46 results about "NdeI" patented technology

The invention discloses a recombinant bacterium and application of the recombinant bacterium to preparation of rebaudioside M2 by catalyzing rebaudioside A. The recombinant bacterium contains a tomato-based glycosyltransferase UGTSL2 gene and a potato-based sucrose synthase StSUS1 gene at the same time; the tomato-based glycosyltransferase UGTSL2 gene is cloned between NdeI and XhoI sites of pRSFDuet-1 and is constructed to obtain a recombinant plasmid pRSFDuet-SL2; then the potato-based sucrose synthase StSUS1 gene is cloned between NcoI and EcoRI sites of the pRSFDuet-SL2 and is constructed to obtain a recombinant plasmid pRSFDuet-SL2-SUS1; the recombinant plasmid pRSFDuet-SL2-SUS1 is transferred into a host cell to obtain the recombinant bacterium. After the recombinant bacterium is subjected to induced expression, crude enzyme liquid is taken and is added into a reaction mixture to catalyze the rebaudioside A to generate the rebaudioside M2; in a reaction process, the crude enzyme liquid obtained by crushing the recombinant bacterium is utilized and separation and purification of an enzyme are avoided; lyophilized powder is also not needed and substrates including rebaudioside D and UDP or UDP-glucose and any cell penetration agent or other chemical reagents do not need to be added, so that the environmental friendliness is better. The yield of the rebaudioside M2 reaches 11.09g / L.

The invention provides a method for preparing soluble genetic engineering human serum amyloid A1 (SAA1) and an expression vector and genetic engineering bacteria thereof. The expression vector provided by the invention is a plamid vector pET28a(+) containing a human SAA (hSAA1) gene sequence, and the hSAA1 encoding sequence is positioned between restriction enzyme cutting sites of NdeI and Xhol I on the pET28a(+) vector. The genetic engineering bacteria provided by the invention are colibacillus pET28a (+)-hSAA1 / Rosetta-gami, which can express soluble target protein. The preparation method of the invention uses a nickel cross-linking affinity-column one-step method, has simple operation, high yield and short consumed time, and is suitable for industrial large-scale production, and the purity of the obtained target protein is larger than 90%.

The invention discloses a PPK2 protein, and an application of the PPK2 protein as a polyacrylamide gel electrophoresis 35kd standard substance. A ppk2 gene is inserted into a prokaryotic expression vector pET30a by using NdeI and Hind III, the obtained recombinant expression vector is transferred into an Escherichia coli BL21 (DE3) strain through a physical method, IPTG is used to induce trial expression of the target protein PPK2 at 37 DEG C, 25 DEG C and 16 DEG C, and the obtained expression product is identified and analyzed through SDS-PAGE electrophoresis and Western blotting; and finally, amplification culture is carried out by utilizing 3 L of an expression bacterial liquid, and the expression product is separated and purified through a Ni-IDA affinity chromatographic column, an anion exchange chromatographic column and a gel filtration chromatographic column in sequence. Results show that a large intestine prokaryotic expression system can be stably and efficiently expressed under the induction of the IPTG with the temperature of 16 DEG C and the final concentration of 0.2 mM.L<-1>, and the PPK2 protein has the advantages of specific charged property, existence in a monomerform in a solution, stable properties, non-degradability and long half-life period, and has an application prospect as the polyacrylamide gel electrophoresis 35 kd standard substance.

The invention discloses a method for catalytically synthesizing curcumin glycoside compounds by a biological method. A glycosyl transferase gene CaUGT2 and a sucrose synthase gene AtSUS1 are constructed on an expression vector and are introduced into escherichia coli to obtain a recombinant strain, the soluble expression quantity of the recombinant strain after induced expression is improved compared with that of glycosyl transferase from other plants, and a substrate curcumin can be efficiently catalyzed. A recombinant plasmid is pRSF-CaUGT2-AtSUS1, a sucrose synthase AtSUS1 gene is inserted into Nco I and EcoR I, glycosyl transferase CaUGT2 is inserted into Xho I and NdeI, a co-expression recombinant plasmid pRSF-CaUGT2-AtSUS1 is formed, the recombinant plasmid is transformed into escherichia coli BL21 (DE3) competent cells, and a recombinant strain CaUGT2-AtSUS1 is obtained. After the recombinant strain is subjected to induced expression, the soluble expression quantity of the glycosyl transferase CaUGT2 is increased compared with that of glycosyl transferase from other plants, the conversion rate of catalytic synthesis of curcumin glycoside compounds reaches 98%, curcumin is catalyzed to generate curcumin monoglucoside and curcumin diglucoside, the water solubility of the curcumin monoglucoside and curcumin diglucoside is superior to that of curcumin, and the problem that curcumin is poor in water solubility is solved. The concentration of the substrate curcumin is 75 mM, the concentration of a catalytic substrate is relatively high, and the method is more suitable for food and medicine industries in industrialization.

