyces cerevisiae CENPK2, so that the construction of the high-expression gene engineering bacterium of the antibacterial peptide Apidaecin IB is completed, and multiple copies are realized.

The invention discloses an EmAGO2 truncated recombinant protein, an echinococcosis multilocularis indirect ELISA (enzyme-linked immuno sorbent assay) diagnostic kit based on the truncated recombinant protein and a use method of the kit. The method comprises the following steps: designing a specific primer by selecting a sequence of 1-738bp at the N end of an Em-AGO2 gene, carrying out PCR (Polymerase Chain Reaction) amplification by taking reverse transcription echinococcosis multilocularis DNA (Deoxyribonucleic Acid) as a template, connecting an amplification product with a pCE2-TA / Blunt vector, and then transforming into a competent cell for culturing, so as to obtain a pCE2-T-EmAGO2 plasmid; respectively carrying out double enzyme digestion on the plasmid and an expression vector pET-28a (+), and connecting enzyme digestion fragments to obtain a recombinant protein expression plasmid pET-28a-EmAGO2; the preparation method comprises the following steps: taking EmAGO2 as a template, transforming the EmAGO2 into competent cells, selecting positive clones for induced expression, and carrying out ultrasonication treatment, separation and purification, concentration and desalination treatment on expressed thalli to obtain the EmAGO2 truncated recombinant protein. Furthermore, a mouse anti-EmAGO2 truncated recombinant protein and goat anti-rabbit IgG are respectively used as an antigen and an antibody to establish an indirect ELISA diagnostic kit for the echinococcosis multilocularis, and the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of the echinococcosis multilocularis.

The invention belongs to but not limited to the technical field of genetic engineering technology, and discloses a construction method and application of a BspE gene deletion strain of Brucella effect protein, the BspE gene deletion strain is constructed by using homologous recombination and SacB reverse screening technology, Brucella abortus A19 vaccine strain liquid is used as a template, PCR amplification is carried out to obtain upstream and downstream homologous arms of BspE gene, and the BspE gene deletion strain is constructed by using the upstream and downstream homologous arms of the BspE gene. The method comprises the following steps: carrying out fusion PCR (Polymerase Chain Reaction) amplification on upstream and downstream homologous arms of a BspE gene subjected to gel recovery, and connecting and transforming an obtained fusion fragment and a pUC19-SacB plasmid to obtain a recombinant plasmid pUC19-delta BspE. The invention relates to a bovine brucella BspE deletion vaccine strain, which is characterized in that a plasmid is obtained, the plasmid is electrically transformed into a bovine brucella A19 competent cell, and the positive transformant is a bovine brucella BspE deletion strain through kanamycin resistance and sucrose pressure screening, PCR (polymerase chain reaction) identification and sequencing, and the deletion vaccine strain improves the levels of antibacterial cytokines and specific brucella antibody IgG, and can be used for preparing the bovine brucella BspE deletion vaccine strain. The Brucella attenuated vaccine strain is a novel Brucella candidate attenuated vaccine strain and has a good market prospect.

The invention discloses a nano antibody for resisting a listeria monocytogenes MurA protein and application of the nano antibody, and relates to the technical field of biology. The amino acid sequence of the nano antibody is as shown in SEQ ID NO.3. MurA protein of prokaryotically expressed listeria monocytogenes is used as an immunogen to immunize alpaca, total RNA of peripheral blood lymphocytes is extracted, VHH fragments are amplified through reverse transcription and two rounds of nested PCR, the VHH fragments and a pComb3Xss vector are converted into TG1 competent cells after enzyme digestion and connection, and a phage display technology is used for three rounds of elutriation, so that the total RNA of the listeria monocytogenes is obtained. The nanometer antibody for resisting the listeria monocytogenes MurA protein is successfully screened out. The nano antibody can specifically recognize MurA protein and whole listeria monocytogenes, and the immunocolorimetric sensor based on thiolated bacteriophage induced colloidal gold aggregation, which is established by utilizing the nano antibody, is strong in specificity, high in sensitivity and low in detection limit, and can greatly shorten the detection time.

The invention discloses an engineering strain for soluble expression of human Cu / Zn superoxide dismutase as well as a construction method and application of the engineering strain, and relates to the technical field of biology. The construction method of the engineering strain comprises the following steps: cloning a coding gene of the human Cu / Zn superoxide dismutase into an expression vector to construct a recombinant plasmid; transforming the recombinant plasmid into escherichia coli competent cells to construct the engineering strain; the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 2. The invention provides a new engineering strain for improving the expression abundance of the human Cu / Zn superoxide dismutase in escherichia coli and the proportion of a soluble expression part, and has important industrial application value.

