161 results about "Competent cell" patented technology

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 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 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 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 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 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 relates to a method for regulating and controlling a flower type by silencing a peony organ gene based on a transient expression system, which belongs to a peony flower type regulation and control method and comprises the following steps of: designing a specific primer aiming at a target gene PsuAG, obtaining a target fragment through PCR (Polymerase Chain Reaction) amplification, and inserting the target fragment into a transient expression vector to construct a gene silencing fusion expression vector; introducing the gene silencing fusion expression vector into an agrobacterium competent cell, preparing an infection solution to infect a peony plant, and injecting the infection solution into a pedicel by using an injector when a flower bud of the peony plant develops to an initial stage of a large wind chime period; when the flower buds grow to the initial stage of the round peach stage, injecting the infection liquid into pedicels and sepal bases; after each time of injection, weak light culture is carried out under a shading condition. The efficient silencing of the key gene PsuAG for regulating stamen petals is realized, and the double petals rate of the peony flower type is improved. The method does not need to depend on a stable genetic transformation system, operation is directly carried out on floral organs of a complete plant, and the gene function verification period is remarkably shortened.

The present application belongs to the technical field of genetic engineering, and relates to a CrtI enzyme mutant, a recombinant plasmid, a recombinant strain and application thereof. The CrtI enzyme mutant provided by the present application is obtained by random mutation of a wild-type CrtI gene through error-prone PCR, and the amino acid sequence thereof is shown as SEQ ID NO. 1. The obtained mutant has multiple site variations in the amino acid sequence, thereby significantly improving the catalytic efficiency and substrate affinity of the enzyme. The present application further constructs a recombinant expression vector containing the CrtI enzyme mutant gene, and transforms the recombinant expression vector into a red Phaffia yeast competent cell, and obtains a high-yield astaxanthin recombinant strain through resistance screening and pigment phenotype screening. Compared with the wild-type CrtI enzyme, the CrtI enzyme mutant of the present application significantly improves the conversion rate of beta-carotene to astaxanthin in the red Phaffia yeast.

The invention discloses a green fluorescence report system for screening a Listeria monocytogenes quorum sensing inhibitor as well as a construction method and application of the green fluorescence report system. The report system is constructed through the following steps: synthesizing a DNA fragment according to a listeria monocytogenes AGR system pII promoter sequence, connecting the DNA fragment with a green fluorescent protein pEGFP plasmid, amplifying an EGFP-pII target fragment through PCR, and connecting the target fragment to a pKSV7 shuttle vector plasmid through homologous recombination to obtain a pKSV7-EGFP-pII plasmid, and then transferring the gene into a competent cell of listeria monocytogenes ATCC BAA-679, and screening to obtain a report system. The report system disclosed by the invention can be used for intuitively evaluating the expression condition of the quorum sensing system of the listeria monocytogenes and screening out the quorum sensing inhibitor.

This invention discloses an engineered bacterium expressing the sqr gene and its application in aerobic remediation of hexavalent chromium pollution. The construction method of the engineered bacterium includes the following steps: S1. Constructing the pEcgRNA-sqr plasmid; S2. Obtaining donor DNA; S3. Preparing electrocompetent cells; S4. Co-transforming the pEcgRNA-sqr plasmid and donor DNA fragment into the electrocompetent cells, performing resistance screening, and obtaining engineered bacteria integrating the sqr gene. This invention utilizes the constructed engineered bacterium expressing SQR coupled with nano-ferrous sulfide to establish a "sulfur metabolism-FeS regeneration" cycle system, accelerating Cr(VI) reduction. Under aerobic / microaerobic conditions, the removal rate of 50 mg / L Cr(VI) in 1 hour can reach over 95%, while simultaneously alleviating the competitive pressure of intracellular reducing power caused by heterologous expression of the sqr gene, improving remediation efficiency and strain growth stability, and achieving a synergistic effect of biochemistry.

This invention constructs pET-32a-CP1 and pET-32a-CP2 expression vectors based on the CP1 and CP2 protein genes of Giardia duodenalis, transforms them into Rosetta (DE3) competent cells, and induces expression using IPTG. After SDS-PAGE analysis of the recombinant protein expression, Ni-NTA affinity chromatography is used to purify the proteins. Two indirect ELISA methods are then established using these as coating antigens. The indirect ELISA methods based on the recombinant proteins CP1 and CP2 of this invention exhibit good specificity, sensitivity, and repeatability, with concordance rates of 98% and 94% with microscopic detection results, respectively.

