6 results about "XhoI" patented technology

The invention belongs to the field of molecular biology and genetic engineering, and discloses a chemiluminescent reporter plasmid pBBR402 and application thereof in detection of the transcription level of a target gene of acetobacter pasteurianus. The plasmid comprises a replication element (oriV and rep) from a pBBR1MCS-2 plasmid, a kanamycin resistance gene from a pMS402 plasmid, a promoter insertion site (BamHI / AvaI / XhoI), a luxCDABE reporter gene cluster and a T1 transcription terminator, and the kanamycin resistance gene, the promoter insertion site (BamHI / AvaI / XhoI), the luxCDABE reporter gene cluster and the T1 transcription terminator are obtained from the pBBR1MCS-2 plasmid. The plasmid can be inserted into a to-be-detected gene promoter of acetobacter pasteurianus to drive luxCDABE expression. During application, the recombinant plasmid is transferred into acetobacter pasteurianus, the OD value and the LUM value of the living bacteria liquid are directly detected, and the transcription level is quantified by the LUM / OD value. The method is easy to operate, high in sensitivity and suitable for gene regulation and control research of acetobacter pasteurianus.

The invention discloses application of transgenic zebrafish in preparation of an animal model capable of visually monitoring mast cells. The transgenic zebrafish is prepared by the following method: respectively carrying out XhoI and BamHI double enzyme digestion on a cpa5 gene promoter sequence (SEQ ID NO: 1) and plasmids with fluorescent protein genes and Tol2 transposon recognition sites, and then connecting to obtain recombinant plasmids; and then introducing the recombinant plasmid and Tol2 transposase mRNA into a zebra fish embryo in a microinjection mode in a 1-cell period of zebra fish embryo development, and culturing at constant temperature to obtain the transgenic zebra fish. The cpa5 promoter is constructed to drive an expression framework of the fluorescent protein, so that the mast cells naturally emit fluorescence in the zebra fish body, and an effective animal model is provided for mechanism research and drug screening of mast cell related diseases.

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 relates to the technical field of biological products, and particularly discloses an XhoI restriction enzyme freeze-drying protective agent and a preparation method and application thereof.The freeze-drying protective agent is composed of trehalose, polyvinylpyrrolidone, arginine, HSA and EDTA in parts by mass and volume, and a solvent is sterilized purified water. A co-amorphous substance is formed through interaction of all the components to construct a multi-level protection network for enzyme, in the freezing and drying process, hydroxyl of trehalose interacts with the surface of enzyme molecules through hydrogen bonds, the position of water molecules is effectively replaced, the enzyme is forced to maintain the natural hydration conformation of the enzyme, and therefore expansion and denaturation caused by dehydration are avoided. The amorphous state of the polyvinylpyrrolidone and the trehalose are in synergistic interaction, so that the glass transition strength of the mixture can be enhanced, the physical stability of the product can be improved, collapse can be prevented, the contact between enzyme molecules can be blocked through the huge steric hindrance effect, and the inactivation caused by aggregation can be effectively prevented.

The present application relates to a kind of H5N1 nucleoprotein prokaryotic expression vector and the method for its nucleoprotein induced expression, belong to the field of bioengineering, the H5N1 nucleoprotein base sequence optimized by in vitro synthesis in the present application, by enzyme cutting site BamHI and XhoI, the H5N1 nucleoprotein optimized base sequence is constructed into PGEX-6P-1 plasmid and obtains H5N1 nucleoprotein prokaryotic expression vector;After being transformed into BL21 competent bacteria by H5N1 nucleoprotein prokaryotic expression vector, inoculate in LB solid culture medium with ampicillin resistance and culture, pick single bacterial clone and inoculate into LB liquid culture medium with ampicillin resistance and culture, finally, IPTG culture induction is obtained, and the nucleoprotein fusion protein of induced expression is obtained, and the nucleoprotein of H5N1 is well expressed, and the fusion protein is obtained, which provides the basis for studying more significant, more durable immunity and effective H5N1 vaccine.

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.