The invention discloses a micro-ring plasmid synthesis method, and belongs to the technical field of gene editing. Two pairs of primers are designed, eukaryotic expression plasmids containing selection markers and target fragments are used as templates for PCR amplification to obtain two target fragments, vector plasmids loxp-R6K with specific sequences and the target fragments form recombinant plasmids, the recombinant plasmids contain sequences such as the target fragments, restriction enzyme cutting sites and R6K replication start sites, and the recombinant plasmids are used for preparing the recombinant plasmids. The recombinant plasmid is transformed in Escherichia coli GT115 competent cells, the recombinant plasmid is verified to have the recombination efficiency of 81% after bacterial colony PCR amplification, a correctly cloned seed solution is screened and subjected to induced expression to form a bacterial solution containing a screening-label-free micro-plasmid, the micro-ring plasmid is separated and purified, and the screening-label-free micro-ring plasmid is obtained. The sequence of the microring plasmid only contains a target fragment and an R6K replication start site, the accuracy of the size sequence of the microring plasmid in a bacterial colony cultured and cloned by the microring plasmid is as high as 90%, and the microring plasmid can be efficiently and stably obtained.

The invention is applicable to the technical field of genetic engineering and biological medicine, and provides a construction method and an expression system of recombinant protein for preventing infectious bronchitis of chicken and application of the recombinant protein. Coding DNA of multi-antigen epitope tandem protein from infectious bronchitis virus N, S1 protein is connected to obtain a fusion recombinant protein IBV: NS1 coding gene, a recombinant expression plasmid pET28a-IBV: NS1 is constructed and transferred into E. coli Rosetta (DE3) competent cells to obtain an E. coli genetic engineering expression bacterium Ec-RD-pET28a-IBV: NS1, and the fusion recombinant protein IBV: NS1 is obtained through induced expression and massive purification. And mixing with an immunologic adjuvant to prepare the subunit vaccine. The subunit vaccine has the advantages of high antigen expression quantity, long antibody maintenance time, high purity, good safety, strong immunogenicity and strong protectiveness, and can effectively prevent infectious bronchitis.

This application discloses a feline allergen Fel d1 egg yolk antibody and its preparation method and application, comprising: obtaining PCR amplification products through PCR amplification; constructing a recombinant plasmid by ligating the PCR amplification products with pET-28a(+); transforming the recombinant plasmid into Rosetta competent cells to obtain transformants; obtaining recombinant antigens by extracting proteins from the transformants; obtaining a subunit vaccine by mixing and emulsifying the recombinant antigens with white oil adjuvant; administering the subunit vaccine to laying hens to obtain immunized egg yolk liquid; adding Tris-HCl buffer and polyethylene glycol to the egg yolk liquid and centrifuging to collect the protein aqueous phase; filtering the protein aqueous phase to obtain a filtrate, adding polyethylene glycol, mixing, centrifuging to collect the precipitate; mixing the precipitate with PBS buffer, adding ammonium sulfate, and centrifuging to obtain the egg yolk antibody. This invention solves the problem that the safety and economy of existing feline allergy treatment methods cannot be simultaneously achieved.

The invention relates to an expression cassette and a method for producing humanized triple helix collagen by using the expression cassette, and belongs to the technical field of genetic engineering. The expression cassette sequentially comprises a promoter sequence, a fusion gene and a terminator sequence from upstream to downstream, the fusion gene comprises nucleotide fragments A, B and C, each fragment comprises an immutable region and a variable region, and the variable region is selected from I, II, III, IV and XVII type human collagen. A kex2 protease recognition site KR is introduced into an expression box, so that an expression product can be subjected to trisection cutting, and three equal-length peptide chains with a triple-helix basic structure are formed. The invention also provides a recombinant humanized collagen with a self-assembled triple helix structure and a production method thereof, and the method comprises the following steps: preparing a recombinant expression vector containing the expression cassette and a proline hydroxylase expression vector, and transferring the two expression vectors into competent cells for co-expression. The method can be used for producing the recombinant humanized collagen with the self-assembled triple-helix structure, and has a good application prospect.

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

The invention provides recombinant escherichia coli for preparing soluble EC-SOD and application of the recombinant escherichia coli, and relates to the technical field of biological engineering. According to the present invention, SOD and KGF2 genes are connected by using a connecting peptide coding sequence containing a protease cleavage site, and are cloned to a pET28a expression vector; the pET28a expression vector is transformed into competent cells of escherichia coli BL21 (DE3), and recombinant escherichia coli is constructed. The obtained recombinant Escherichia coli can solve the problem that an inactive inclusion body is formed when EC-SOD is expressed in Escherichia coli. The method is suitable for preparing soluble EC-SOD.

The invention discloses a preparation method and application of a quail CENP-A polyclonal antibody, and the preparation method of the quail CENP-A polyclonal antibody comprises the following steps: constructing a recombinant expression vector containing a CENP-A target gene segment as shown in SEQ ID NO.1; transforming the recombinant expression vector into a competent cell, inducing expression of a fusion protein, collecting and purifying to obtain a CENP-A recombinant protein; carrying out animal immune treatment by taking the CENP-A recombinant protein as an antigen, and collecting and separating an antibody in animal serum as a CENP-A polyclonal antibody. The invention also develops a method and a device for CUAMP; the quail CENP-A antibody sequenced by Tag fills up the blank of a quail commercial CENP-A antibody, is also an important basic work for further constructing a quail genome complete map, and provides a basic support for subsequently revealing how centromere related genes of vertebrates such as birds evolve among different species.

