299 results about "Shuttle vector" patented technology

In vitro methods for making a recombinant adenoviral genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from first and second vectors. The first vector includes an adenoviral genome having an E region deletion and three different, non-adenoviral restriction endonuclease sites located in the E region. The second vector is a shuttle vector and includes an insertion nucleic acid flanked by two of the three different non-adenoviral restriction endonucleases sites present in the first vector. Cleavage products are prepared from the first and second vectors using the appropriate restriction endonucleases. The resultant cleavage products are then ligated to produce the subject recombinant adenovirus genome. The subject adenoviral genomes find use in a variety of application, including as vectors for use in a variety of applications, including gene therapy.

Helicobacter based preparations comprising a pharmacologically active molecule of interest are disclosed, as well as methods of preparing and using said preparations. In particular, Helicobacter pylori vectors, vector plasmids and recombinant cells that include a sequence encoding a pharmacologically active molecule of interest useful in therapeutic treatments and/or vaccination against disease are provided. Delivery of the pharamacologically active molecules is provided at the mucosal surface, such as the gastric mucosa or nasal membranes, to provide effective and continuous delivery of a pharmacologically active agent. In some embodiments, the Helicobacter provides exposure of a desired molecule of interest though the surface of the Helicobacter, providing exposure of the antigen to the host at the gastric mucosa. Live Helicobacter pylori vaccines are also provided. Vectors and shuttle vector constructs of the Helicobacter are also disclosed.

The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described, that can be used to clone large regions of DNA by homologous recombination. The important feature of present invention is the presence of the a bacterial replication origin, which allows large DNA insert capacity. The utility of this vector lies in its ability to isolate, manipulate and maintain large fragments in bacteria and yeast, allowing for mutagenesis by yeast genetics and simplified preparation of plasmid DNA in bacteria.

The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described that can be used to clone large regions of DNA by homologous recombination. Also described is a method for isolating entire genomes, including chloroplast genomes, or large portions thereof, and manipulating the same. Also described are methods for determining minimal genomes, minimal pathway requirements, and minimal organelle genomes.

The invention discloses porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as a preparation method and application thereof. Sequences of VP0, VP1 and VP3 genes are artificially synthesized by referring to an FMDV (Foot And Mouth Disease Virus) O-type epidemic strain gene sequence; the VP0, VP1 and VP3 genes are connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHmVP013 is obtained. The baculovirus transfer vector pFBDPHmVP013 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant shuttle vector Bacmid; the shuttle vetcor Bacmid is transferred with a sf9 cell, and the recombinant baculovirus QP-Ac-FVLP is obtained by collecting the cell supernatant. The recombinant baculovirus can be used for efficiently expressing FMDVVP0, Vp1 and Vp3 proteins and forming virus-like particles. And the virus-like particles are used for preparing subunit vaccine, so that the organism is induced to generate specific immunity response after the mouse is immunized.

The present invention relates to pig reproduction and respiratory syndrome virus recombined adenovirus and vaccine, and belongs to the field of biological high-tech. Through RT-PCR process to proliferate whole PRRSV GP5 sequence, cloning the gene sequence to the shuttle vector pShuttle-CMV of adenovirus carrier system, cotransforming colibacillus BJ5183 strain together with the skeleton vector of adenovirus carrier system to obtain recombinant plasmid, transfecting HEK293-A cell to obtain recombinant adenovirus and plaque purification, and RT-PCR and indirect immunofluorescence technique inspection, the recombinant adenovirus rAd-GP5 expressing PRRSV GP5 protein is constituted. The recombinant adenovirus can set ahead the expression of PRRSV GP5 protein and raise the expression amount to simulate the immune protecting reaction of body effectively.

