13 results about "PUC19" patented technology

The invention relates to the technical field of nucleic acid detection, and discloses a detection method based on LAMP (loop-mediated isothermal amplification) and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas and application. The detection method comprises the following steps: carrying out key amino acid mutation on a PAM structural domain of an eBrCas12b protein; the preparation method comprises the following steps: preparing Cas12b sgRNA through in-vitro transcription and purification; the cis-cleavage capability of the eBrCas12b, the eBrCas12b-G and the eBrCas12b-GN on the target sequence is evaluated, and the cis-cleavage capability of the Escherichia coli carrying pUC19-blaNDM5 plasmids is constructed, and an eBrCas12b-GN one-tube method detection system is applied to detection of the NDM drug-resistant escherichia coli. According to the detection method based on LAMP and CRISPR / Cas and the application, a traditional LAMP-Cas12b one-tube method detection system is optimized, and the sensitivity and speed of one-tube method detection are remarkably improved.

The invention relates to a method for splitting, assembling and site-specific integration of large-fragment exogenous DNA (deoxyribonucleic acid) in Trichoderma reesei. Comprising the following steps: (1) constructing an arg:: Ptcu-Cas9-TtrpC-hph strain containing a Cas9 protein; (2) constructing a pUC19 <-> integration site: a pyr4 plasmid; (3) obtaining a plurality of target gene segments containing integration site homologous arms and having overlapping regions at the tail ends of adjacent DNA segments; (4) obtaining a gRNA expression cassette of a targeted genome integration site; (5) arg conversion: the arg is converted into a Ptcu-Cas9-TtrpC-hph strain; and (6) screening and verifying. According to the method, the CRISPR / Cas9 system is combined with the TAR in the trichoderma reesei for the first time, the exogenous large-fragment DNA is split into a plurality of small fragments, then the plurality of small-fragment exogenous DNA are assembled in the trichoderma reesei and are subjected to fixed-point integration, one-step editing of the large-fragment exogenous DNA is realized, and compared with a traditional genetics method, the gene editing efficiency in the trichoderma reesei is greatly improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention discloses a replication-limited recombinant feline herpesvirus as well as a construction method and application thereof, and relates to the technical fields of genetic engineering and preventive veterinary medicine, exogenous feline parvovirus VP2 protein is expressed in the recombinant feline herpesvirus, and meanwhile, a gL gene in the feline herpesvirus is deleted. The construction method comprises the following steps: 1, constructing a QD-1 gL gene stable expression F81 cell line by using a lentivirus packaging three-plasmid system; 2, constructing a PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid, and carrying out GFP FPV VP2 recombinant plasmid construction; 3, an sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the gL gene is constructed, and the sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the And 4, transfecting the recombinant vectors in the step 2 and the step 3 to an F81-gL cell line to obtain the QD-1 gL-FPV VP2 recombinant feline herpesvirus. The recombinant feline herpesvirus can be used as a candidate vaccine to prepare a strain so as to resist feline herpesvirus and feline parvovirus infection.

The present invention relates to the field of nucleic acid detection technology, and discloses a detection method and application based on LAMP and CRISPR / Cas. The detection method comprises the following steps: mutating key amino acids in the PAM domain of the eBrCas12b protein; preparing Cas12b sgRNA by in vitro transcription and purification; evaluating the cis-cleavage ability of eBrCas12b, eBrCas12b-G, and eBrCas12b-GN on the target sequence; constructing a pUC19- without NDM5 The present invention adopts the above-mentioned detection method and application based on LAMP and CRISPR / Cas, optimizes the traditional LAMP-Cas12b one-tube detection system, and significantly improves the sensitivity and speed of one-tube detection.

The application discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is constructed by introducing an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, a MCS (multiple cloning site), a 3'-UTR sequence and a T7 terminator sequence on the basis of part of a sequence of an E. coli plasmid pUC19. The pSY1A plasmid vector has a MCS into which an exogenous gene can be inserted, and the MCS and a transcription assembly constitute a transcription module, so that the transcription module can be preserved by preserving the plasmid, and in-vitro transcription of the wheat germ cell-free expression system becomes more convenient and faster.

The invention discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is characterized in that on the basis of partial sequence of escherichia coli plasmid pUC19, an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, MCS multiple cloning sites, a 3 '-UTR sequence and a T7 terminator sequence are introduced to construct the plasmid vector pSY1A. According to the pSY1A plasmid vector provided by the invention, the vector has multiple cloning sites into which exogenous genes can be inserted, and the multiple cloning sites and a transcription component form a transcription module, so that the transcription module can be stored by storing plasmids, and the in-vitro transcription of a wheat germ cell-free expression system becomes more convenient and quicker.

The application discloses a chimeric Kunjin virus of tamabi virus prM-E and a preparation method and application thereof. On the basis of a reverse genetic manipulation system of the Kunjin virus KUNV, the KUNV prM / E gene is replaced by the DTMUV prM / E gene to construct a chimeric DTMUV virus infectious clone plasmid with the KUNV as a skeleton, which is named as pUC19-KUNV-DTMUV prM / E. Then, the chimeric tamabi virus prM-E Kunjin virus is obtained through virus rescue, and is named as rKUNV-CHv-prM / E (CHv). Analysis on the virus characteristics shows that the CHv can effectively proliferate in BHK-21 and DEF cells, and the duck embryo virulence is weaker than that of the KUNV and the DTMUV. The animal experiment results show that the CHv only detects obvious virus levels in the brain, and does not produce viremia; the CHv after immunization can produce similar IgG antibody levels to those after immunization of the KUNV and the DTMUV. Therefore, the recombinant virus of the chimeric DTMUV prM / E with the KUNV as a skeleton prepared by the application can induce the duckling to produce sufficient immune protective neutralizing antibody levels, and can be applied to preparation of a duck tamabi virus vaccine.

