46results about How to "High expression activity" patented technology

The invention provides a method for culturing microzyme of secretory expression proteolytic enzyme. The method is characterized in that an inorganic salt culture medium is adopted for fermenting and culturing a recombinant methylotrophic yeast. Fermentation and culture comprise the growth period of the microzyme and the secretory expression period of the proteolytic enzyme. In the secretory expression period of the proteolytic enzyme, the feed flow of methanol can be controlled in real time according to the variation of oxygen dissolved in fermentation liquid, and the PH value of the fermentation liquid is adjusted with ammonia, thus expressing the proteolytic enzyme at high efficiency. Compared with the prior art, the method for culturing the microzyme of the secretory expression proteolytic enzyme has the characteristics of low culture cost, convenient and accurate reaction control mode and high expression activity of the proteolytic enzyme.

The invention discloses a heat and ultrasound tolerant mutant nitrile hydratase which belongs to the technical field of enzyme engineering and industrial microbes. The mutant nitrile hydratase is obtained by displacing at least one residue of amino acid residues in the nitrile hydratase with the amino acid sequence represented by SEQ ID NO:1 and adding 1-2 residues to a position behind the terminal residue of the amino acid sequence represented by the SEQ ID NO:1, and the displaced residue corresponds with 141Ser, 143Ser and 144Leu in the amino acid sequence represented by the SEQ ID NO:1. The stress resistance of the mutant nitrile hydratase is good, and the heat tolerance, the survivability and the ultrasonic survivability of the mutant nitrile hydratase are substantially improved.

The invention discloses a construction method of double knockout recombinant rhodococcus. The construction method comprises the following steps: (1) primers are designed according to nitrile hydratase gene sequence of the rhodococcus for respective amplification of an upstream sequence and a downstream sequence of a nitrile hydratase gene, and an upstream fragment and a downstream fragment of the nitrile hydratase gene are obtained; (2) the upstream fragment and the downstream fragment of the nitrile hydratase gene are inserted into a suicide plasmid, and a recombinant suicide plasmid is obtained, wherein the suicide plasmid carries a kanamycin resistant gene and a levansucrase gene; (3) competent cells of recombinant rhodococcus of an amidase gene are knocked out through transformation of the recombinant suicide plasmid, and the double knockout recombinant rhodococcus is obtained: first screening is performed through kanamycin resistance, and a first recombinant colony is obtained and subjected to second screening through a sucrose plate. The invention further discloses recombinant rhodococcus with high nitrilase expression, a construction method of the recombinant rhodococcus and a method for preparing acrylamide.

The invention belongs to the field of plant genetic engineering and discloses an MYB type transcription factor GmMYB29 of Glycine max as well as an encoding gene and an application of the transcription factor GmMYB29. The transcription factor GmMYB29 has an amino acid sequence shown as SEQ ID NO.2. An open reading frame of the gene has a DNA sequence shown as SEQ ID NO.1. The transcription factor GmMYB29 increases the content of isoflavones of the Glycine max by promoting driving expression activity of a key structure gene promoter for an isoflavone biosynthetic pathway. The transcription factor GmMYB29 has significant value in breeding of a new Glycine max variety with high content of isoflavones.

The invention relates to the field of gene expression, in particular to efficiently expressed series porcine alpha and gamma interferon genes. The nucleotide sequence of the interferon genes is shown as a sequence 2. The genes of the sequence 2 are cloned to eukaryotic expression vectors to obtain recombinant plasmids. A synthesis method of the series porcine alpha and gamma interferon genes comprises the following steps of: inserting Rabbit beta-GlobinIntronII, a pCMV immediate early promoter and a T7 promoter into pVAX1 to obtain pMVAX1, and inserting fusion protein genes pIFN-alpha / gamma into pMVAX1 to obtain pMVAX1-pIFN-alpha / gamma. The invention also relates to application of expression protein of the interferon genes in preparation of a medicament for treating porcine reproductive and respiratory syndrome. Through the interferon genes, the limitations of low expression quantity and poor activity when pIFN-alpha and pIFN-gamma are expressed through the pVAX1 are broken, the activity reaches 1*10<8.0>U / 0.1mL, the multiplication of high pathogenic porcine reproductive and respiratory syndrome viruses (PRRSV) can be obviously inhibited, and the economic loss of a pig farm is reduced.