The invention discloses a rapid assembling method of multi-fragment DNA yeast. The rapid assembling method comprises the following steps: (1) performing co-transformation on a plurality of DNA molecules with homologous arms, and linearized yeast shuttle vectors, so as to obtain yeast; (2) eluting all the transformed yeast colonies on a whole screening culture plate, centrifuging, and discarding the supernate to obtain transformed yeast cells; (3) extracting plasmid DNA of the yeast cells obtained in step (2), transforming the competent cells of colon bacillus; and (4) screening colon bacillus, and cloning to obtain large-fragment DNA. The method for assembling large-fragment DNA molecules is fast in speed, simple, convenient and feasible, high in success rate, low in cost, high in efficiency, easy to operate, beneficial in enlarging the industrialization scale and wide in application, and a plurality of small-fragment DNA molecules can be assembled into one large-fragment DNA molecule.

The present invention relates to method for obtaining optimum expressed proteins in a transformed gram positive bacteria by specific integration of T7 RNA polymerase gene into the chromosome of a gram positive bacteria exhibiting resistance to aminoglycosides, said method comprising:- digesting an E. coli plasmid with a restriction enzyme- digesting the genomic DNA of said gram positive bacteria- ligating the said digested plasmid to the digested genomic DNA of said gram positive bacteria- transforming the said gram positive bacteria protoplasts with the said ligation mixture of step 2 to yield transformed gram positive bacteria (transformants),- screening the said transformants for kanamycin resistance and aminoglycoside sensitivity to ensure that the targetting of the said plasmid vector into the chromosome of the said gram positive bacteria is successful- cloning of the desired gene in the said vector—culturing the transformant in a suitable culture medium- isolating the expressed proteins from the culture medium

The invention discloses a method for expression of a PCV 2 (Porcine circovirus type2) Cap protein by a pFast Bac Dual baculovirus. The method comprises the following steps of: amplifying a gene fragment of an encoded PCV 2 Cap protein with a His tag; connecting the gene fragment to a pFast Bac Dual plasmid so as to obtain a recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2; transforming Escherichia coli DH10Bac with the recombinant transfer plasmid, and carrying out blue-white selection to obtain a recombinant shuttle vector Bac-p10-ORF2-pH-ORF2; transfecting an insect cell with the recombinant shuttle vector so as to obtain a recombinant baculovirus Ac.Dual-Cap; and poisoning the insect cell with the recombinant baculovirus, performing cultivation, then collecting the insect cell, and purifying an expression product so as to obtain the recombinant Cap protein. The method disclosed in the invention solves the problem of low expression level of the Cap protein in eukaryotic cells. The recombinant Cap protein in the invention is designed with a His tag, thus being beneficial to the follow-up purification. And the recombinant Cap protein has biological activity superior to that of a Cap protein expressed by a prokaryotic expression system, thus being applicable to establishment of epidemiological diagnosis methods and reseach as well as development of PCV2 subunit vaccines.

The invention relates to construction and application of recombinant nucleocapsids of Cap genes of porcine circovirus expression type 2 nucleocapsid protein, belonging to the genetic engineering bacterin field. C-terminal gene fragments of porcine parvovirus (PPV) VP2 genes are cloned into a type 5 adenovirus shuttle vector of the human beings, and recombinant adenovirus rAd-deltaVP2 is obtained; deltaVP2 proteins are expressed successfully and highly efficiently and can be self-assembled into the nucleocapsids [PPV:VLPs]; the PPV VP2 nucleocapsids are used as antigen transport vectors and 165 to 200 sites of amino acid (deltaCap) genes of the porcine circovirus type 2 (PCV2) nucleocapsid proteins (Cap) are embedded into an N-terminal (deltaVP2) of the PPV VP2, and then recombinant adenovirus rAd-deltaCap-deltaVP2 is obtained; embedded VP2 (deltaCap-deltaVP2) proteins are expressed successfully and highly efficiently and can be self-assembled into nucleocapsids [PPV:VLP(PCV2)]. The invention also relates to application of the recombinant virus and recombinant PPV VP2 nucleocapsids of the expression Cap genes of the recombinant virus in the aspects of bacterin immunity and so on.

