65 results about "TA cloning" patented technology

The invention discloses a method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout and belongs to the field of gene knockout. According to the method, construction of a gRNA expression vector and gRNA in-vitro synthesis are performed through design of a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) gene knockout target site, micro-injection is performed on an embryo of the zebra fish, the effectiveness of the target site is detected, tail cutting identification is performed, TA cloning is performed on a target sequence, plasmids are subjected to Sanger sequencing, an F1 generation of heritable zebra fish mutants is obtained, the same mutant female fish and male fish are picked from mutants of the F1 generation, hybridization is performed, an F2 generation of the zebra fish mutants is obtained, F2 generation homozygote is picked from the F2 generation of the zebra fishmutants, F3 generation pure-line inheritance is performed, and an rmnd5b gene deletion type zebra fish strain is obtained. The method is lower in off-target rate and has good medical research value inresearch of the correlation between rmnd5b gene deletion and development of other organs.

The invention belongs to the technical field of biological engineering and synthetic biology, and particularly relates to a multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on BT1 integrase and mutation recognition sites thereof. In the method, sixteen pairs of mutational integrase recognition sequences are provided through mutation screening and verification, on the basis, a complete set of related carriers is constructed and comprises a framework carrier and seven TA cloning vectors, the framework carrier can realize escherichia coli-streptomyces coelicolor shuttle, the seven TA cloning vectors comprise mutation substrates and are used for DNA element construction and screening, and a concrete method for multi-fragment external series connection recombination reaction is provided. The method of the invention is simple, fast and efficient and can be used for the multi-fragment fast series connection recombination assembly, and the amplification of series connection products is realized through escherichia coli transformation. The method can be widely used for combination and assembly of standard modules and elements in the synthetic biology.

The invention discloses an HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau. The gene is defined by a nucleotide sequence of SEQ ID NO:1 and has a regulatory sequence defined by SEQ ID NO:2. A method of acquiring the gene comprises the steps of designing an electron probe based on an OsCIPK (CIPK, CBL-interacting protein kinases) homologous gene sequence of paddy rice; searching homologous fragments from an existed nucleotide sequence database of various barley such as an EST (expressed sequence tags) database; obtaining a coding sequence of a targeted gene by methods of cluster analysis, electronic splicing, etc.; conducting composition analysis and function prediction of the targeted gene by the bioinformatics; extracting a total RNA of the Hordeum spontoneum C. Koch; preparing a cDNA and designing a PCR primer; cloning the targeted gene of HsCIPK2 via a PCR technology; conducting TA clone sequencing and sequence analysis on the obtained targeted gene; and on the above basis, designing three primers in a 5'non-coding region of an HsCIPK2cDNA and further cloning the HsCIPK2 gene by utilizing six arbitrary degenerate primers and a Tail-PCR technology. The regulatory sequence is of significance to the research on tissue-specific expression of the HsCIPK2 gene and on response to stresses.

The invention relates to a temperature-controlled zero background T-vector precursor and a construction method and application thereof. The temperature-controlled zero background t-vector precursor is a vector pFLX107, and a sequence of the same is shown in SEQ ID NO.1. Volume of the successfully constructed vector is 3029bp, volume of a T-vector prepared via Xcml enzyme digestion is only 2747bp and approximate to those of various commercial T-vectors in the market at present, and the T-vector can be widely applied to TA cloning of various exogenous PCR (polymerase chain reaction) fragments. Due to presence of temperature controlled lethal gene, positive clone selection of recombined transformants by 100% can be achieved at the temperature of 37 DEG C. Meanwhile, since universal sequencing primer sequence binding sites which can be provided by biological companies are integrated on two sides of a cloning site in the vector, primers are not required to be provided by oneself during sample presentation and sequencing, and cost is further lowered.

The invention discloses a vector for non-background directed cloning of PCR products, a preparation method thereof and an application thereof. The vector is characterized in that an XcmI box is introduced before the vector screens and labels gene, and the XcmI box is characterized in that a protruded dT end is formed after XcmI enzyme digestion, an inactive ribosome binding site is formed before gene screening and labeling and after sequence splicing, and the screened and labeled gene cannot express during self-joining of the vector. The protruded dT end formed after the XcmI enzyme digestionof the vector prepared in the invention can be applied to TA cloning, and can be filled in by a T4DNA polymerase to form blunt ends, so as to be applied to blunt end joining; the method of the invention has the advantages of easy implementation, and low cost; and effects of no background and directed cloning are reached in the application of two cloning methods.

