66 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 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 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 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 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.