Disclosed are bacillus licheniformis engineering bacteria for nattokinase production and a method for producing nattokinase by using the bacillus licheniformis engineering bacteria, which belong to the technical fields of microorganism genetic engineering and enzyme engineering. According to the invention, a nattokinase gene of bacillus subtilis MBS04-6 is obtained through PCR technology augmentation. Bacillus licheniformis BL10 is utilized as an expression host; promoter P43 of bacillus subtilis 168 is taken as a promoter; a signal peptide of extracellular serine protease Vpr of bacillus licheniformis WX-02 is taken a signal peptide; and a terminator sequence of alpha-amylase of bacillus licheniformis WX-02 is taken as a terminator. The expression elements are connected to shuttle vector pHY300PLK to form an expression vector, and the expression vector is transformed into the bacillus licheniformis BL10 to obtain the bacillus licheniformis engineering bacteria BL10 (pP43SNT) for nattokinase production. The bacterial strain has been deposited with the China Center for Type Culture Collection (CCTCC) at September 10th, 2013, and the accession number is CCTCC NO:M2013401. The invention also provides a method of the bacterial strain for producing nattokinase, and the maximum enzyme activity can reach 11.37 FU/mL in a liquid fermentation medium.

An Actinobacillus succinogenes plasmid vector which provides a means to overexpress proteins in A. succinogenes. The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in A. succinogenes. The plasmid comprises at least one marker gene, operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of Actinobacillus succinogenes, including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant Actinobacillus succinogenes is described, including a method of transformation. Additionally, a recombinant Actinobacillus succinogenes is disclosed and a method for producing succinate utilizing this recombinant Actinobacillus succinogenes is described.

The invention relates to high heat resisting phytase yeast engineering bacteria and a constructing method thereof. Reported phytase AppA gene is analyzed by a biological information method to find a potential thermal stable associated locus. Site-specific mutagenesis is performed to the key locus of the gene by a site-specific mutagenesis technology to obtain a mutant gene Appa-M2. The mutant gene is constructed to a yeast shuttle vector pPIC9 and converted to a pichia pastoris GS115 so as to screen heat-resisting phytase strains with efficient expression. The heat resisting performance of phytase expressed by recombinant yeast engineering bacteria screened is good. The residual enzyme activity of the yeast engineering bacteria insulated for 10 minutes at 85 DEGC can reach 60%. The yeast engineering bacteria has wide application prospect in feed and food industry.

The invention discloses a recombinant baculovirus vector, a virus like particle, a preparation method and use and aims at providing tandem expression and accurate cutting expression of a plurality of genes with a single promoter and successfully packaging the virus like particle. The recombinant baculovirus vector is simple to operate, high in expression quantity and high in cutting efficiency. The obtained virus like particle is good in immunogenicity and does not have an exogenous sequence. The technical essential is that picornaviridae 3C/3CD gene and P1/P1op gene are expressed under the single promoter, the 3C/3CD gene and the P1/P1op gene are connected by a 2A sequence to build a 3C/3CD-2A-P1/P1op expression frame, then the 3C/3CD gene is placed under the promoter of baculovirus vector plasmid to obtain a recombinant shuttle vector, a host cell is transfected to obtain recombinant baculovirus, and the host cell is infected to obtain the virus like particle.

The invention relates to respiratory syncytial virus-like particles, and establishes a novel method of preparing respiratory syncytial virus-like particles for animal immunization. The preparation method comprises the following steps: according to codon bias of mammalian cells, carrying out codon optimization for M, F and G genes which are respectively cloned to a shuttle vector to obtain recombinant adenovirus plasmids, and transfecting to obtain the recombinant adenovirus FGAd-Msyn, FGAd-Fsyn and FGAd-Gsyn; infecting the recombinant adenovirus by use of Vero cells to obtain the virus-like particles; purifying the virus-like particles by adopting a sucrose density gradient centrifugation method; applying the particles to animal immunization. According to the invention, the virus-like particles are prepared by adopting the adenovirus vector, and a novel virus-like particle preparation method is established. The protein expression quantity is improved and the yield of the virus-like particles is improved through codon optimization, so that a better immune effect is obtained, and the respiratory syncytial virus-like particles are relatively safe.

