Application of lettuces as host to expression of Hepatitis B vaccines
A host and vaccine technology, applied in applications, microorganisms, biochemical equipment and methods, etc., can solve problems such as hindering the development of plant exogenous protein drugs and increasing costs
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Embodiment 1
[0060] The construction of embodiment 1 plant transient expression vector
[0061] In order to provide foreign protein for expression in plants, HBV (NC_003977.2) in the present invention is synthesized by GenScript. A Xbal restriction site was added at the 5' end of the HBV sequence, a Sacl site was added at the 3' end, and cloned into the pUC57 vector by GenScript. Human HBV gene fragments were isolated from pUC57-INS by Kpnl / Sacl and cloned into the binary plant vector pCam35S to generate the transient expression vector p35S-HBV, respectively. The plant expression constructs were individually transformed into Agrobacterium tumefaciens EHA105 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany). The resulting bacterial strain was evenly spread on the selective YEP plate containing kanamycin antibiotic (50mg / L). After incubating at 28°C in the dark for 48 hours, pick a single colony and inoculate 200 mL of YEB (yeast extract broth, 5 g / L sucrose, 5 g / L trypt...
Embodiment 2
[0062] Example 2 Agrobacterium-mediated vacuum infiltration
[0063] The invention optimizes the method of vacuum infiltration of Agrobacterium. 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. It can be clearly seen that the permeate diffuses obviously 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 covered with plastic film. Keep the treated samples in the dark for 4 ...
Embodiment 3
[0065] Example 3 Protein Extraction and Separation
[0066] 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 ...
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