Application of Plants as Hosts in the Expression of Hemoglobin
A hemoglobin and plant technology, applied in hemoglobin/myoglobin, application, plant gene improvement, etc., can solve problems such as increasing costs and hindering the development of plant exogenous protein drugs
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Embodiment 1
[0047] Example 1 Construction of plant transient expression vector
[0048] In order to provide high-efficiency expression of foreign aid proteins in plants, the present invention optimizes the codons of the human hemoglobin Hb-α+2A+Hb-β fusion protein sequence to plant-preferred codons, designed by Integrated DNA Technology (IDT) and designed by Kings Synthesized by Rui Company. An Xbal restriction enzyme site was added to the 5' end of the optimized Hb sequence, and a Sacl site was added to the 3' end, and cloned into pUC57 vector by GenScript. The human hemoglobin gene fragment Hb was isolated from pUC57-Hb by Xbal / Sacl and cloned into the binary plant vector pCam35S to generate the transient expression vector p35S-Hb, respectively. The plant expression constructs were individually transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany). The resulting strains were spread evenly on selective LB plates containin...
Embodiment 2
[0049] Example 2 Agrobacterium-mediated vacuum infiltration
[0050] The present invention optimizes the method of Agrobacterium vacuum infiltration. The prepared Agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. The lab-preserved lettuce was inverted (core up) and gently swirled into the bacterial suspension, sealing the desiccator. The vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keep the pressure state for 30-60s. The system is quickly opened to release pressure, allowing permeate to penetrate the space within the tissue. This process was repeated 2 to 3 times until it was clearly visible that the permeate had diffused in the lettuce tissue. The lettuce tissue was then gently removed from the infiltrate and rinsed three consecutive times with distilled water before being transferred to a container covered with plastic film. Treated samples were kept in the dark...
Embodiment 3
[0052] Example 3 Protein extraction and isolation
[0053]The Agrobacterium vacuum-infiltrated lettuce samples were stirred with a stirrer and homogenized at high speed for 1-2 min in a 1:1 volume ratio of extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM β-mercaptoethanol). 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 cellular debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated on ice for 60 minutes with shaking. Separate again by centrifuge (10,000 g) at 4°C for 15 min. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, and was 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 processed sample precipitated protein was dissolved in 5 mL of buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β-mercaptoethanol) and stored ...
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