The application discloses a method for preparing a bacterial expression liquid by using a Malacosoma americanum V-ATPase D gene hpRNA and application thereof, and is based on a targeted Malacosoma americanum V-ATPase D gene hpRNA fragment and a virus-like particle MS2 protein gene to construct an hpV-ATPase D expression vector or an MS2+hpV-ATPase D expression vector, which is introduced into a bacterial competent cell, and MS2 protein and the targeted Malacosoma americanum V-ATPase D gene hpRNA are continuously and massively expressed by IPTG induction, so as to obtain the bacterial expression liquid, wherein the MS2 protein expressed by the MS2 protein gene is protected from being degraded by a nuclease by encapsulation, and the hpRNA of the Malacosoma americanum V-ATPase D gene has good stability; experiments show that the hpV-ATPase D and the MS2+hpV-ATPase D both have a high lethal rate on the Malacosoma americanum and have a significant inhibiting effect on development and reproduction of the Malacosoma americanum. The application uses the bacterial expression liquid containing the Malacosoma americanum V-ATPase D gene hpRNA to control the Malacosoma americanum, and has the characteristics of strong practicability, convenience, rapidness, high efficiency and sensitivity, and provides a theoretical basis for research on Malacosoma americanum control methods.

The invention discloses a preparation method of high-purity TIR2 and T1R3 recombinant plasmids. The preparation method comprises the following steps: transforming the T1R2 and T1R3 recombinant plasmids into modified Trans1-T1 escherichia coli competent cells through a heat shock method; large-scale amplification of plasmids is carried out by utilizing the efficient cloning characteristic of Trans1-T1 escherichia coli; and extracting and purifying recombinant plasmids from thalli by adopting an optimized alkali cracking combined column purification technology. According to the preparation method of the high-purity TIR2 and T1R3 recombinant plasmids, target DNA fragments or recombinant DNA plasmids constructed by the target DNA fragments are introduced into competent cells, amplification of genetic materials is achieved through a replication and expression system of host cells, finally the high-purity DNA plasmids are obtained through extraction and purification, and the high-purity TIR2 and T1R3 recombinant plasmids are obtained by improving transformation, amplification and purification processes. The yield and the purity of the T1R2 / T1R3 recombinant plasmid are remarkably improved, and the high-standard requirement of a transfection experiment is met.

The invention provides a separated nucleic acid molecule. The nucleotide sequence of the separated nucleic acid molecule is shown as SEQ ID NO. 1. The invention also provides an antibody coded by the nucleic acid molecule. The invention also provides a carrier which contains the nucleic acid molecule. The invention also provides a method for producing the antibody. The method comprises the following steps: transfecting antibody expression plasmids in competent cells, culturing, collecting cell supernatant, centrifuging to remove precipitate, purifying antibody protein, collecting eluent, and concentrating to obtain the antibody. The invention also provides an application of the antibody, the carrier or the pharmaceutical composition in preparation of drugs for treating autoimmune diseases. The antibody disclosed by the invention can be specifically combined with FcRn and Fc gamma Rs, is a potent inhibitor for FcRn-mediated IgG recirculation and Fc gamma R-dependent immune complex activation, is a therapeutic drug for autoimmune diseases, and has huge potential.

The invention provides a genome editing method for animal bifidobacterium. The genome editing method comprises the following steps: constructing plasmids capable of being expressed in bifidobacterium; carrying out demethylation on the plasmid, and carrying out in-vitro methylation modification; the plasmid subjected to methylation modification is electrically transformed into competent cells of animal bifidobacterium; a gene editing mutant strain is obtained through resistance screening and homologous recombination, a'totipotent 'plasmid which can express Cas9 and sgRNA in bifidobacteria and carries a homologous repair template is constructed, the plasmid is protected by simulating a methylation modification system of bifidobacteria in vitro, then the plasmid is electrically transferred into a thallus, and the homologous repair template is obtained. A gene editing mutant strain is obtained through resistance screening and homologous recombination, gene knockout is achieved in animal bifidobacterium, flow gene editing is achieved, methylation modification is conducted on non-methylation plasmids in vitro, and a mutant strain without a selection marker can be obtained. In addition, the invention also provides a genome editing system, electronic equipment and a computer readable medium.