The present invention discloses a kind of fusion expressed product of vascular inhibine and endosatin in colibacillus and its preparation process, and belongs to the field of genetic engineering and peptide-containing medicine preparation. Through extraction of RNA from liver tissue of animal, RNA reverse transcription, PCR amplification of ES and AS segment and recovering ES and AS segment, and the connection reaction of introducing NdeI, NheI and XholI cleavage site to the primer via the SacI cleavage site in pGEM-T Easy vector to connect vascular inhibine and endosatin, fusion expressed product of vascular inhibine and endosatin in colibacillus is obtained. The fusion expressed product has not only the functions of both vascular inhibine and endosatin but also the synergistic effect of antagonizing angiogenesis and tumor.

The invention discloses a PPK2 protein and application thereof as a 30kd standard substance for polyacrylamide gel electrophoresis. Ppk2 genes are inserted into a prokaryotic expression vector pET30aby virtue of NdeI and HindIII, a recombinant expression vector is transferred into an escherichia coli BL21(DE3) strain by virtue of a physical method, a target protein PPK2 is induced to realize trial expression at 37 DEG C, 25 DEG C and 16 DEG C by virtue of IPTG, and an expression product is authenticated and expressed by virtue of SDS-PAGE electrophoresis and Western Blotting; and finally, amplification culture is carried out by virtue of 3.2L of expression bacteria liquid, and the expression product is sequentially separated and purified by virtue of Ni-IDA affinity chromatography, cation-exchange chromatography and gel filtration chromatography. A result shows that a large intestine prokaryotic expression system can be induced to realize stable and efficient expression by virtue of the IPTG with a final concentration of 0.2mM.L<-1> at 16 DEG C, the PPK2 protein has an exclusive charging property, stable performance and a long half-life period, exists in a solution in a monomer manner, is unlikely to degrade and has an application prospect as the 30kd standard substance for the polyacrylamide gel electrophoresis.

The invention discloses a recombinant bacterium and application of the recombinant bacterium to preparation of rebaudioside M2 by catalyzing rebaudioside A. The recombinant bacterium contains a tomato-based glycosyltransferase UGTSL2 gene and a potato-based sucrose synthase StSUS1 gene at the same time; the tomato-based glycosyltransferase UGTSL2 gene is cloned between NdeI and XhoI sites of pRSFDuet-1 and is constructed to obtain a recombinant plasmid pRSFDuet-SL2; then the potato-based sucrose synthase StSUS1 gene is cloned between NcoI and EcoRI sites of the pRSFDuet-SL2 and is constructed to obtain a recombinant plasmid pRSFDuet-SL2-SUS1; the recombinant plasmid pRSFDuet-SL2-SUS1 is transferred into a host cell to obtain the recombinant bacterium. After the recombinant bacterium is subjected to induced expression, crude enzyme liquid is taken and is added into a reaction mixture to catalyze the rebaudioside A to generate the rebaudioside M2; in a reaction process, the crude enzyme liquid obtained by crushing the recombinant bacterium is utilized and separation and purification of an enzyme are avoided; lyophilized powder is also not needed and substrates including rebaudioside D and UDP or UDP-glucose and any cell penetration agent or other chemical reagents do not need to be added, so that the environmental friendliness is better. The yield of the rebaudioside M2 reaches 11.09g / L.

The invention relates to a modified cloning vector and an application thereof. The modified cloning vector is an annular cloning vector, and is obtained by replacing multiple cloning sites of a pUC57 vector by employing three flush end cloning sites of EcoRV, SmaI and StuI and removing an NdeI enzyme digestion site at the LacZ downstream in the pUC57 vector, and the modified cloning vector only comprises three cloning sites of EcoRV, SmaI and StuI. The vector can completely replace any one cloning vector and is convenient to use and is wide in application.

Belonging to the technical field of goat genetic marker preparation, the invention in particular relates to a molecular marker related to goat growth traits and application thereof. The molecular marker is obtained by cloning of a sixth intron sequence of a goat TR alpha gene, and the nucleotide sequence of the gene is shown as a sequence table SEQ ID NO:1. Base mutation of C230-T230 occurs at a 230th base of the sequence shown as the sequence table SEQ ID NO:1, and results in NdeI-RFLP polymorphism. The invention also discloses a preparation method of the molecular marker and application of a polymorphic detection method, and provides a new genetic marker for marker assisted selection of goat.

