A kind of potassium ion channel toxin-derived polypeptide and its preparation method and application
A potassium ion channel and toxin technology, applied in the field of potassium ion channel toxin-derived polypeptides and its preparation, can solve the problems of inability to perform subsequent work, incorrect folding, host mismatch, etc., to achieve low production costs, prolong life, and produce The effect of early egg timing
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Embodiment 1
[0030] Embodiment 1 Design primers and PCR amplification
[0031]The cDNA sequence of the potassium channel toxin was screened from the constructed scorpion venom gland cDNA library, and the cDNA sequence and amino acid sequence of the potassium channel toxin-derived polypeptide were designed with computer aid. The design is based on many factors that affect the interaction between the polypeptide and the potassium ion channel toxin, such as the distribution of functional residues of the polypeptide, the polarity of the residues, and the stability of amino acids. The amino acid sequence of the designed potassium ion channel toxin-derived polypeptide BmKT4 and the nucleotide sequence of the coding gene are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.
[0032] Artificially synthesize the gene fragment shown in SEQ ID NO.2, use the gene fragment as a template, and carry out PCR reaction with the designed upstream and downstream primers. Among them, the upstream and downstr...
Embodiment 2
[0036] Example 2 Construction of recombinant expression vector (pGEX-BmKT4)
[0037] The PCR product obtained in Example 1 was recovered using a gel recovery kit (Omega, USA). The recovered PCR product and the expression vector pGEX-4T-1 were digested with restriction endonucleases BamHI and XhoI. Digestion reaction: BamHI (14U / μL) and XhoI (20U / μL) each 0.5μL, 10×buffer 2.5μL, PCR product or pGEX-4T-1 plasmid 50-100ng, add ddH 2 O to a total volume of 25 μL in a 37 °C water bath for 3 h.
[0038] The enzyme-digested product was recovered by the gel recovery kit and ligated with T4 DNA ligase. The construction process is shown in the attached figure 1 . Ligation reaction: T4 DNA ligase (1U / μL) 1 μL, the molar ratio of digested PCR product to expression vector pGEX-4T-1 is 3:1, 10×buffer 2 μL, add ddH 2 O to a total volume of 20 μL at 16 °C overnight.
Embodiment 3
[0039] Example 3 Obtaining recombinant Escherichia coli
[0040] Transform the ligation product in Example 2 into Escherichia coli Rossetta (DE3) competent cells, mix well, and bathe in ice for 30 minutes, then in water bath at 42°C for 90 seconds (do not shake), then in ice bath for 1-2 minutes; add 500 μL of LB medium, Incubate on a shaker at 37°C for 50 minutes; shake the bacterial solution evenly, take 100 μL and spread it on an ampicillin agar plate (LB / AP + ), after the bacterial solution was blotted dry, place it upside down at 37°C and incubate for 12-16 hours, and observe the results.
[0041] LB / AP + A single colony was picked on the agar plate as a template, and PCR was performed with the primers P1 and P2 in Example 1. The positive clones screened by PCR were further identified by restriction enzyme digestion reaction. Both clones were positive results for sequencing analysis. The sequencing primer is a universal sequencing primer for the pGEX-4T-1 plasmid. If...
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