The invention discloses a recombinant yeast strain and application thereof. The recombinant yeast strain expresses lycopene synthesizing genes crtE, crtB and crtI, the genes crtE, crtB and crtI come from deinococcus gobiensis, and all the genes are put under a growth-coupling dynamic controlling element HSP26 promoter, and are integrated onto a saccharomyces cerevisiae genome in a series mode; anHMGR gene is dynamically controlled through the overexpression rate-limiting step of the recombinant yeast strain, the HMGR gene is put under a growth-coupling dynamic controlling element Cit1 promoter, a competitive metabolic pathway ERG9 promoter is replaced with a growth-coupling dynamic controlling element PDC1 promoter, an HMG1 gene promoter and a control area of the HMG1 promoter are replaced with the growth-coupling dynamic controlling element Cit1 promoter, and an Ald6 gene is knocked out. The recombinant yeast strain can be applied to synthesis of carotenoid.

The invention relates to a myogenin (MyoG) gene enhancer, belonging to the technical field of genetic engineering. The MyoG gene enhancer consists of regulatory elements, namely E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1 and TEF-1. The MyoG gene enhancer is characterized in that the sequence of the regulatory elements of the MyoG gene enhancer is E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1 and TEF-1; and the SE nucleic acid sequence of the MyoG gene enhancer is shown in Seq ID No: 1 and has the length of 147 bp. Due to an artificially-synthesized enhancer sequence, the expression of a MyoG gene can be promoted at the proliferation and differentiation stages of myoblasts, and the expression activity of the MyoG gene can be remarkably improved.

The invention relates to a method for increasing the activity of a myogenin (MyoG) gene promoter, and belongs to the technical field of genetic engineering. The method for increasing the activity of the myogenin (MyoG) gene promoter is characterized in that a deletion fragment which is 373 bp in length and has higher muscle specific promoter activity, namely pGL3-MyoGpro373, is obtained by adopting a method for analyzing the activity of a promoter deletion fragment by means of cloning a nucleotide sequence, which is at the 5' end control region of the MyoG gene and is 2125 bp in length; a promoter element therein having the SP1 positive regulation effect is cloned; the two fragments are connected in series, so that an expression carrier containing two copying numbers of promoter regulatory elements is constructed; the expression carrier is called as pGL3-MyoGpro373-double, namely a promoter sequence having double characteristics; and the base composition of the expression carrier is represented by Seq ID No:2.

The invention discloses a Bt protein as well as a coding gene and application thereof. An amino acid sequence of the Bt protein is as shown in SEQ ID No.1, and a base sequence of the coding gene of the Bt protein is as shown in SEQ ID No.2. The application refers to application of the Bt protein in improving insect resistance of plants. Expression activity of an mBt gene of mBT transgenic rice obtained by transferring rice through the coding gene is 20%-50% stronger than that of a wBT gene; resistance on lepidopteron ice leaf folder and striped rice borer is also greatly improved as follows: the rice leaf folder can form a certain withered and yellow hazard symptoms on leaves of the wBt gene ruce, but the leaves of the mBt transgenic gene rice are not formed or only formed with withered and yellow hazard symptoms or only extremely small withered and yellow symptoms are formed near a shear mouth; on the wBt transgenic rice, a white head rate caused by striped rice borer is 1.1 + / -0.1%, and the white head rate on the mBt transgenic rice is 0.

The invention discloses a recombinant saccharomyces cerevisiae expressing CBDAS and a construction method and an application thereof. The CBDAS is positioned on a net membrane of endogenous endoplasmic reticulum positioning peptide of the saccharomyces cerevisiae to be expressed, the recombinant saccharomyces cerevisiae expressing the CBDAS is obtained, firstly, a genome of the saccharomyces cerevisiae serves as a template, and an upstream homologous arm 416d-Up fragment and a downstream homologous arm 416d-Down fragment are obtained through PCR amplification; then, by taking a plasmid pZF048 or pZF049 as the template, PCR amplification is carried out, so as to obtain a Gal1-CBDAS-CEN1-tADH1 fragment or a Gal1-CBDAS-CYB5-tADH1 fragment; the recombinant saccharomyces cerevisiae for expressing the CBDAS is obtained by transforming the fragments into the saccharomyces cerevisiae, and the heterologous protein CBDAS is expressed by using endoplasmic reticulum positioning peptide of the saccharomyces cerevisiae, so that the expression activity of the enzyme is improved, and the yield of the product CBDA is increased.

