86 results about "Cloning Site" patented technology

The invention discloses a double-fluorescence-labeled engineering bacterium for in-situ tracing of horizontal transfer of plasmids in pseudomonas among bacteria as well as a construction method and application of the double-fluorescence-labeled engineering bacterium, pseudomonas is taken as an original strain, a green fluorescence strong expression component is integrated into a pseudomonas chromosome genome, and green fluorescence-labeled pseudomonas is obtained; a red fluorescence strong expression element is inserted into a multiple cloning site of a broad-host zygote, and is converted into the green fluorescence labeled pseudomonas to form a double-fluorescence labeled engineering bacterium. The double-fluorescence-labeled strain disclosed by the invention can generate corresponding green fluorescence or red fluorescence after being excited by a green or red fluorescence excitation wavelength; when the plasmids enter other bacteria under the action of conjugation or transformation, the recipient bacteria can only present red fluorescence under the excitation of the red fluorescence excitation wavelength, and the horizontal transfer condition of the plasmids in the pseudomonas bacteria among the bacteria under the in-situ condition can be accurately tracked through the intuitive method.

The invention provides an hpRNA interference vector for laver and application of the hpRNA interference vector, and belongs to the technical field of genetic engineering and algae biology. According to the hpRNA interference vector, a binary vector pBI121 is used as a skeleton vector, a laver endogenous Actin promoter and a multiple cloning site segment are inserted, the multiple cloning sites allow target gene segments to be cloned in a forward and reverse repetition mode, the target gene segments are spaced by introns, and therefore an efficient hpRNA structure is formed after transcription, and the efficient hpRNA interference vector is obtained. The gene can be directly used for silencing important genes related to growth and development, metabolic regulation, stress response and the like in the laver. The invention further provides a construction method of the hpRNA interference vector and application of the hpRNA interference vector in silencing laver genes and provides a method for silencing laver gene expression, the gene silencing effect is remarkable, experimental results can be repeated, and a powerful tool is provided for molecular breeding and genetic improvement of laver.

The invention belongs to the field of genetic engineering and biological catalysis, and discloses carboxylase, engineering bacteria and application of the carboxylase in production of aromatic carboxylic acid. The invention discloses a strain of escherichia coli expressing a carboxylase coding gene from marinicaulia flavus, and a construction method of the escherichia coli comprises the following steps: inserting a target protein carboxylase coding gene into a multiple cloning site of a vector pET-28a (+), so as to obtain a recombinant plasmid pET28a-CW354; and introducing the recombinant plasmid into escherichia coli BL21 (DE3) to obtain the engineering bacterium. The invention also protects a method for producing aromatic carboxylic acid, the carboxylase can be used for carboxylating aromatic phenols obtained by depolymerizing lignin, has a wide substrate spectrum, does not need high temperature and high pressure in the whole process, and has the advantages of mild conditions, simplicity in operation, environmental friendliness and the like.

The invention discloses a method for constructing an engineered bacterium of Clostridium aeruginosa, comprising the following steps: preparing a desired gene in a biosynthetic pathway of a target product, namely a pathway gene, to form a pathway gene cluster; embedding the pathway gene cluster into a transposase recognition site of a mariner transposon to form a pathway gene expression cassette; selecting a constitutive strong promoter commonly used in Clostridium jundal for driving transposase expression, loading the pathway gene expression cassette into a multiple cloning site of a plasmid, and inserting an inducible repressor element upstream of the replicon to obtain a transposition plasmid; transforming the transposition plasmid into Clostridium aeruginosa to obtain an engineered bacterium of Clostridium aeruginosa. The yield of 3-hydroxybutyric acid expressed by the engineered bacterium constructed by the above method is as high as 3g / L.

The invention discloses an escherichia coli-bacillus subtilis shuttle plasmid and application, and belongs to the field of gene engineering. The shuttle plasmid comprises the following elements: a promoter sequence, a coding sequence of bacillus subtilis signal peptide, a replication initiation sequence of bacillus subtilis, a replication initiation sequence of escherichia coli and an antibiotic resistance gene. According to the plasmid design, a modular structure is adopted, and quick replacement of key elements such as a promoter, a signal peptide and RBS is supported through optimized enzyme cutting site layout; a 6 * His tag introduced at the tail end of a multiple cloning site (MCS) can be adapted to nickel column affinity chromatography, and the purification process of the recombinant protein is simplified. The plasmid has the characteristics of small molecular weight and convenience in operation, can be efficiently cloned and constructed in escherichia coli and then directly converted into bacillus subtilis for protein expression, is particularly suitable for development of a gram-positive bacterium secretory expression system, and provides an efficient tool for large-scale production of industrial enzyme preparations.