The invention relates to specifically relates to a plasmid vector capable of realizing directional TA cloning, background sticky end-free cloning and expression, and a construction method thereof, belonging to the field of gene engineering. The plasmid vector is a closed circular double-stranded DNA and contains two multiple cloning sites, wherein a ccdB expression cassette exist between the two multiple cloning sites, and the ccdB expression cassette can realize constitutive expression of toxic protein ccdB, which is used as a negative selection marker, in most Escherichia coli strains. The multiple cloning sites are designed on the basis of the plasmid vector; and the plasmid vector is applicable to directional TA cloning and improved background sticky end-free cloning. Moreover, the plasmid vector is directly applicable to expression and purification of proteins. Each function of the plasmid vector is verified. The plasmid vector provided by the invention is expected to play an important role in the field of gene engineering due to its convenience and high efficiency.

The invention discloses a T carrier and a preparation method thereof. A known carrier is a carrier after an improvement of a starting carrier which is pBlueScript II SK(-), pUC18, pUC19, pUC118, pUC119, pUC19, or pGEM serial carriers the polyclone sites of which are replaced with XcmI cases, wherein the XcmI cases comprise ccdB gene sequences and an XcmI restriction enzyme cutting site sequence which is connected to each of two sides of the ccdB gene sequences, and resistance genes in the starting carrier are lengthened by inserting an antisense gene with the length of 1000-2000bp in front and/or back of the antisense gene. The T carrier provided by the invention has higher TA clone efficiency, the TA clone efficiency can reach 100% especially for fragments which are difficult to distinguish for cloning conventional business clone carriers and have the lengths between 2500bp and 3500bp, and the T carrier plays an important role in the filed of genetic engineering.

The invention relates to a recombinant strain of a mink IFN-Gamma gene, which is characterized by being an IFN-Gamma gene sequence which is cloned from peripheral mink bloodlymph cells (PMBC) simulated by phytohemagglutinin (PHA) and being a recombinant plasmid vector containing a nucleotide sequence; i.e., using a plasmid vector pGM-T which is formed by the cutting of a cloning vector by taking an EcoR V enzyme as the cutting point and then adding a 'T' from the '3' end at both sides; and TA cloning is carried out on the nucleotide sequence of a coding Gamma-interferon which is amplified from the peripheral mink bloodlymph cells so as to construct the recombinant plasmid of the nucleotide sequence of the mink Gamma-interferon. In addition, Escherichia coli conversed from the plasmid vector is used as a host. The recombinant strain aims at laying down foundation for researching and developing genetically engineered mink recombinant interferons biological products with anti-viral activity and immunomodulatory activity.

The invention provides a recombinant bacillus calmette guerin (BCG) vaccine for toxoplamasis and a preparation method thereof. The preparation method for the recombinant bacillus calmette guerin vaccine comprises the following steps of: performing target (TA) cloning on an obtained toxoplasma gondii cyclophilin gene; sequencing and identifying correctly, performing enzyme cutting and recovering target fragments; connecting the target fragments with an Escherichia coli-mycobacterium shuttle expression vector pMV261 and an integrated expression vector pMV361 which are subjected to enzyme cutting reaction respectively; and converting recombinant plasmids to BCG, and screening by resistance and polymerase chain reaction (PCR) to obtain the recombinant bacillus calmette guerin vaccine for the positive toxoplasma gondii. The vaccine is high in heat stability and easy to transport, store and produce, is not needed to be purified and can be directly used for immune protection tests, and a complex process of protein aftertreatment is avoided, so the cost is reduced greatly, and the recombinant bacillus calmette guerin vaccine is suitable for vast rural areas.

The invention discloses a directional cloning method, a transformation method of a carrier and a carrier T, wherein the directional cloning method includes the steps of designing and synthesizing an enzyme digestion kit, constructing the linear carrier T with two ends provided with protruded dT, generating PCR (Polymerase Chain Reaction) segment of the target gene, and constructing a recombinant carrier containing the target gene. According to the directional cloning method, the directional TA cloning technology can be used for preparing efficient directionally-cloned carrier T which can be widely applied to cloning and expression of a PCR product in molecular biology experiment. As an expression carrier is reconstructed, the PCR product can be directly connected to the expression carrier, the connecting direction of the target gene can be determined after identification of single enzyme digestion, expression can be performed in cells, the connecting direction is determined without complex steps such as conventional sequencing and the like, the direction cloning can be simply and rapidly realized, engineering bacteria can be constructed through one step, and the application prospect is wide.