The invention discloses a CPC acylation enzyme whose encode gene is DNA sequence shown as SEQ IN NO: 4 and DNA sequence having at least 80154000mology with the above DNA sequence. The invention also discloses carriers and transformants containing coding CPC acylation enzyme and the use of the enzyme. The enzyme has high expressing activity, high catalytic activity and high outcome tolerance to catalysis bottom object in high efficiency to prepare outcome 7-ACA.

The invention discloses a sea island cotton receptor albuminoid kinase gene promoter and an application thereof. The length of the promoter is 3183bp, nucleotide sequences are shown as SEQ ID NO.1. Receptor albuminoid kinase gene encoding protein products are relevant to the signal conduction of plant stress response, the gene promoter is the promoter with special vascular bundles and is expressed through biological or abiological inducement, the expression level of target genes in the vascular bundles by a gene engineering method through the promoter is enhanced, and the resistance of cotton on greensickness and abiotic stress is improved.

A method for determining whether a substance can increase the expression level of a fatty acid binding protein (FABP) in an animal. The method includes using a cell that includes an expression construct that comprises a FABP promoter operably linked to a polynucleotide sequence encoding a reporter molecule, wherein the cell is contacted with a candidate substance and then cultivating the cell under conditions conducive to expression of the reporter molecule. Increased expression of the construct in the presence of the candidate substance as compared to a control leads to improved sleep and long-term memory in the subject.

The invention discloses a technology for producing lycopene by biological fermentation and relates to the field of biological pharmacy. The technology for producing the lycopene by the biological fermentation, disclosed by the invention, comprises the following steps: inoculating positive fungus spores and negative fungus spores into saline solutions respectively to obtain a positive fungus sporesuspension solution and a negative fungus spore suspension solution; inoculating the positive fungus spore suspension solution and the negative fungus spore suspension solution into a seed culture medium according to the concentration ratio of 2 to (3 to 5) to obtain a seed solution; inoculating the seed solution into a fermentation culture medium and carrying out fermentation culture; after carrying out the fermentation culture for 12h, supplementing a fermentation culture medium which is equivalent to an original fermentation culture medium into a fermented solution. By adopting the technology disclosed by the invention, a form of the seed solution is improved; the positive spore suspension solution and the negative spore suspension solution are directly inoculated into the seed culturemedium to carry out conjugation; produced thalli are extremely small fungus pellets or thalli and pelletized thalli are not generated, so that the absorption of oxygen and nutrient substances in a liquid culture medium is facilitated; fermentation conditions are improved so that the metabolism of the thalli and the expression activity of a product are enhanced; the accumulation of the lycopene isfacilitated and the yield of the lycopene is improved.

The invention relates to the field of gene expression. The nucleotide sequence of a porcine alpha-interferon gene with efficient expression and high antiviral activity is shown as a sequence 2. Genes of the sequence 2 are cloned to a eukaryon expression vector to obtain a recombinant plasmid. A synthetic method of the porcine alpha-interferon gene comprises the following steps of: inserting a Rabbit beta-Globin Intron II, a pCMV immediate early promoter and a T7 promoter into pVAX1(C) to obtain pMVAX1(C); and inserting a maturation protein gene of pIFN-alpha into the pMVAX1(C) to obtain pMVAX1(C)-pIFN-alpha. The expression protein of the porcine alpha-interferon gene is applied to the preparation of a medicine for treating a porcine reproductive and respiratory syndrome. The porcine alpha-interferon gene with efficient expression and high antiviral activity breaks through the limitation that the expression amount is low and the activity is poor while being used for expressing the pIFN-alpha by the pMVAX1(C) carrier and the activity unit can be up to 2*10<7.0> U / 0.1 mL; and through the porcine alpha-interferon gene provided by the invention, the proliferation of PRRSV (Porcine Reproductive and Respiratory Syndrome Viruses) with high pathogenicity can be obviously inhibited and the economic loss of a pig farm can be reduced.

