5 results about "NdeI" patented technology

This invention relates to a novel truncated mutant apolipoprotein E and its calibrators and quality control methods. The amino acid sequence of the truncated mutant apolipoprotein E is shown in SEQ ID. The diluent for the calibrators and quality control methods for detecting the truncated mutant apolipoprotein E includes a preservative, a protein protection composition, a surfactant composition, an ion stabilizer composition, and a buffer solution. The method for preparing the apolipoprotein E mutant includes the following steps: 1. Determining the specific sequence of the truncated mutant; 2. Determining that the mutation site is located at position 78 of wild-type apolipoprotein E; 3. Containing the NdeI site and the mutation sequence; locking the mutation; artificially optimizing the apolipoprotein E nucleotides using E. coli; 4. Extracting the corresponding truncated mutant by electrophoresis; 5. Obtaining the truncated mutant apolipoprotein E through recombination with E. coli. This invention solves the problems of discrepancies between detection results and actual clinical applications, as well as the poor stability of apolipoprotein E calibrators.

The invention provides a preparation method and a detection method of a recombinant cat-omega interferon antiviral preparation, and the preparation method comprises the following steps: carrying out reverse transcription on total RNA (Ribonucleic Acid) of cat spleen to obtain cDNA (Complementary Deoxyribose Nucleic Acid); by taking the cDNA as a template, executing a PCR amplification program on the cat IFN-omega gene sequence by using the specific primer to obtain a PCR product; carrying out connection operation on the PCR product and a pGEM-T-Easy vector, and transferring the PCR product and the pGEM-T-Easy vector into DH5alpha competent cells; the method comprises the following steps: extracting positive clone plasmids which are verified to be correct from DH5alpha competent cells by using specific primers to obtain recombinant expression plasmids, carrying out double enzyme digestion on the recombinant expression plasmids and a pET30a vector by using NdeI and XhoI, and transferring obtained recovered target fragments into BL21 (DE3) competent cells to identify positive recombinant plasmids containing target genes; transferring the positive recombinant plasmid containing the target gene into BL21 (DE3) competent cells for bacterial colony culture, and performing induced expression on the obtained culture thalli; and after the induced expression is completed, preparing a preparation corresponding to the cat interferon from the cultured thalli. The preparation efficiency is at least improved.

The invention discloses a DNA structure for saccharomyces cerevisiae single-cell barcode labeling and chromatin state conjoint analysis and application thereof, and belongs to the technical field of molecular biology and single-cell multi-omics analysis. From the 5'end to the 3 'end, a fluorescent protein gene, a Tn5ME structure, a reverse transcription primer binding region, a CYC1 terminator fragment and a linearized shuttle plasmid skeleton are subjected to homologous recombination, and a pCM carrier is obtained; after NdeI / BamHI enzyme digestion, oligonucleotides with random sequences, which are subjected to annealing, amplification, Klenow filling-in and enzyme digestion, are connected, and transformation extraction is carried out, so that the DNA structure for saccharomyces cerevisiae single-cell barcode labeling and chromatin state combined analysis is obtained. On the basis of the constructed DNA structure, ATAC-seq and a bar code reverse transcription method are combined, important data related to the transcription state and chromatin state of a single cell can be obtained at the same time through bar code information, single cell pedigree tracking of the saccharomyces cerevisiae is achieved, and research on single cell epigenomics of the saccharomyces cerevisiae and other microorganisms is promoted.

This invention relates to the field of bioengineering technology and discloses a method for preparing a short-chain dehydrogenase that catalyzes the conversion of cycloartenol to cycloargentone and its application, comprising the following steps: Step 1: Cloning of the short-chain dehydrogenase gene, the KhSDR1 gene is cloned from the cDNA of Schisandra chinensis; Step 2: Construction of the recombinant expression vector, the PCR product is digested with NdeI and SalI, ligated to the similarly treated pET-28b vector, and transformed into E. coli BL21(DE3) to construct the recombinant expression vector pET-28a-KhSDR1; Step 3: The recombinant expression vector pET-28a-KhSDR1 is transformed into Escherichia coli BL21(DE3) for induced expression; Step 4: Enzyme-catalyzed reaction is performed; Step 5: Product detection, the reaction product is analyzed by gas chromatography-mass spectrometry (GC-MS) and confirmed to be cycloargentone. Under optimized conditions, the substrate conversion rate of this invention can reach over 50%; the reaction is carried out in an aqueous phase at room temperature and pressure, making it green and environmentally friendly; the reaction system is simple and easy to scale up for production, providing an efficient and environmentally friendly route for the industrial production of cycloarpineone.

The application provides a SAA recombinant protein and a preparation method and application thereof, and the preparation method comprises the following steps: S10, constructing a recombinant plasmid: a gene sequence of an amino acid sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO: 2 is inserted into a pET-28a(+) plasmid which is double-digested by NdeI and XhoI after being double-digested by NdeI and XhoI, so that a recombinant plasmid is obtained; S20, transforming and culturing the recombinant plasmid; S30, protein expression and purification. The application solves the technical problem that the stability of the currently recombinantly expressed SAA protein in vitro is poor, and achieves the technical effect of improving the stability of the SAA recombinant protein in vitro.