Ternary expression element, eukaryotic expression vector containing same and use thereof
A technology of expression elements and expression vectors, applied in the field of genetic engineering, can solve the problems of low eukaryotic stable expression level, cumbersome selection process, time and manpower, etc., to facilitate the screening process, improve expression level, save time and cost Effect
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Embodiment 1
[0054] Embodiment 1: Construction of ternary eukaryotic expression vector
[0055] The ternary eukaryotic expression vector of the present invention is composed of a series of gene elements, and its core component is a ternary expression element. The structure of the ternary expression element specifically used in this embodiment is MCS-IRES-DHFR-IRES-EGFP, Its nucleotide sequence is shown in SEQ ID No.1, and the nucleotide sequence of the ternary expression vector constructed based on it in this example is shown in SEQ ID No.3.
[0056] Also provided in this embodiment on the basis of the above elements, the ternary expression element after the sequence exchange of DHFR gene and EGFP gene, the structure is MCS-IRES-EGFP-IRES-DHFR, its nucleotide sequence sequence As shown in SEQ ID No.2. The nucleotide sequence of the ternary expression vector constructed based on it in this example is shown in SEQ ID No.4.
[0057] In this embodiment, the vector of the present invention is...
Embodiment 2
[0059] Example 2: Small-scale preparation of carrier
[0060] The carrier 1 of the present invention was added to the competent cells, and ice bathed for 30 minutes. Put the bacterial solution into a water bath at 42°C for 90 seconds, and immediately place it in an ice bath for 2 minutes. Add culture medium and incubate at 200 rpm on a constant temperature shaker at 37°C for 1 hour. Spread the cells on the surface of the agar plate containing kanamycin, and culture the plate upside down at 37°C overnight. The next day, the colonies on the picked plate were inoculated in the medium containing kanamycin, and cultured overnight at 37°C on a shaker. The next day, the bacterial solution was collected, and a small amount of carrier 1 was prepared by the alkaline method for future use.
[0061] Using the above method, a small amount of carrier 2 was also prepared for future use.
Embodiment 3
[0062] Example 3: Enzyme digestion and identification of vectors
[0063] Take an appropriate amount of vector 1, and select two restriction enzymes, XbaI and XhoI, and matching Buffer. Add the following components to the centrifuge tube: 10×Buffer (1 μl), sample to be cut (2 μl), XbaI and XhoI Dicer enzymes (0.5 μl each), add water to make up 10 μl. Mix the sample well and centrifuge briefly to sink the sample to the bottom of the tube. After incubating the centrifuge tube at 37°C for 3 hours, carry out 1% agarose electrophoresis to identify the result of enzyme digestion. The results show that after double digestion with XbaI and XhoI, the target bands appear at about 2500bp and 4000bp, which is in line with expectations (see figure 2 ). The same method was used to identify vector 2 by enzyme digestion, and the result was also in line with expectations.
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