Optimized gene of salmonella typhimurium flagellin and preparation method of flagellin
A Salmonella, flagellin technology, applied in biochemical equipment and methods, chemical instruments and methods, botanical equipment and methods, etc., can solve problems such as low production of flagellin
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Embodiment 1
[0028] Example 1 Optimization of Salmonella typhimurium flagellin gene
[0029] According to the published amino acid sequence of Salmonella typhimurium flagellin protein (accession number: P06179), the gene sequence optimization design was carried out. In order to facilitate purification, a 6×His tag was added to the N-terminus of the amino acid sequence of the flagellin protein, which enables the resulting recombinant protein to be affinity-purified with a Ni column. In order to remove the 6×His tag from the final product protein, a TEV protease cleavage site was added after the 6×His tag. In order to prevent the flagellin protein expressed in Pichia pastoris from being N-glycosylated, amino acid substitutions were performed on 6 potential N-glycosylation point sequences (N-X-S / T, X being any amino acid) in the flagellin protein sequence, and In the N glycosylation point sequence, N is replaced by Q, and the amino acid sequence of the Salmonella typhimurium flagellin protei...
Embodiment 2
[0033] Example 2 Synthesis of Salmonella typhimurium flagellin flagellin optimized gene sequence and construction of expression vector
[0034] The optimized sequence of the flagellin flagellin gene of Salmonella typhimurium can be sent to any gene synthesis company for sequence synthesis and cloned into the SnaB I and EcoR I sites of the Pichia pastoris expression vector pPIC9K, so that the optimized sequence is compatible with the α- The factor signal peptide sequence is in the same reading frame. In this example, the optimized sequence synthesis and cloning of the flagellin gene of Salmonella typhimurium into the expression vector pPIC9K of Pichia pastoris was completed by Wuhan Jinkairui Bioengineering Co., Ltd. The obtained plasmid was named pPIC9K-flagellin, and the vector diagram is shown in attached figure 1 . The recombinant plasmid was digested with SnaB I and EcoR I to identify whether the flagellin gene exists in the constructed vector, and the results of agarose...
Embodiment 3
[0035]Example 3: Expression of Salmonella typhimurium flagellin in Pichia pastoris
[0036] 1. Obtain electroporation plasmid
[0037] 1) Pipette 1ul (concentration: 50-1000ng / ul) of pPIC9K-flagellin plasmid DNA into competent Escherichia coli TOP 10', stir gently with a sterile pipette tip, ice bath for 20-40min, 42°C Heat shock for 60-90s, add LB liquid medium without ampicillin and incubate at 220r / min at 37°C for 0.5-1h, then spread evenly on LB solid plates containing ampicillin, and culture overnight at 37°C to obtain transformants.
[0038] 2) Pick a single colony on a plate, inoculate it into 10ml LB liquid medium with ampicillin, culture it in a shaker at 37°C and 220r / min for 12-16h, and extract the plasmid according to the general method for plasmid extraction.
[0039] 2. The pPIC9K-flagellin plasmid was linearized with Sal I, and the restriction enzyme digestion conditions for linearization were carried out according to the manual, and the linearized plasmid was ...
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