Application of plant as host in expression of insulin
A technology of insulin and human insulin, applied in the direction of insulin, application, plant gene improvement, etc., can solve problems such as hindering the development of plant exogenous protein drugs and increasing costs
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Embodiment 1
[0048] The construction of embodiment 1 plant transient expression vector
[0049] In order to provide high-efficiency expression of foreign aid proteins in plants, the present invention optimizes the codons of human insulin (GenBank Accession number: AH002844.2) to plant-preferred codons, provided by GeneArt TM GeneOptimizer TM (ThermoFisher) and synthesized by GenScript. The Xbal restriction site was added to the 5' end of the optimized insulin sequence, and the Sacl site was added to the 3' end, and cloned into the pUC57 vector by GenScript. The human insulin gene fragment was isolated from pUC57-INS by Kpnl / Sacl, and cloned into the binary plant vector pCam35S to generate the transient expression vector p35S-INS respectively. The plant expression constructs were respectively transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany). The resulting bacterial strains were evenly spread on selective LB plates con...
Embodiment 2
[0051] Example 2 Agrobacterium-mediated vacuum infiltration
[0052] The present invention optimizes the method of Agrobacterium vacuum infiltration ( figure 2 ). The prepared Agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. The lettuce kept in this laboratory was turned upside down (core up) and gently swirled in the bacterial suspension, and the desiccator was sealed. The vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was seen in the leaf tissue. Maintain the pressure state for 30-60 seconds. The system is quickly opened to release the pressure and allow permeate to seep into the spaces within the tissue. This process was repeated 2 to 3 times until it was clearly visible that the permeate diffused significantly in the lettuce tissue. The lettuce tissue was then gently removed from the permeate and rinsed three times consecutively with distilled water before being transferred to a container co...
Embodiment 3
[0054] Example 3 Protein Extraction and Separation
[0055] Lettuce samples vacuum-infiltrated with Agrobacterium were stirred with a stirrer and extracted with extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM at a ratio of 1:1 by volume). -Mercaptoethanol) high-speed homogenization in a blender for 1 to 2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 min at 4°C to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated with shaking on ice for 60 minutes. Centrifuge again (10,000 g) for 15 min at 4°C. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, shaken and suspended on ice for 60 min, and centrifuged again at 10,000 g for 15 min at 4°C. Then, the supernatant was discarded, and the protein precipitated from the treated samples was dissolved in 5 mL of buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β...
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