The application discloses a fusion protein and application thereof in preparation of semaglutide precursor polypeptide, wherein N (N>=1) semaglutide fragments GLP-1 (11-37) are connected through KR, and then sequentially fused with an enterokinase enzyme cutting site and a fusion protein label to realize tandem expression, so that the fusion protein is a fusion protein label-enzyme cutting site-N associated semaglutide fragment GLP-1 (11-37), and the fusion protein label is an albumin affinity peptide (SEQ ID NO. 1). The fusion protein is expressed in a heterologous manner in an E. coli BL21 (DE3) competent cell, an engineering strain is constructed, and then fermentation expression is carried out by using a flask, so that the fermentation density of the bacterial body is greatly improved, expression of the target protein in an inclusion body is promoted, the yield of the target protein is improved, and the fusion protein has a good industrial application prospect. The fusion protein can be successfully recognized and cut by commercial enterokinase, KEX2 enzyme and carboxypeptidase B to release the semaglutide precursor polypeptide.

The invention discloses a urate oxidase gene derived from aspergillus cristatus and application thereof, and the urate oxidase gene is characterized in that a natural urate oxidase gene coding region sequence is obtained from the aspergillus cristatus, and the gene is optimized according to the codon preference of escherichia coli to obtain an optimized sequence. Connecting the optimized gene to a carrier through NdeI and BamHI restriction enzyme cutting sites, and constructing a wild type uox recombinant plasmid; the method comprises the following steps: replacing a sequence of uox of aspergillus cristatus for coding 243-site tyrosine (Y) with a sequence for coding lysine (K) through site-specific mutagenesis, constructing mutant Y243K recombinant plasmids, respectively transforming the recombinant plasmids into competent cells, culturing, and adding IPTG to induce target protein expression. And collecting the induced thalli, carrying out ultrasonication and centrifugation to collect the supernatant so as to obtain a crude enzyme, and purifying the crude enzyme through a nickel affinity column so as to obtain the purified UOX protein. Uric acid is used as a substrate to measure the enzyme activity of the purified UOX, and the result shows that the specific enzyme activity of the mutant Y243K is obviously higher than that of a wild type, and the activity is improved by 2.75 times, so that the mutant Y243K has higher application value.

The invention discloses a group of nocardia seriolae mycolic acid synthase genes and a construction and application method of a deletion strain of the nocardia seriolae mycolic acid synthase genes, and belongs to the technical field of disease prevention and control. Comprising the following steps: strain culture and whole genome extraction, specific primer design and gene fragment cloning, deletion plasmid construction, competent cell preparation, electrotransformation and deletion strain screening, and deletion strain characteristic verification. By analyzing the synergistic function of nocardia seriola seriola mycolic acid synthesis key genes, the core effect of the nocardia seriola seriola mycolic acid synthesis key genes in a synthesis pathway is clear to fill the blank of multi-gene association research, and a core theoretical basis is provided for revealing the pathogenic mechanism of nocardia seriola seriola mycolic acid.

The invention provides a method for prokaryotically expressing cat allergen Fel d 1 recombinant protein, which comprises the following steps of: cloning a cat allergen Fel d 1 gene to multiple cloning sites of an expression vector pGEX-4T-1 to obtain a recombinant expression vector pGEX-4T-1-Fel d 1, transforming the recombinant expression vector into escherichia coli Rosetta (DE3) competent cells to construct recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), and carrying out prokaryotic expression on the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) to obtain the cat allergen Fel d 1 recombinant protein. IPTG (isopropyl-beta-d-thiogalactoside) is used for inducing protein expression, and a GST affinity chromatography medium is used for capturing high-purity soluble GST-Fel d 1 fusion protein. According to the invention, a pGEX-4T-1-GST label system is combined with Escherichia coli Rosetta (DE3), so that the problem of soluble expression of the cat allergen Fel d 1 protein is fundamentally solved.