An uropathogenic escherichia coli recombinant protein composition LS, a construction, expression and purification method therefor, and a use thereof, relating to the technical field of genetic engineering. The method comprises: carrying out PCR amplification on LpcA and SurA genes, and respectively inserting the LpcA and SurA genes into multiple cloning sites of a cloning vector to construct a recombinant vector; then converting the recombinant vector into an escherichia coli expression protein; and finally, carrying out purification to obtain a composition LS of an LpcA protein and an SurA protein, wherein the composition LS can be used in a urinary tract infection vaccine. The present invention has the following advantages: a protein vaccine can induce relatively strong cell and humoral immune response of animals; and animal challenge experiments show that the protein vaccine has the protection effect of reducing the bacterial loads of the bladder and the kidney.

The present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

The present application relates to the field of genetic engineering, and in particular to a vector for analyzing plant promoter expression specificity, a preparation method and application thereof. The T-DNA region of the vector comprises, from 5' end to 3' end, a callus-specific promoter-driven selection marker gene expression cassette and a reporter gene expression cassette with a multiple cloning site; the callus-specific promoter-driven selection marker gene expression cassette comprises a callus-specific promoter, a selection marker gene coding sequence and a terminator. The vector of the present application utilizes a rice callus-specific promoter to drive the expression of a selection marker gene, can greatly reduce the non-specific interaction with the promoter of a target gene, improve the specificity of the expression of the target gene, and effectively reduce the biological safety risk caused by the selection marker gene in a transgenic plant, and has important application value in basic theoretical research and molecular breeding.

The application provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

To provide recombinant nucleic acids containing alphaherpesvirus promoter sequences.SOLUTION: The present invention provides a recombinant vector for expression of a target gene in a host cell, comprising: a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for the expression of the target gene in the host cell.SELECTED DRAWING: None

The invention discloses a method for expressing a specific domain of a Mouse SPO11 protein. The method comprises the following steps: using a sequence of the Mouse SPO11 protein as a template, designing primers for PCR amplification, and obtaining a Mouse SPO11 gene fragment after PCR amplification; in an Alp_PET_StHis_SUMO empty vector, sequentially designing a Strep tag II affinity tag, an 8x his affinity tag, and a SUMO solubilizing tag at the N-terminus of a multiple cloning site; subjecting the Mouse SPO11 gene fragment after PCR amplification to gel electrophoresis, followed by gel excision recovery, and then simultaneously subjecting the Mouse SPO11 gene fragment to BamHI and EcoRI double enzyme digestion with the expression vector Alp_PET_StHis_SUMO, and then subjecting the Mouse SPO11 gene fragment to gel excision recovery, connection, transformation, colony PCR, colony culture, and plasmid extraction to obtain a successfully constructed plasmid; inducing expression of the plasmid to obtain an expressed bacterial solution; and purifying the expressed bacterial solution to obtain the Mouse SPO11 protein specific domain. The invention can express the specific structural domain of Mouse SPO11 protein and obtain high-purity Mouse SPO11 protein.

The invention discloses a preparation method and application of a biological herbicide based on an RNA (Ribonucleic Acid) interference technology, which specifically comprises the following steps: designing a specific siRNA (Small Interference Ribonucleic Acid) sequence through a targeted glutamine synthetase gene (GS2), cloning a reverse repeat structure of the specific siRNA sequence to multiple cloning sites of an L4440 vector, and transforming the reverse repeat structure to RNase III defective escherichia coli HT115 for induced fermentation. By optimizing IPTG concentration and induction time, the thalli efficiently express target dsRNA, and a dsRNA aqueous solution is prepared after splitting decomposition and purification. Foliage spraying is adopted during spraying treatment, dsRNA can permeate into plant cells through pores, GS2 mRNA is specifically degraded, ammonium ions in the cells are accumulated, a photosynthetic system collapses, and leaves wither, wither and yellow and finally die within 72 hours. Compared with a traditional chemical herbicide, the herbicide has the advantages of being accurate in targeting, safe, environmentally friendly, free of residues and the like, and an efficient and sustainable biological solution is provided for agricultural weeding.

The invention discloses a high-efficiency expression and purification method of 5 '-3' exonuclease, which comprises the following steps: based on a Bac-to-Bac system, adding a tag and a TEV restriction enzyme cutting site to optimized 5 '-3' exonuclease Xrn1, then connecting the 5 '-3' exonuclease Xrn1 to a multiple cloning site behind a Polyhedrin promoter of a pFastBac expression vector to prepare expression baculovirus plasmids, and carrying out purification on the expression baculovirus plasmids to obtain the 5 '-3' exonuclease. Then transfecting the recombinant baculovirus particles into insect cells to generate recombinant baculovirus particles; finally, the 5 '-3' exonuclease Xrn1 is obtained through nickel column affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the 5 '-3' exonuclease Xrn1, and a technical foundation is laid for large-scale production of the 5 '-3' exonuclease.

