Application of plants as hosts in expressing rituximab
A technology of rituximab and an expression vector is applied in the application field of plants as hosts in the expression of rituximab antibodies, and can solve the problems of high production cost, high price, low production capacity of animal cells and the like
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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 protein in plants, the amino acid sequence of human rituximab antibody heavy chain, light chain (https: / / www.drugbank.ca / drugs / DB00073) was reverse-translated software (https: / / www .idtdna.com / CodonOpt) to obtain the nucleotide sequence and optimize its codons to plant-preferred codons, which were synthesized by GenScript (Nanjing, China). An Xbal restriction site was added to the 5' end of the optimized rituximab heavy chain sequence, and a SacI site was added to the 3' end. A Xbal restriction site was added to the 5' end of the rituximab light chain sequence, and a Sacl site was added to the 3' end. and cloned into the pUC57 vector by GenScript Company to obtain pRit-H and pRit-L cloning vectors respectively ( figure 1 ), the gene fragments were separated from the cloning vector by Xbal / Sacl, and cloned into the binary plant vector, p...
Embodiment 2
[0050] Example 2 Agrobacterium-mediated vacuum infiltration
[0051] Mix the prepared Agrobacteria containing p35S-Rit-H and p35S-Rit-L in equal amounts until the O.D.600 is 0.5. The 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. Hold the pressure 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 the penetration of the permeate into the lettuce tissue was clearly visible. 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. The...
Embodiment 3
[0052] Example 3 Protein Extraction and Separation
[0053] Lettuce samples vacuum-infiltrated with Agrobacterium were stirred with a stirrer and homogenized in a mixer at high speed for 1-2 minutes with an extraction buffer (100mM KPi, pH7.8; 5mM EDTA; 10mM β-mercaptoethanol) with a volume ratio of 1:1 . The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 minutes 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 (10,000g) for 15 minutes at 4°C. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended by shaking on ice for 60 minutes, and centrifuged again at 10,000 g at 4°C for 15 minutes. 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-m...
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