The invention relates to a method for screening sensitive genes to tumor chemotherapy medicament by using an RNA interference library. The method comprises the step of using the RNA interference library with reduced gene expression to reduce medicament sensitive gene expression of tumor cells, so that the medicament sensibility of the tumor cells is weakened, but the medicament tolerance is raised; therefore, the tumor cells survive in high-concentration medication condition and form cell clone; finding and screening the medicament sensitive gene through gene clone; and extracting genome DNA from survived cells, amplifying a DNA fragment inserted from the RNA interference library by RCR, using a TA cloning method to clone the DNA fragment amplified by the RCR, selecting the clone, purifying the DNA, and finally measuring the sequence of the gene to determine screened genes. The method makes first screening of the medicament sensitive genes of liver cancer cells, first determines a batch of real medicament sensitive genes in the world, and is used for (1) guiding medicament selection of tumor chemotherapy, that is, when the sensitive genes aiming at certain medicament expresses normally, recommending the medicament, and when the sensitive genes aiming at certain medicament reduces expression or undergoes mutation, avoiding using the medicament, and (2) developing and applying the genes as biological markers of tumor for evaluating risk and preliminarily surveying pathogeny before diagnosis, classifying and defining the level of the tumor at the diagnosis, and evaluating treatment effect and detecting recurrence after the diagnosis.

The invention relates to the field of genetic engineering, and in particular to a plasmid vector, which is capable of flexibly implementing non-background TA cloning, cohesive end cloning and blunt end cloning and is capable of implementing temperature-induced protein expression, and a construction method of the plasmid vector. The plasmid vector is closed-ring shaped double-stranded DNA, which contains a gene, which is capable of coding toxic protein ccdB, as a negative selection marker. A flexible cloning function and a temperature-induced expression function of the plasmid are verified. Theplasmid, which is more convenient and efficient, can play an important role in the field of genetic engineering.

The invention discloses a preparation method of a recombinant panda IL-6 immunological adjuvant. The preparation method comprises the following steps of: cloning A1 panda IL-6 whole-genome; culturing A11 panda peripheral blood mononuclear cells in vitro; extracting an A12 total RNA (Ribonucleic Acid); synthesizing Al3cDNA; designing and synthesizing an A14 primer; carrying out PCR (Polymerase Chain Reaction) amplification on an A15 target gene; carrying out TA (trophoblast antigen) cloning on a PCR product of an A16 target gene; carrying out preliminary identification on A17 recombinant plasmids; and carrying out prokaryotic expression of A2 panda interleukin 6, and preparing a polyclonal antibody. The canine distemper is known as a destructive infectious disease and is the main fulminating infectious disease which threatens the population quantity and the life safety of pandas and seriously affects the healthy development of the pandas. The recombination panda IL-6 immunological adjuvant can be used for remarkably enhancing the immune effect of canine distemper vaccines and has important significance for the prevention and treatment of the canine distemper.

The present invention discloses a multifunctional cloning vector, wherein the construction method comprises adopting a resistance gene expression self-saving manner and directly adopting the conventional antibiotic to screen positive clones, and specifically comprises: designing a pair of primer sequences with 5' terminal phosphorylation, adopting PUC19 plasmid as a template, and adopting high-fidelity PCR amplification to obtain the multifunctional cloning vector. With the vector prepared by using the method, various PCR products can be efficiently cloned, efficient cloning efficiency of the blunt-ended PCR product and the general Taq enzyme amplification product can be achieved, and the vector of the present invention can be adopted to completely replace the blunt-ended cloning vector and the TA cloning T vector. The method for preparing the multifunctional cloning vector has advantages of general positive rate of greater than 90%, low false positive rate, low test cost, less experiment operation steps, convenience and rapidness. The invention further discloses the use method of the multifunctional cloning vector.