The invention discloses a product-resistant inhibitory mutated cephalosporin C acylase, a gene carrier and a transformant of the enzyme, and application of the enzyme in one-step enzymatic production of 7-aminocephalosporanic acid, belonging to the technical field of enzyme engineering and biotechnology industry. The amino acid sequence of the enzyme is obtained by conducting deletion mutation on the amino acid sequence of cephalosporin C acylase coded by the gene sequence SEQ ID NO:1 to obtain enzyme CPCAcy-D2 and CPCAcy-D4. The amino acid sequences are shown as SEQ ID NO:2 and SEQ ID NO:3, respectively. The invention also discloses the gene carrier, the transformant, and the application of the enzyme. The enzyme has high expressing activity, and also the tolerance of the product is improved significantly, so that CPC is catalyzed efficiently to produce 7-ACA.

The invention relates to a transgenic construct and application of the same in preparation of an epididymis head gene conditional knockout mouse model. Specifically speaking, it is discovered by the inventor that an Lcn5 gene promoter sequence with a length of 1.8 kb can drive expression of an exogenous gene in head (especially middle-and-far-end specific) areas of epididymis of a mouse and an exogenous Cre gene expression vector driven by the promoter is thus constructed. The transgenic vector can drive specific expression of an exogenous Cre gene at the head of the epididymis of an animal and expression of other genes like CreERT2 recombinase. Usage of such a transgenic system for preparation of a transgenic mouse, a high genomic integration rate (greater than 25%) can be obtained, and expression of an exogenous gene in medium-and-far-end areas of epididymis heads can be accurately and highly efficiently driven. The invention further establishes an epididymis head specific area conditional gene knockout or overexpression technology platform.

A method for determining whether a substance can increase the expression level of a fatty acid binding protein (FABP) in an animal. The method includes using a cell that includes an expression construct that comprises a FABP promoter operably linked to a polynucleotide sequence encoding a reporter molecule, wherein the cell is contacted with a candidate substance and then cultivating the cell under conditions conducive to expression of the reporter molecule. Increased expression of the construct in the presence of the candidate substance as compared to a control leads to improved sleep and long-term memory in the subject.

The invention discloses a method for reinforcing meat chicken cold stress resistance, and belongs to the technical field of agricultural cultivation. The method comprises the following steps: (1) illumination control, (2) chicken house environmental control, and (3) feeding control. According to the invention, the method can effectively guarantee normal development of meat chickens under cold stress conditions, improves the meat yield of the meat chickens and the development degree of intracorporeal organs, reduces the cultivation risk, and has extremely-good economic benefits and promotion value.

The invention provides lipase CALB mutant and a preparation method and application thereof. An error-prone PCR technology in a gene in-vitro directed evolution strategy is utilized, mutagenesis in-vitro is conducted on a wild type lipase gene from Klebsiella sp., screening is conducted by distinguishing a culture medium, and a strain for efficiently producing lipase is obtained. The obtained lipase CALB mutant is high in expression activity, the expression activity is 8.7 times catalytic activity of the wild type lipase, and the lipase CALB mutant can be used for industries related to lipid hydrolysis. Batched production of the lipase CALB mutant can be achieved through large-scale fermentation of engineering bacteria containing the gene for encoding the CALB mutant.

The invention discloses a method for improving the expression activity of aquaporin in a vesicle. The method comprises the following steps: 1, performing phosphorylation on the aquaporin and preparing a phospholipid vesicle system solution; and 2, introducing the phosphorylated aquaporin. The invention also provides an aquaporin reverse osmosis membrane. The aquaporin reverse osmosis membrane is prepared by using the phosphorylated aquaporin vesicle prepared by the above method. The expression activity of the aquaporin is improved through the phosphorylation of the aquaporin; and the phosphorylation modified aquaporin is embedded into the vesicle and applied to the reverse osmosis membrane, so under the condition of achieving water flux and desalinization rate with the same level, the ratio of the used aquaporin to the vesicle is greatly reduced.

The invention relates to a transgenic construct and application of the same in preparation of an epididymis head gene conditional knockout mouse model. Specifically speaking, it is discovered by the inventor that an Lcn5 gene promoter sequence with a length of 1.8 kb can drive expression of an exogenous gene in head (especially middle-and-far-end specific) areas of epididymis of a mouse and an exogenous Cre gene expression vector driven by the promoter is thus constructed. The transgenic vector can drive specific expression of an exogenous Cre gene at the head of the epididymis of an animal and expression of other genes like CreERT2 recombinase. Usage of such a transgenic system for preparation of a transgenic mouse, a high genomic integration rate (greater than 25%) can be obtained, and expression of an exogenous gene in medium-and-far-end areas of epididymis heads can be accurately and highly efficiently driven. The invention further establishes an epididymis head specific area conditional gene knockout or overexpression technology platform.

