Soluble expression preparation method of Patatin sample phospholipase III
A phospholipase, soluble technology, applied in the field of genetic engineering, can solve the problems of difficult purification, complex and expensive protein, difficult practical application and research, etc., and achieves the effect of saving time and effort, and low cost.
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Embodiment 1
[0027] Embodiment 1, contains Mal E Construction and identification of the first expression vector of the gene sequence.
[0028] According to E. coli Mal E Design primers for the gene sequence, and introduce endonuclease Nde1 and Sac1 sequences into the upstream and downstream primers respectively, the primers are as follows Mal E -F: CGC CATATG AAAATAAAAACAGGT (SEQ ID NO: 5) and Mal E -R:CTCGTATCACCAAG GAGCTC ATA (SEQ ID NO: 6). Extract the genomic DNA of Escherichia coli as a template, and obtain it through high-fidelity PCR enzyme amplification Mal E Gene coding sequence. Treat the gene with Nde1 and Sac1 double digestion Mal E and expression vector pCold1. Digested products were run on a gel and recovered, ligated with T4 DNA ligase Mal E and pCold1, transformed into DH5α competent cells, spread the bacterial solution on a solid medium containing ampicillin, cultivate overnight at 37°C, then pick a positive single colony for cultivation, and identify the...
Embodiment 2
[0029] Example 2. Construction and identification of prokaryotic expression vectors.
[0030] The total RNA of the duck was extracted according to the instructions of TRIzol from TaKaRa Company. Take 100 mg of pectoralis muscle, extract total RNA, detect by electrophoresis, and measure OD with Eppendorf nucleic acid quantitative analyzer 260 / 280For the ratio and concentration, 1 μg of total RNA was used as a template, and reverse transcription was performed according to the instructions of the cDNA synthesis kit. Reverse transcription program: 37°C for 15 minutes, 85°C for 5 minutes, store at 4°C, and store at -20°C for long-term storage. design PLP3 Gene-specific primers, upstream primer F: 5'-cgc GGATCC ATGCAGCCCTGT-3' (SEQ ID NO: 3; the underline is the restriction site of BamH1); downstream primer R: 5'-ATTCCAGAAAA TGTCGAC gtc-3' (SEQ ID NO: 4; the underline is the Sal1 restriction site). Using cDNA as a template for PCR amplification, amplified Hsp90α The full l...
Embodiment 3
[0031] Example 3. Prokaryotic expression and solubility analysis of fusion protein.
[0032] Transform Escherichia coli Transetta2 competent cells with positive recombinant plasmids, pick a single colony and inoculate it into 5 mL of LB medium containing 100 μg / mL ampicillin, culture at 37°C with shaking at 200 r / min, when the concentration of the bacteria is OD 600 When the value is 0.6-0.8, add IPTG to a final concentration of 0.1-0.5mM (0.25mM is the best), 18-25°C (22°C is the best), 200r / min shaking culture induction for 12-16 hours. Use the bacterial solution induced without IPTG as a control, collect the precipitate by centrifugation, resuspend the precipitate with PBS, crush it on an ultrasonic cell disruptor for 5 minutes, and centrifuge at 12000r / min for 5 minutes, take out the supernatant and precipitate respectively, add SDS-PAGE electrophoresis and load the sample Buffer solution, heated at 95°C for 10 min, and checked whether the fusion protein was soluble by 12%...
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