The invention relates to the fields of molecular biology and immunology, in particular to a vibrio harveyi secretory type vaccine and a structure and application thereof. Particularly, the secretory type vaccine is a base sequence in a sequence table SEQ ID No.1, and the construction process thereof is as follows: utilizing vibrio harveyi T4 as a template and F11 and R8 as primers for PCR amplification, connecting the product with pBS-T at room temperature, utilizing NdeI / XhoI double-enzyme cleavage to recover 1.2kb segment from plasmids pBSVhP1, simultaneously utilizing NdeI / XhoI double-enzyme cleavage to recover 4.3kb segment of plasmids pBT3, connecting the two segments at room temperature by ligase T4DNA for 2-4h, transforming connection liquid into colon bacillus DH5 alpha, culturingon an LB solid medium containing Ap for 24h to screen out white transformant, i.e. the Vibrio harveyi secretory type vaccine of base sequence in the expression sequence table SEQ ID No.1. The obtained vaccine has immune and protective functions on Vibrio harveyi. The obtained vaccine has the immune and protective effects on the Vibrio harbeyi, and the immune and protective efficiency of the vaccine of the invention on the Vibrio harveyi can reach to 90%. The preparation process is simple, devitalization and other steps do not needed, and no adjuvant is needed.

The invention provides a replication-type recombinant adenovirus HAdV‑5 vector system, which includes a backbone plasmid pKAd5, an intermediate plasmid pMDXE3YA and a shuttle plasmid pKNXE3M. Clone the coding region of the target gene into the multiple cloning site of the shuttle plasmid, digest the shuttle plasmid carrying the target gene with EcoRI, and replace the corresponding part of the intermediate plasmid with the resulting fragment; then digest the newly constructed intermediate plasmid with NdeI, and replace it with the resulting fragment The corresponding part of the backbone plasmid can obtain the recombinant adenovirus plasmid; the PacI linearized recombinant adenovirus plasmid transfects 293 cells, and can prepare the recombinant HAdV‑5 adenovirus controlled by the HAdV‑5 major late promoter (MLP) to express the target gene . The recombinant adenovirus can replicate and proliferate in various human cells, and is a replication-type virus. It is expected to have broad application prospects in the research and development of oral vector vaccines for animals.

The invention relates to the technical field of potassium ion channel regulatory proteins, in particular to a recombinant vector, an expression vector, a genetically engineered bacterium and application thereof. The recombinant vector is obtained by inserting a DNA (Deoxyribose Nucleic Acid) sequence as shown in SEQ ID NO.1 between NruI and NdeI sites of a pPrey-PartnerSUS-2in1-Dest vector, and is named as pKY2in1-Dest, and the expression vector is obtained by transferring a potassium channel gene and other regulatory protein genes into the pKY2in1-Dest in one step through Gateway cloning reaction. The genetically engineered bacterium is obtained by transferring the expression vector into a recipient bacterium. The genetically engineered bacterium can be applied to large-scale screening of regulatory proteins affecting the potassium absorption function of the potassium ion channel, influences on the potassium channel function are directly shown by observing yeast growth under a low-potassium condition, and the genetically engineered bacterium has the advantages of being simple in condition, low in cost and short in experimental period.

The invention provides a recombinant vector CTE528, and a construction method and application thereof, and belongs to the field of gene engineering. The invention provides a vector system construction method capable of being efficiently used in dunaliella salina gene engineering by aiming at the defect that research of microalgae transgenosis mostly focuses on mode algae Chlamydomonas reininhardtii. The vector system construction method comprises the following steps of: after NdeI is added to two sides of a CAT gene, cloning the CAT gene to a pBI221-GFP vector to serve as an intermediate vector CTE527, splicing Ubi and Omega, adding Sse8387I / BamH I to two sides, and cloning the Sse8387I / BamH I to the CTE527.

The present invention discloses a kind of fusion expressed product of vascular inhibine and endosatin in colibacillus and its preparation process, and belongs to the field of genetic engineering and peptide-containing medicine preparation. Through extraction of RNA from liver tissue of animal, RNA reverse transcription, PCR amplification of ES and AS segment and recovering ES and AS segment, and the connection reaction of introducing NdeI, NheI and XholI cleavage site to the primer via the SacI cleavage site in pGEM-T Easy vector to connect vascular inhibine and endosatin, fusion expressed product of vascular inhibine and endosatin in colibacillus is obtained. The fusion expressed product has not only the functions of both vascular inhibine and endosatin but also the synergistic effect ofantagonizing angiogenesis and tumor.