The invention provides a construction method and an application method of a wheat yeast two-hybrid cDNA library, and belongs to the technical field of biology. The invention provides a construction method of a yeast two-hybrid cDNA library, which comprises the following steps: extracting total RNA, separating mRNA, converting into cDNA, transferring into escherichia coli competent cells through a series of reactions to obtain a cDNA primary library bacterial liquid, separating primary library plasmids, and obtaining a yeast two-hybrid cDNA library bacterial liquid through an LR recombination reaction and conversion of the escherichia coli competent cells. A high-quality wheat variety Zheng wheat 158 is taken as a research material, wheat gliadin genes Tagli-gamma-1 and Tagli-gamma-11 are cloned, a corresponding bait vector is constructed, the interaction protein is screened from a cDNA library of the Zheng wheat 158 by utilizing a yeast two-hybrid technology, and a foundation is laid for carrying out interaction protein screening of genes related to wheat quality and analyzing a regulation mechanism of the interaction protein in a later period.

The present invention discloses an application and method of a SPILR protein for improving plant resistance to Pseudomonas syringae, belonging to the field of agricultural science and technology. Specifically, the method comprises using Pseudomonas syringae to induce expression of the SPILR protein in Arabidopsis leaves, constructing an expression vector pCambia1300-SPILR containing the nucleotide sequence of the SPILR protein, transforming the expression vector into Agrobacterium tumefaciens GV3101 competent cells, culturing the cells, and infecting plant leaves to obtain Pseudomonas syringae-resistant plants. Therefore, genetic engineering techniques can be used to cultivate resistant varieties.

The invention discloses a preparation method of a novel composite carbon source. The preparation method comprises the following steps: S1, preparing a solution; s2, preparing a culture medium; s3, extracting a genome; s4, extraction of plasmid DNA (deoxyribonucleic acid); s5, preparation of agarose gel electrophoresis; s6, constructing a linearized vector plasmid; s7, preparing an electrotransfection competent cell; and S8, electric conversion treatment. According to the preparation method of the novel composite carbon source, pfkA and pfkB can be respectively, independently and simultaneously knocked out when the carbon source is prepared by utilizing a gene editing technology, the consumption of a glucosamine precursor 6-fructose phosphate in an EMP pathway can be reduced, more glucose is used for synthesizing a target product, and by optimizing the preparation process of the composite carbon source, the yield of the target product is improved. The shake-flask fermentation yield of the glucosamine can be obviously improved, the conversion rate of the composite carbon source is also improved, and cell growth and synthesis of the target product glucosamine can be reasonably balanced, so that the yield of the glucosamine is improved.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No: 1 and is named as L8. The preparation method of the antibacterial peptide L8 comprises the following steps: S1, obtaining an L8 target gene segment; connecting an L8 target gene segment with a pLC vector to obtain a connection product; transforming the connection product into an escherichia coli DH5-alpha competent cell, and screening positive clones to obtain a pLC-L8 recombinant plasmid; s2, taking the pLC-L8 recombinant plasmid as a substrate, and carrying out cell-free protein synthesis reaction to obtain the antibacterial peptide L8, the cell-free protein synthesis system comprises 175 mM of potassium glutamate, 10 mM of ammonium glutamate, 2.7 mM of potassium oxalate, 33 mM of PEP, 38 mM of compound amino acid, 10 mM of magnesium glutamate, 2% (w / v) PEG-8000, 5 nM of pLC-L8 recombinant plasmid, 11.25 mM of lactose, 33.33% (v / v) of crude extract, 100 mg / mL of escherichia coli tRNA and 2.5 nM of T7 RNA polymerase. The antibacterial peptide L8 is obtained by optimizing the antibacterial peptide LRGG, and compared with the LRGG, the antibacterial peptide L8 subjected to cell-free protein synthesis reaction expression has better antibacterial activity and biological safety.

The invention discloses a type 1 goose astrovirus virus-like particle and a preparation method and a pharmaceutical composition thereof, and the preparation method comprises the following steps: cloning a gene segment for coding type 1 goose astrovirus ORF2 protein to a baculovirus transfer vector, and constructing to obtain a recombinant plasmid; transforming the recombinant plasmid into a DH10Bac competent cell to obtain a recombinant baculovirus rod grain; transfecting the recombinant baculovirus stem into an insect baculovirus expression system to obtain a recombinant baculovirus; and inoculating the recombinant baculovirus into an insect baculovirus expression system, culturing, collecting a cell culture, and purifying to obtain the purified goose astrovirus type 1 virus-like particles. The GAstV-1 VLPs and the vaccine thereof provided by the invention not only solve the technical problem of research and development of the GAstV-1 vaccine, but also have the advantages of clear structure, high safety, good immunogenicity and exact protective efficacy, and have remarkable creativity and industrial application value.