Recombination cytohesin antagonism polypeptide TAT-Sec 7 and preparation method thereof
A tat-sec7, antagonistic technology, applied in the fields of genetic engineering and biopharmaceuticals, can solve problems such as difficult treatment methods
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0027] Design of recombinant Cytohesin antagonistic polypeptide fusion gene.
[0028] According to the amino acid sequence of the ARNO of the Cytohesin family, find Sec7 reported in the literature, optimize the nucleotide sequence according to the preferred codons of E. coli, and then add a transmembrane peptide (TAT) sequence at its 5' end, and add a 6His tag at its 3' end sequence. sequence design as figure 1 shown.
Embodiment 2
[0030] PCR synthesis of recombinant Cytohesin antagonistic polypeptide fusion gene and construction of pET-20b-recombinant Cytohesin antagonistic polypeptide expression vector.
[0031] In order to obtain the sequence of the TAT-Sec7 gene sequence, design primers as shown in Table 1, use P1 and P2 as primers (see Table 1 for primer sequences), and synthesize the TAT-Sec7 sequence by PCR method.
[0032]
[0033] Reaction program: pre-denaturation at 98°C for 5min; denaturation at 98°C for 10min; annealing at 55°C for 15s; extension at 72°C for 1min, 30 cycles, and finally extension at 72°C for 5min. After no non-specific bands were detected by 1% agarose gel electrophoresis, the DNA fragment of the product was purified and recovered with a DNA gel recovery kit, and then the fragment was double-digested with NdeI and XhoI, and then digested with the same two enzymes. The pET-20b plasmid was subjected to enzyme reaction to obtain the pET-20b-TAT-Sec7 plasmid. The result is a...
Embodiment 3
[0037] Recombinant Cytohesin antagonizes the acquisition of polypeptide fusion protein.
[0038] 1. Induced expression
[0039]Shake culture pET-TAT-Sec7-his transformed Escherichia coli BL21(DE3) host cells at 37°C for 16 hours, then transfer to fresh LB medium at a ratio of 5%. Under induction for 3 hours, ultrasonic (200w, ultrasonic 5s, interval 5s, cycle 180 times) to break the bacteria, the recombinant protein can account for more than 70% of the soluble bacterial protein, the results are as follows image 3 shown. Among them: a is the whole bacteria before induction; b, c, d are the expression of TAT-Sec7 after induction.
[0040] 2. Purification of Recombinant Proteins
[0041] Load the sample onto the Ni-NTA affinity chromatography column, equilibrate the Ni-NTA chromatography column with buffer solution 50mmol / LTris-HCl, pH8.0, 0.5mol / LNaCl, 10mmol / L imidazole, and drain the lysate to the equilibrium well In the column, wash with buffer solution 50mmol / L Tris-HCl...
PUM
| Property | Measurement | Unit |
|---|---|---|
| purity | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More