This invention relates to genetic engineering technology and aims to provide a method for preparing and applying bacteriophage capsid protein MS2-armored PRRSV antigen mRNA particles. The method includes: inserting the MS2 bacteriophage capsid protein gene and an mRNA expression cassette into a double multiple cloning site expression vector; transforming the recombinant expression plasmid into host bacteria; inducing expression to allow the MS2 capsid protein to express and self-assemble into virus-like particles; utilizing the specific recognition of the MS2 capsid protein on the packaging stem-loop structure, packaging the transcribed mRNA inside the virus-like particles to form MS2-armored PRRSV antigen mRNA particles; lysing the host bacteria, separating the supernatant, and enriching and purifying the particles. This invention significantly enhances the resistance of mRNA to nuclease degradation and improves formulation stability; it can increase the efficiency of mRNA entering antigen-presenting cells and enhance immunogenicity; it can be mass-produced with controllable processes and relatively low cost.

The invention discloses a multi-reporter gene expression cassette for plant promoter expression pattern and activity analysis, the expression cassette sequentially comprises a multiple cloning site MCS and a terminator from 5'end to 3 'end, at least two reporter genes are arranged between the MCS and the terminator, and the adjacent reporter genes are spaced by a 2A sequence, the reporter genes are different types of reporter genes, the types of the reporter genes include but are not limited to fluorescent protein reporter genes and enzyme reporter genes, the 2A sequence is a 2A self-cleavage peptide sequence, and the terminator has the capability of terminating transcription started by the Pol II type promoter. The MCS in the expression cassette facilitates cloning of various promoters and other functional elements, and the expression cassette contains fluorescent protein reporter genes and enzyme reporter genes at the same time, has the advantages of the fluorescent protein reporter genes and the enzyme reporter genes, and has the advantages of being wide in application range and diverse in detectable means.

The invention discloses an alkali-resistant application of a soybean GmNPF7.13 gene. The nucleotide sequence of the soybean GmNPF7.13 gene is as shown in SEQ ID NO. 1. The plant GmNPF7.13 gene silencing vector comprises a recombinant GmNPF7.13 gene silencing vector, the gene GmNPF7.13 segment is inserted into multiple cloning sites of a no-load VIGS-EV of the silencing vector, and GmNPF7.13 gene silencing is realized in a plant. According to the invention, two independent verification systems, including two mutants and a virus-induced GmNPF7.13 gene silencing material, both show enhanced alkali resistance and are strong in verification consistency. The GmNPF7.13 gene disclosed by the invention not only facilitates analysis of a response mechanism of soybeans to alkali stress, but also can be used as a target gene for creating alkali-resistant plants and expanding planting of the soybeans in soda saline-alkali soil, and has important theoretical significance and breeding application prospects.

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.

The invention relates to the technical field of biomedicine, and particularly discloses a dynamic monitoring method for ATP (adenosine triphosphate) level in cardiac muscle cells, recombinant cardiac muscle cells and application. The preparation method comprises the following steps: cloning an iATPSnFRs gene as shown in SEQ ID NO.3 into multiple cloning sites of a pLVX-AcGFP1-N1 plasmid, so as to construct an iATPSnFR1 recombinant plasmid; then transfecting myocardial cells with the iATPSnFR1 recombinant plasmid to obtain recombinant myocardial cells; and monitoring the ATP level change in the iATPSnFR1 recombinant myocardial cells in real time by using a high content imaging system. The recombinant myocardial cell containing the iATPSnFR1 is obtained through construction, so that the iATPSnFR1 can be specifically combined with ATP in the cell and generate a fluorescence signal, real-time and dynamic monitoring of the ATP level can be realized, and the recombinant myocardial cell has the advantages of non-invasiveness, high spatial resolution and the like.

The present invention belongs to the field of genetic engineering and molecular biology, and specifically relates to a simple, fast and efficient method for constructing an RNA interference vector and its application. The method specifically includes designing specific primers with homology arms and restriction sites, amplifying exogenous DNA fragments using the specific primers, and using restriction endonucleases to Bam HI and Kpn Ⅰ Enzyme cut the RNA interference empty vector, and use the homologous recombination method to connect the exogenous DNA fragment to the RNA interference empty vector to form the RNA interference final vector. This method introduces a lethal gene on the RNA interference backbone vector. Combined with the homologous recombination method, only one homologous recombination is needed to simultaneously connect two exogenous DNA fragments into the two multiple cloning sites of the RNA interference vector, which greatly shortens the RNA interference vector construction cycle, and the vector construction positive rate reaches 100%. In addition, the selection of exogenous fragments is no longer limited by the restriction site, and can be freely selected, which broadens the range of targets.