The invention discloses a method for constructing a recombinant virus vector based on iridovirus PCNA protein. The method comprises the following steps: amplifying upstream and downstream fragments of an ORF068 gene by taking iridovirus as a template, cloning a puro gene fragment from a pLVX-puro plasmid, inserting the puro gene fragment into a pEGFP-N3 plasmid to construct a pEGFP-puro-GFP vector, respectively connecting the upstream and downstream fragments of the ORF068 with a CMV-puro-GFP fragment in the pEGFP-puro-GFP vector, and inserting the connected fragments into a pUC19 plasmid to construct a recombinant virus vector. The plasmid construction method provided by the invention is simple and strong in operability; and the constructed plasmid has strong stability in cells.

The invention belongs to the technical field of genetic engineering and microorganisms. The invention provides a plasmid pAHC9 and an aspergillus niger unitary editing system. The plasmid pAHC9 is obtained by connecting a DNA (deoxyribonucleic acid) fragment containing a Cas9 gene to a Sma I site of a plasmid pUC19-AMA1-Hyg; the plasmid pUC19-AMA1-Hyg is obtained by assembling a trpC promoter and a DNA (Deoxyribose Nucleic Acid) fragment of a hyg gene onto the plasmid pUC19-AMA1, and the plasmid pUC19-AMA1 is obtained by the plasmid pUC19-AMA1; the plasmid pUC19-AMA1 is obtained by inserting a DNA (deoxyribonucleic acid) fragment containing AMA1 into a Hind III site of the plasmid pUC19. By utilizing the aspergillus niger unary editing system provided by the invention, the albA gene, the pkaC gene, the amyA gene and the csA gene are respectively subjected to targeted editing by utilizing the unary editing system under the condition of not depending on homologous recombination. The editing efficiency on pkaC is 46.8%, the editing efficiency on other genes exceeds 90%, and the one-step blocking efficiency on double genes (albA and pkaC) reaches 24.7%. According to the technology, the editing efficiency of the aspergillus niger genome is greatly improved, and an efficient gene editing means is provided for construction and optimization of a chassis strain based on aspergillus niger.

The invention belongs to the technical field of microbial genetic engineering and gene editing. The invention provides a Cas9 enzyme mediated cytosine base editing plasmid, and compared with a starting plasmid, the Cas9 enzyme mediated cytosine base editing plasmid has the following inserted sequences: a terminator Tcyc1; the base sequence of the terminator Tcyc1 is as shown in SEQ ID NO. 5; a promoter PgpdA; the base sequence of the promoter PgpdA is as shown in SEQ ID NO. 1; the preparation method comprises the following steps: preparing a mini-SDD7 deaminase; the amino acid sequence of the mini-SDD7 deaminase is as shown in SEQ ID NO. 2; an nCas9 (D10A) coding sequence is obtained; the amino acid sequence of the nCas9 (D10A) coding sequence is as shown in SEQ ID NO. 3; a UGI coding sequence; the amino acid sequence of the UGI coding sequence is as shown in SEQ ID NO. 4; the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg, and the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg. The base editing system provided by the invention can be used for gene editing in trichoderma koningii.

The invention relates to a construction method of a recombinant baculovirus shuttle vector Bacmid '. The method comprises the following steps: cloning a lacZ alpha-attTn7 sequence into a baculovirus transfer vector subjected to reverse amplification, so as to obtain a recombinant plasmid pBacPAK8-lacZ alpha-attTn7; the recombinant virus AcNPV-lacZalpha-attTn7 is obtained by co-transfecting Sf9 cells with a defective virus Bacmid, and a recombinant virus AcNPV-lacZalpha-attTn7 is generated; carrying out amplification and ultra-high-speed centrifugation on the obtained recombinant virus, and extracting through phenol chloroform to obtain virus DNA (Deoxyribonucleic Acid); the recombinant baculovirus shuttle vector Bacmid'is obtained by co-transforming competent cells of recombinant virus DNA (deoxyribonucleic acid) and pBvBAC (obtained by cloning Vcat, miniF and Chitinase to pUC19), and carrying out recombinant cloning by utilizing blue-white spot screening. According to the present invention, the miniF replicon co-localized with lacZalpha-attTn7 is separated, such that the recombinant baculovirus shuttle vector Bacmid'with the high gene cloning stability is successfully constructed; according to the technology, the expression stability of the exogenous gene is remarkably improved, and a powerful research tool is provided for vaccine development and virology application based on the baculovirus vector.

The invention provides a protein expression system and application thereof, the protein expression system at least comprises a recombinant expression vector and Escherichia fagenii for transforming the recombinant expression vector, and the recombinant expression vector comprises a target protein gene segment and a pUC57 vector segment. The research finds that the same type of plasmids have significant expression difference in the Escherichia fagenii, for example, the target protein amount expressed by the pUC57 plasmid is significantly higher than that expressed by the pUC19 plasmid, then the pUC57 plasmid and the Escherichia fagenii are combined to form a protein expression system, the protein expression system has a better expression effect on the target protein, and the expression efficiency of the protein expression system is improved. The method has the advantages of simple operation, no need of extra inducers, reduction of the production cost of recombinant proteins, substantial improvement of the yield and enzyme activity of target proteins, and provision of more choices for the production of biological pharmacy, diagnostic reagents and industrial enzymes.