The invention discloses a promoter of a bovine alpha-actin gene and an application thereof, belonging to the technical field of genetic engineering. The promoter is characterized in that the nucleic acid sequence of 170bp containing three SRE positive regulation elements inside the 262bp promoter segment of the bovine alpha-actin gene is subjected to PCR amplification by use of different primers to obtain two nucleic acid sequences of 170bp, and the two nucleic acid sequences of 170bp are connected in series with the upstream of the 262bp promoter segment (namely, 170bp-170bp-262bp) to build a promoter segment of alpha-actin, wherein the nucleotide sequence is shown by Seq ID No:1. The promoter has relatively high promoting activity and skeletal muscle specificity and can remarkably improve the expression activity of the alpha-actin gene in bovine skeletal muscle satellite cell and can be used as an efficient and specific skeletal muscle promoter and applied to the transgenic beef production for increasing the muscle output.

The invention provides a constitutive lactic acid bacteria promoter, a recombinant vector and its recombinant bacteria and application, belonging to the field of genetic engineering. The promoter P provided by the present invention gapA The nucleotide sequence of is shown in SEQ ID No.1. The promoter P gapA It can promote the high-efficiency expression of downstream genes in lactic acid bacteria, and the protein expression activity is significantly higher than that of traditional inducible P NisinA It is an ultra-efficient promoter. The protein expression cassette composed of the promoter PgapA and the target gene of the present invention, the expression vector and the host bacteria can be used for oral drug delivery of peptide and protein drugs, which not only has good application prospects in genetic engineering, but also can improve Patients' medication compliance is expected to generate considerable social and economic benefits.

The invention relates to the field of gene expression. The nucleotide sequence of a porcine alpha-interferon gene with efficient expression and high antiviral activity is shown as a sequence 2. Genes of the sequence 2 are cloned to a eukaryon expression vector to obtain a recombinant plasmid. A synthetic method of the porcine alpha-interferon gene comprises the following steps of: inserting a Rabbit beta-Globin Intron II, a pCMV immediate early promoter and a T7 promoter into pVAX1(C) to obtain pMVAX1(C); and inserting a maturation protein gene of pIFN-alpha into the pMVAX1(C) to obtain pMVAX1(C)-pIFN-alpha. The expression protein of the porcine alpha-interferon gene is applied to the preparation of a medicine for treating a porcine reproductive and respiratory syndrome. The porcine alpha-interferon gene with efficient expression and high antiviral activity breaks through the limitation that the expression amount is low and the activity is poor while being used for expressing the pIFN-alpha by the pMVAX1(C) carrier and the activity unit can be up to 2*10<7.0> U / 0.1 mL; and through the porcine alpha-interferon gene provided by the invention, the proliferation of PRRSV (Porcine Reproductive and Respiratory Syndrome Viruses) with high pathogenicity can be obviously inhibited and the economic loss of a pig farm can be reduced.

An enhancer of myogenin (MyoG) gene, which belongs to the technical field of genetic engineering, is composed of regulatory elements E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1, TEF-1, and its characteristics The sequence of regulatory elements of the MyoG gene enhancer is: E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1, TEF-1; the nucleic acid sequence of the MyoG gene enhancer "SE" is shown in SeqIDNo:1 , with a length of 147bp. The artificially synthesized enhancer sequence can promote the expression of MyoG gene in both the proliferation and differentiation stages of myoblasts, and can significantly improve the expression activity of MyoG gene.

The invention discloses an operon, its carrier and its application, wherein the operon sequence at least includes, the 3' end is located at any base in the 201-257 bp upstream of the ATG coding sequence of the Corynebacterium glutamicum SEQ ID NO: 6 gene The 5' end is located at any base in the upstream 308bp-345bp of the coding sequence ATG of the Corynebacterium glutamicum SEQ ID NO: 6 gene. After the operon of the present invention is induced by the inducer, the protein expression activity is significantly higher than that of the traditional Ptac, and it is an ultra-efficient operon. The operon of the present invention can be used for high-efficiency expression of prokaryote endogenous or exogenous proteins.