The invention provides a restructing adenovirus of 2-type pig-ring virus and constructing method thereof, and the preventing appliances of pigs' relevant diseases caused by PCV2, including: augment and clone of PCV2 ORF2 genes; construction of recombinant shuttle vectors containing ORF2 genes; construction of recombinant adenovirus plasmid vectors; acquirement of recombinant adenovirus. Said recombinant adenovirus could determinately grow inside the bodies and continueously express ORF2 proteins to stimulate the organisms which generate neutralizing antibodies for ORF2 proteins, and offer endurance protection to pigs with promising future in the prevention and cure of PCV2, which could prevent efficiently poor quality of male-pig sperms, abortion, dead fetus, mummy and low production of female-pigs to increase the uniformity of pigs greatly.

Disclosed herein are improved plasmid shuttle vectors, vaccines based on them, and methods related to their construction and use. Particular arrangements of functional elements of such plasmids, namely origins of replication and eukaryotic transcription/translation control elements, which give rise to generally undesirable side-products upon propagation of the plasmids in bacterial culture are disclosed. These side-products apparently arise as terminated replication intermediates. Strategies both to eliminate accumulation of these side-products, and to make them useful as a vaccine adjuvant, are described.

The invention discloses a herpes simplex virus I-type gene recombinant attenuated live vaccine and a preparation method thereof. The attenuated live vaccine is characterized in that: US2, US3, US4 and US5 genes in a genome and other duplicated or infected nonessential gene fragments are jointly knocked out, wherein other duplicated or infected nonessential gene fragments are US9-US12 genes. The method comprises the following steps: separating and identifying wild HSV-1 (herpes simplex virus-1) from blister fluid of a patient with remarkable herpes labialis and herpes progenitalis; propagating and extracting a complete genome of virus; co-transfecting a Vero cell with a shuttle vector containing homologous flanking sequences of 700bp to 2000bp on the left and right sides of a fluorescent expression gene and the HSV gene to be knocked off; and selecting the recombinant virus by using one or combination of multiple fluorescent protein genes serving as a marker under fluorescent microscope, and acquiring the required recombinant attenuated live vaccine after plaque purification.

The invention relates to a method for expressing cecropin AD by virtue of bacillus subtilis and a preparation method of cecropin AD. A fusion protein coding gene (SPSacB)SUMO-CAD is designed and synthesized by taking bacillus subtilis WB800N as a host strain and escherichia coli-bacillus subtilis shuttle vector as an expression vector, the nucleotide sequence of the fusion protein coding gene is represented by SEQ ID NO.1, the amino acid sequence is represented by SEQ ID NO.2, and a recombinant expression vector pGJ148-(SPSacB)SUMO-CAD is successfully constructed. The recombinant expression vector is converted into a host bacteria competent cell in a chemical conversion manner, induced fermentation is carried out by virtue of maltose so as to successfully express foreign proteins, a fusion protein with relatively high purity is obtained through purification through an NI-NTA column and a protein purification analysis meter and is subjected to SUMO digestion so as to obtain recombinant antibacterial peptide, and then fermentation conditions are optimized. The method is low in cost and beneficial to separation and purification.

The invention discloses a recombinant protein containing an SARS virus RBD antigen and a baculovirus displaying an RBD protein. The recombinant protein SP-RBD-TM is formed by connecting an N- end of the RBD protein of an SARS virus with a signal peptide SP of a baculovirus envelope protein GP64, and connecting a C- end with a transmembrane domain TM of the baculovirus envelope protein GP64. The recombinant baculovirus having the surface displaying the SARS antigen RBD protein is the recombinant baculovirus obtained by the steps of inserting a cording gene of the SP-RBD-TM into a donor plasmid, carrying out homologous recombination with a genome of a shuttle vector Bacmid through transposition to obtain a recombinant baculovirus genome, then transfecting a bombyx mori cell with the recombinant baculovirus genome, and packaging in the cell to obtain the recombinant baculovirus. The recombinant baculovirus allows the RBD protein of the SARS virus to be fused with the bombyx mori baculovirus envelope protein GP64, realizes display of the RBD protein on the surface of a viral capsid, and has good immunogenicity.