The invention discloses a functional molecular marker of a brassica chinensis anthocyan synthesis regulation and control gene. The functional molecular marker comprises a brassica chinensis anthocyansynthesis regulation and control gene Pur promoter, coding region InDels markers and InDels marker PCR primer sequences. The invention relates to the technical field of agricultural plant molecular marker assisted breeding. According to the molecular marker technology of the brassica chinensis anthocyan synthesis regulation and control gene, accuracy of the sequences is verified through TA cloning, Sanger method sequencing and the like, a regulation and control gene associated with leaf characters of brassica chinensis is identified, two pairs of Indels markers associated with purple characters are developed according to sequence differences between promoters and coding regions of purple and green brassica parachinensis, two pairs of primers achieve one-step method identification screeningthrough multiplex PCR, materials with the anthocyan synthesis regulation and control gene can be simply, conveniently and rapidly screened and identified from a large amount of brassica parachinensisgerm plasm resources, a technical support is provided for rapidly creating new germ plasm of brassica chinensis, a breeding process can also be accelerated, the consumption of resources is lowered, and the cost is reduced.

The invention relates to a plant expression vector facilitating connection of genes as well as a construction method and applications thereof. The vector is prepared through the following steps: cloning 1#, 2#, 3#, 4# and 5# fragments from starting expression vectors pGreenII0229, pCAMBIA3300-35S-HAK, pGH-X6145G (purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd) and pCAMBIA2300 by using a PCR method; respectively carrying out enzyme digestion on the 1# and 2# fragments by using NotI and MIUI to be connected to the obtained product so as to obtain a vector pJWWD-12; respectively carrying out enzyme digestion on the pJWWD-12 and the 3# fragment by using XhoI to be connected to the obtained product so as to obtain a vector pJWWD-123; respectively carrying out enzyme digestion on the vector pJWWD-123 and the 4# fragment by using SacI to connected to the obtained product so as to obtain an expression vector pJWWD-1234; and respectively carrying out enzyme digestion on the pJWWD-1234 and the 5# fragment by using PstI to be connected to the obtained product so as to obtain an expression vector pJWWD-1718 finally. According to the invention, target genes can be conveniently and quickly inserted, and the target genes can be inserted in the vector through TA cloning, thereby greatly simplifying the operation steps, reducing the cost and improving the operating efficiency. The vector disclosed by the invention can provide a strong support for plant genetic engineering breeding, and lay the foundation for the safety evaluation and commercial planting of genetically modified plants.

The invention relates to the technical field of molecular biology and discloses a method for identifying material sources of meat products based on clone and conventional sequencing. The method comprises the following steps: (1) extracting to-be-detected genome DNA; (2) designing and synthesizing a forward primer and a backward primer; (3) carrying out PCR amplification on the genome DNA, and detecting and recycling amplification products; (4) carrying out TA cloning on the PCR amplification products to obtain positive clones, selecting single clones, and carrying out sequencing; and (5) removing carrier sequences at two ends in a sequencing result to obtain COI sequence information of to-be-detected meat products, and therefore, identifying the material sources of the meat products. By virtue of the method, the material compositions and sources of the meat products can be identified, and species can be well differentiated by virtue of difference among base sequences in the COI gene sequence, so that the method for identifying the material sources of the meat products has relatively high accuracy.

The invention discloses a method of assembling multiple gene fragments to a py-2u vector via a yeast and application and relates to the technical field of bioengineering. Homologous sequences homologous to the py-2u vector are added to the two ends of a target gene to be cloned; the obtained gene is divided into multiple small fragments; independent digestion sites are added to two ends of each fragment; the digestion sites at the two ends of each fragment are identical, or different independent ones; each small fragment is acquired through genetic synthesis or PCR (polymerase chain reaction)amplification; the plain end of each amplified product is connected to a cloning vector or is TA-cloned to one small fragment vector; each fragment is subjected to enzyme digestion via one independentdigestion site; the recycled product is applied to next yeast transformation. The constructed Py-2uL may be induced via arabinose to gain a larger copy number in Escherichia coli, so that genetic operations, such as subsequent plasmid extraction, are facilitated; genetic synthesis and assembly of a growth gene can be achieved stably and quickly; the method is simpler than conventional cloning processes and is economical and efficient.

The invention relates to a preparation method of mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1), and belongs to the technical field of genetic engineering. The specific preparation process of the monoclonal antibodies comprises ten steps: identification of a CD36 gene knockout mice genotype, PCR amplification of human platelet cDNA, TA cloning and blue-white spotscreening, cell transfection and G418 screening, flow identification, Western Blot identification, CD36 gene knockout mice immunization, cell fusion and antibody preparation, identification of anti-human CD36 monoclonal antibodies and purification of the monoclonal antibodies. The method can obtain hybridoma cell lines stably secreting mouse-derived anti-human CD36 mAb, wherein the mAb can specifically bind to a human platelet CD36 antigen and can be used for detecting the expression of CD36 antigens in humans.