A method for preparing human-like sugar chains from Haemophilus influenzae based on the skeleton protein fn3
A technology of Haemophilus influenzae and skeleton protein, applied in the biological field, can solve the problems of loss of activity and difficulty in preparing humanized oligosaccharides, and achieve the effects of efficient preparation, improvement of physical and chemical properties, and improvement of pharmacokinetic properties
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Embodiment 1
[0034] 1. In this example, human fibronectin type III domain (Fn3) protein is used as a model gene (EMBL accession number AJ320527). According to the modeling results, the glycosylation modification site was designed at the C-terminal of the Fn3 backbone protein, the amino acid sequence of the glycosylation site DQNAT was selected, and a separation and purification tag (His-tag) encoding 6 histidines was fused downstream of the gene. ), easy to separate and purify, named as Fn3-Gly, fusion gene structure see figure 1 .
[0035] The fusion gene shown in the sequence list [002] is synthesized (Nanjing KingScript Biotechnology Co., Ltd.), constructed on the Escherichia coli periplasmic cavity expression vector pIG6 with Nco I and Hind III, and obtains the recombinant vector pIG6-Fn3-Gly, fusion gene structure see figure 2 .
[0036] 2. Integrate the prokaryotic oligosaccharide synthesis mechanism and rebuild the glycosylation pathway in E. coli. The specific steps are as fol...
Embodiment 2
[0044] 1. In this example, human fibronectin type III domain (Fn3) protein is used as a model gene (EMBL accession number AJ320527). According to the modeling results, the glycosylation modification site was designed at the C-terminus of the Fn3 backbone protein, the amino acid sequence of the glycosylation site DQNAT was selected, and a separation and purification tag encoding 6 histidines (His-tag ), easy to separate and purify, named as Fn3-Gly, fusion gene structure see figure 1 .
[0045] The fusion gene synthesis (Nanjing KingScript Biotechnology Co., Ltd.) shown in the sequence table [002] was constructed on the E. coli periplasmic cavity expression vector pIG6 with Nco I and Hind III to obtain the recombinant vector pIG6-Fn3-Gly, fusion gene structure see figure 2 .
[0046] 2. Integrate the prokaryotic oligosaccharide synthesis mechanism and rebuild the glycosylation pathway in E. coli. The specific steps are as follows: (1) The oligosaccharidyl transferase gene ...
Embodiment 3
[0054] 1. In this example, human fibronectin type III domain (Fn3) protein is used as a model gene (EMBL accession number AJ320527). According to the modeling results, the glycosylation modification site was designed at the C-terminus of the Fn3 backbone protein, the amino acid sequence of the glycosylation site DQNAT was selected, and a separation and purification tag encoding 6 histidines (His-tag ), easy to separate and purify, named as Fn3-Gly, fusion gene structure see figure 1 .
[0055] The fusion gene synthesis (Nanjing KingScript Biotechnology Co., Ltd.) shown in the sequence table [002] was constructed on the E. coli periplasmic cavity expression vector pIG6 with Nco I and Hind III to obtain the recombinant vector pIG6-Fn3-Gly, fusion gene structure see figure 2 .
[0056] 2. Integrate the prokaryotic oligosaccharide synthesis mechanism and rebuild the glycosylation pathway in E. coli. The specific steps are as follows: (1) The oligosaccharidyl transferase gene ...
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