244 results about "Competent cell" patented technology

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 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 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.

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 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.

The invention discloses a yeast clone with a pAbAi reporter gene as well as a construction method and application thereof, the yeast clone comprises a bait sequence Bit as shown in SEQ ID NO: 1 and an original promoter Pmin as shown in SEQ ID NO: 2, the promoter of the pAbAi reporter gene is formed by the inserted Bit and Pmin, a pAbAi / -CAAT plasmid as a no-load plasmid as shown in SEQ ID NO: 3 is constructed, the bait sequence as shown in SEQ ID NO: 1 is connected, and the pAbAi reporter gene is obtained. BstBI is used for carrying out single enzyme digestion on pAbAi-Bait / CAAT plasmids, Y1Hgold yeast competent cells are converted, SD / -Ura plates are coated, after the Y1Hgold yeast competent cells grow for 3-5 days, monoclonal colonies are selected and subjected to colony PCR detection, it is confirmed that a bait sequence is successfully integrated into the Y1Hgold yeast competent cells, and subculture is carried out, so that the Y1Hgold yeast is obtained.

The invention relates to an XVII type collagen, a recombinant bacterium and a construction method and application of the XVII type collagen. The XVII type collagen comprises a polypeptide of which the amino acid sequence is as shown in SEQ ID NO. 1. In the XVII type collagen, the glycine of the polypeptide with the amino acid sequence as shown in SEQ ID NO.1 accounts for 26% or above, and the XVII type collagen is high in structural stability, easy to express and high in yield. Experiments prove that the yield of the recombinant bacteria obtained by transforming the XVII type collagen into competent cells to the XVII type collagen is as high as 4.5 g / L, and the yield of the XVII type collagen is as high as 9.3 g / L after plasmid pSB1a-mtk (Mca)-mcl (Rsp)-aceAK is transferred into the recombinant bacteria.

The application discloses a construction method and application of engineering escherichia coli for producing humulone, and the pTrcHis2B-IDI plasmid and pACYDuet-1-CCL4-VPS-PT1-linker-PT2 plasmid are transferred into engineering escherichia coli Trc-low competent cells to obtain the engineering escherichia coli for producing humulone. The engineering humulone bacteria are constructed based on the Trc-low competent cell host, the pACYDuet-1-CCL4-VPS-PT1-linker-PT2 is combined with the pTrcHis2B-IDI, the side chain precursor activation function of CCL4 is matched with the IDI / DMAPP donor module, the VPS-mediated skeleton construction and the PT1 / PT2-mediated continuous isoprenylation process are further connected, the humulone target path can be more completely operated in the same host, and therefore the specificity, continuity and directional guiding ability of the product biosynthesis are improved.

The application discloses a bacteriocin synthesis regulation gene plnC overexpression and a construction method and application of a recombinant plant lactis bacillus, and has the characteristics that the nucleotide sequence is shown in SEQ ID No:1 in the sequence table, and the construction method steps are as follows: 1) taking the plant lactis bacillus genomic DNA as a template, designing specific primers and performing PCR amplification; 2) purifying and double enzyme cutting the plnC gene fragment, and connecting the linear carrier pMG36e which is also double enzyme cut to obtain a pMG36e-plnC recombination cloning carrier; (3) through an electric transformation method, the pMG36e-plnC is transferred into plant lactis bacillus B1 competent cells to obtain a plnC overexpression strain B1-plnC, and the advantages are that the synthesis of the plant lactis bacillins is promoted, the plnC gene expression amount of the overexpression strain B1-plnC is increased by more than 4.5 times, and the PlnC protein content is increased by more than 1.5 times.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

The invention is applicable to the technical field of genetic engineering, and provides a vaccine for preventing and treating staphylococcus aureus mastitis and application of the vaccine. A recombinant bacillus subtilis construction method comprises the following steps: taking a DNA sequence as shown in SEQ ID NO.3 as a template, and performing PCR amplification to obtain a PCR product sequence; connecting the PCR product to a pHT43 vector, and converting the pHT43 vector into a competent cell DH5alpha, so as to obtain a recombinant plasmid pHT43-cCsa; the method comprises the following steps: transforming a recombinant plasmid pHT43-cCsa into a competent cell of bacillus subtilis WB800N, taking pHT43-F and pHT43-R as primers, and screening a positive transformant through a PCR (Polymerase Chain Reaction) technology; the recombinant bacillus subtilis for expressing the staphylococcus aureus Csa protein is constructed by utilizing a homologous recombination technology, so that pathological injury caused by staphylococcus aureus infection can be reduced, and staphylococcus aureus mastitis of dairy cattle can be prevented.

The invention discloses a crassostrea hongkongensis NKA-alpha protein polyclonal antibody as well as a preparation method and application thereof. The preparation method comprises the following steps: constructing a recombinant expression vector containing an NKA-alpha target gene segment; transforming the recombinant expression vector into competent cells to construct a recombinant expression strain, inducing the recombinant expression strain to express fusion protein, and collecting and purifying to obtain NKA-alpha recombinant protein; carrying out animal immune treatment by taking the NKA-alpha recombinant protein as an antigen, collecting and separating antiserum, and purifying to obtain the Crassostrea hongkongensis NKA-alpha protein polyclonal antibody. The prepared Crassostrea hongkongensis NKA-alpha polyclonal antibody is high in affinity, can specifically recognize NKA-alpha protein in Crassostrea hongkongensis, and can be widely applied to Crassostrea hongkongensis biological immunodetection.

The invention provides a method for synthesizing melanin nanoparticles by using escherichia coli, and belongs to the technical field of biological genetic engineering. According to the method, a bacillus megaterium-derived tyrosinase gene Tyr1 (SEQ ID NO.2) after codon optimization is taken as a target gene, a recombinant expression plasmid pET-24a-Tyr1 is constructed, and the recombinant expression plasmid pET-24a-Tyr1 is transformed into an escherichia coli BL21 (DE3) competent cell to obtain the genetically engineered bacterium. After the engineering bacterium induces expression of tyrosinase through IPTG (isopropyl-beta-d-thiogalactoside), polymerization of an L-tyrosine substrate is catalyzed in the presence of CuSO4 to obtain high-purity melanin nanoparticles. The melanin nano-particles synthesized by the method are uniform in particle size, have wide-spectrum strong absorption characteristics in a wavelength range of 400-800nm, show excellent photo-thermal performance, free radical scavenging capacity and biocompatibility, and have wide application prospects in the fields of biomedicine and functional materials.

The invention provides antigen protein HPA of bovine-derived A-type pasteurella multocida and a preparation method and application of recombinant protein of the antigen protein HPA. The amino acid sequence of the antigen protein HPA is shown as SEQ ID NO.3. The preparation method comprises the following steps: cloning a coding gene sequence, shown as SEQ ID NO.1, of the antigen protein HPA after codon optimization into a prokaryotic expression vector; a recombinant plasmid containing the antigen protein HPA coding gene is obtained; transforming the recombinant plasmid into escherichia coli competent cells to obtain a recombinant expression strain; the antigen protein HPA is obtained through fermentation culture, inducible expression and purification. According to the invention, mice are immunized, the protection rate can reach 100%, and the problem of poor protection effect of the A-type pasteurella subunit vaccine for bovine pneumonia at present is solved.