Preparation method of recombinant human oligoadenylate synthetase-1
A technology of chemical synthesis and recombinant engineering bacteria, applied in the direction of transferase, etc., can solve the problems of complex steps, low process yield, limited improvement of OAS1 protein purity, etc.
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Embodiment 1
[0035] Embodiment 1: Construction of expression vector
[0036] The pBluescript II SK(+)-OAS1 synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. was digested with Nde I and EcoRI, and the digested product was subjected to 1% agarose gel electrophoresis. The results were as follows: figure 1 shown. The target gene was recovered with Agarose Gel DNA Purification Kit, and then ligated with the expression vector pET23b(+) that had undergone the same digestion. The ligation reaction conditions were: 10x T4 buffer 2 μl, T4 DNA Ligase 1 μl, target fragment 5 μl, vector 12 μl, overnight at 16°C connect. Thus, the pET23b(+)-OAS1 recombinant plasmid was constructed. The recombinant plasmid was identified by agarose gel electrophoresis after double digestion with Nde I and EcoRI. figure 2 shown.
Embodiment 2
[0037] Example 2: Transformation and screening of competent Escherichia coli BL21(DE3)pLys
[0038] Add 1-2ng of the supercoiled recombinant plasmid pET23b(+)-OAS1 into 100μl Escherichia coli BL21(DE3)pLys competent cells, swirl gently to mix well, let stand in ice bath for 30s, heat shock at 42°C for 60-90s, and put in ice bath Let stand and cool for 2-3min, add 900μl LB medium (without antibiotics), culture with shaking at 37℃ for 45min, pipette 100μl after mixing and spread on LB plate (containing 1μg / ml ampicillin sodium), incubate upside down at 37℃ for 12- 16h.
[0039] A single colony was picked for culture, and the clones identified as positive by enzyme digestion and expression detection were automatically sequenced by the ABI377 sequencer with T7 universal primers, and the sequencing results were consistent with the theoretical sequence.
Embodiment 3
[0040] Example 3: LB medium fermentation culture of OAS1 and acquisition of inclusion bodies
[0041] After the positive engineered bacteria obtained by screening were activated by spreading on the plate, a single colony was selected and inoculated in LB medium containing ampicillin (prepared with 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl per liter, and the pH was adjusted to 7.0), at 37 ° C, 220 R / min Shaker Flask Culture to OD 600nm Reach 0.6-0.8. Then inoculate in the LB culture medium of 50L with 5% volume inoculum amount, carry out fermentation culture (every liter contains the ampicillin of 0.1%) in 80L fermenter, culture temperature is 37 ℃, regulates pH with ammonia water in the cultivation process Between 6.5-7.5, the dissolved oxygen value is controlled between 3-5% by adjusting the stirring speed. in OD 600nm After reaching 1.0, 10 g of IPTG was added at a mass volume ratio of 1:5000 to continue the induction culture for 3 hours. The induction cultur...
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