The invention discloses an acid-resistant microalgae construction method based on bicarbonate ion transporter protein, and belongs to the field of biology and molecular biology. The method specifically comprises the following steps: taking Phatr3J33543 with a sequence structure as shown in SEQ ID No.1, Phatr3J50516 with a sequence structure as shown in SEQ ID No.2 or Phatr3Jdraft1806 with a sequence structure as shown in SEQ ID No.3 as a target gene, removing a termination codon of the target gene, and connecting a fluorescent protein gene with an initiation codon removed through a DNA (Deoxyribose Nucleic Acid) sequence for coding glycine to obtain a recombinant gene; inserting the recombinant gene into multiple cloning sites of a pPhaNR plasmid to obtain a vector plasmid; introducing the carrier plasmids into diatom cells through electroporation, and inoculating the diatom cells to an ESAW solid selective culture medium containing bleomycin to grow brown algal colonies; selecting the algal colonies with fluorescent protein signals from the algal colonies, namely the acid-resistant engineering algae. Compared with wild algae, the acid resistance of the engineering algae obtained by the invention is obviously improved. The method has important reference significance on acid-resistant diatom cultivation.

The invention relates to a process for transferring DNA (Deoxyribose Nucleic Acid) into a plant green pigment (chloroplast) to produce protein, which specifically comprises the following steps of: cloning a gene to be transferred after a chloroplast specific promoter, embedding the artificial gene and an antibiotic resistance gene into a transformation vector, the cloning site is located between two adjacent DNA sequences in a plant pigment genome to be transformed, then in a deionized protoplast liquid culture medium with the osmotic pressure identical to that in plant cells, cell walls of the plant cells are digested with appropriate cell wall degrading enzymes (cellulase, polygalacturonase and the like), protoplast spheres and protoplast are obtained, and the plant pigment genome is transformed. The method comprises the following steps: preparing a protoplast culture medium containing enzyme, mixing plasmid DNA carrying coding genes with the protoplast culture medium containing no enzyme, adding the DNA into the protoplast, putting the mixture into an electroporation cuvette, impacting with current pulse, and finally transferring cells with resistance to selective antibiotics into the selective culture medium to proliferate, thereby regenerating the whole plant.

The invention discloses a CAR (chimeric antigen receptor) recombinant lentiviral expression vector suitable for CIK (cytokine-induced killer) cells and NK (natural killer) cells to express EpCAM antibodies, IL-21 and CCL19, and belongs to the technical field of genetic engineering and biology. According to the expression vector, a CAR (chimeric antigen receptor) gene structure, an IL-21 (interleukin-21) gene expression structure and a CCL19 (chimeric antigen receptor 19) gene expression structure are connected between multiple cloning sites Nhe1 and Not1 of a vector pCHD-CMV-MCS-EF1-copGFP-T2A-puro, so that a recombinant plasmid pCDH-21 * 19 EpCAM-CAR is obtained. And transforming the recombinant plasmid into escherichia coli DH5alpha to obtain the recombinant microbial cell. The pCDH-21 * 19 EpCAM-CAR recombinant lentiviral expression vector is packaged, lentiviral particles are concentrated, CIK cells and NK cells are infected, and eukaryote transgenic cells are obtained. The CAR vector aiming at the solid tumor colorectal cancer is constructed, the design of the vector aims at improving the activating and killing capacity of CIK cells and NK cells and solving the problems of tumor microenvironment immunosuppression and the like, and a foundation is further laid for the research of treating solid tumors by applying a CAR technology.

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 present invention discloses a plant gene editing vector and its application. The key point is that the plant gene editing vector contains an NC cloning frame structure (NC universal linker 1-SfiI-ccdB gene-SfiI-NC universal linker 2) at the cloning site. Gene fragments containing one or more gene editing target sequences can be quickly cloned into the vector through a one-step ligation reaction called Nimble cloning (NC cloning) for plant gene editing research. The vector provided by the present invention can simply, quickly, and efficiently clone plant gene editing target sequences, facilitating plant gene editing research.

The invention relates to the technical field of biology, in particular to construction and application of an lnc30 overexpression strain. Comprising the following steps: (1) carrying out enzyme digestion on an original plasmid pLN-ENR-GFP by utilizing Hind III restriction enzyme, recovering a larger fragment, and then carrying out fragment self-connection to obtain a pBSD plasmid; (2) construction of a pBSD-30 plasmid: carrying out PCR (Polymerase Chain Reaction) to obtain and purify a U6 promoter gene, and connecting the gene between a 5'cloning site ApaI and a 3 'cloning site Avr II of the PLN-ENR-GFP plasmid; the method comprises the following steps: synthesizing an lnc30 full-length gene, and connecting the lnc30 gene between a 5'cloning site Avr II and a 3 'cloning site Afl II of a PLN-ENR-GFP plasmid; the lnc30 is connected with a U6 promoter, so that high expression of the lnc30 in cells is ensured. Experiments prove that lnc30 overexpression can significantly enhance the growth advantage of plasmodium falciparum and the ability of merozoite invading erythrocytes and participate in regulation of the erythroid stage development process of plasmodium, but knock-down of lnc30 may cause death of plasmodium, which indicates that the lnc30 overexpression strain is successfully constructed and can be used for research of a plasmodium falciparum gene expression regulation mechanism, and the lnc30 overexpression strain can be used for preparing the plasmodium falciparum gene expression regulation mechanism. The method is used for screening and researching drugs for inhibiting growth and development of plasmodium falciparum, and has remarkable scientific value and application prospect.

Provided is a precursor plasmid used for preparing a screening-tag-free plasmid, the precursor plasmid comprising: (1) a conditioned replication initiation site having a plasmid replication initiation capacity that is dependent on regulatory proteins, wherein the conditioned replication initiation site has a first replication initiation state in the presence of a first regulatory protein and a second replication initiation site in the presence of a second regulatory protein, and has a stronger ability to initiate plasmid replication in the second replication initiation state than in the first replication initiation state; 2) a first regulatory protein expression cassette expressing the first regulatory protein; 3) a sequence encoding a repressor protein; 4) a screened tag gene; 5) a target gene, or a cloning site for inserting the target gene; and 6) paired recombination sites. Also provided are a host cell suitable for the recombination of the precursor plasmid and the production of a screening-tag-free plasmid, and a method for large-scale production of the screening-tag-free plasmid.

The invention discloses an EpCAM gene high expression vector based on a lentiviral vector and a cell strain thereof, and belongs to the technical field of gene engineering and biology. According to the vector, an EpCAM gene expression structure is connected between multiple cloning sites Xba1 and Nhe1 of a vector pCHD-CMV-MCS-EF1-RFP-T2A-puro, high-level expression is carried out under the driving of a CMV (cytomegalovirus) promoter, and a recombinant plasmid pCDH-OE-EpCAM is obtained. And transforming the recombinant plasmid into escherichia coli DH5alpha to obtain the recombinant microbial cell. The recombinant plasmid is respectively transfected with colorectal cancer cell strains HT-29 and HT-115 through a Lipo2000 transfection reagent, and then monoclonal cells are screened, so that transgenic cells are obtained. The vector for overexpression of the human EpCAM gene and the colorectal cancer cell strain for overexpression of the EpCAM gene are constructed, and a very important foundation is laid for molecular mechanism research of generation and development of the EpCAM gene and colorectal cancer.

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 invention belongs to the field of plasmid vector construction, and provides a plasmid vector as well as a construction method and application thereof. The plasmid vector is constructed by modifying pAN7-1, a trichoderma viride promoter gene and a resistance gene are inserted into multiple cloning sites of the pAN7-1 of the plasmid vector, and the resistance gene is located at the downstream of the trichoderma viride promoter gene. According to the invention, the selected trichoderma viride promoter gene segment can efficiently start the gene connected behind, so that the transformation efficiency of the plasmid vector is greatly improved. The invention establishes a method for efficiently transforming trichoderma viride by using an agrobacterium tumefaciens-mediated exogenous gene, and can provide help in the aspects of genetic modification of trichoderma viride, development of useful genes, separation of drug target genes and the like.

The application discloses a gene expression vector pJXNU for actinomycetes, a construction method and application thereof. The sequence of the vector is shown as SEQ ID NO. 2. The vector is based on a pSET152 plasmid, and loaded with kanamycin and thiostrepton antibiotic resistance genes, and a 533bp DNA fragment containing three gene expression element sequences and two sets of multiple cloning sites (MCS) shown as SEQ ID NO. 1. The vector has two autonomously expressed strong gene expression elements and one thiostrepton-induced expression element, can realize autonomous high-efficiency expression of two or two groups of genes and / or induced expression of one or one group of genes on one vector, and can realize multi-type functional gene research and application in an actinomycete host.