Staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof
A technology of capsular polysaccharides and staphylococci, which is applied in the field of Staphylococcus aureus capsular polysaccharides and protein conjugates and preparations, can solve the problems of inability to induce animal immunity, and achieve the effect of promoting immune response and improving immune response
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[0060] The preparation method of staphylococcus aureus capsular polysaccharide and protein conjugate comprises the following steps:
[0061] (1) Preparation of polylysine Salmonella typhimurium flagellin
[0062]Introduce 6 consecutive lysine amino codon DNA fragments into the upstream of Salmonella typhimurium flagellin gene by overlap extension PCR method: AAAAAGAAAAAGAAAAAG, this DNA fragment is connected to the expression vector pET-His, and the obtained vector pET-His-6 ×Lys-Flic was transformed into Escherichia coli to obtain engineering bacteria expressing poly-lysine Salmonella typhimurium flagellin. The expression level of the target protein reaches 15-25% of the total bacterial protein;
[0063] (2) Chemical modification of capsular polysaccharide of Staphylococcus aureus
[0064] The capsular polysaccharide of Staphylococcus aureus was formulated into 4-6 mg / ml physiological saline solution. In each milliliter of this solution, add 0.03-0.07 g of adipic acid dihy...
Embodiment 1
[0072] The preparation of embodiment 1 Salmonella typhimurium flagellin (Flic)
[0073] 1. Materials
[0074] Salmonella typhimurium gene DNA was donated by Zhejiang Center for Disease Control and Prevention; E. coli competent cells BL21(DE3), DH5α, plasmid pMD-18T, prokaryotic expression vector pET-his were preserved by our laboratory; DNA gel recovery kit, λ -EcoT14 I digest DNA standard, restriction endonucleases EcoR I, BamHI and Nhe I, T4 DNA ligase were purchased from Dalian Bao Biological Company; IPTG, Pfu DNA polymerase were purchased from Shanghai Sangong; protein purification column HisTrapTMFFcrude was purchased from GE, USA Company; Ampicillin (Amp), Agarose II was purchased from Beijing Pubo Biotechnology Co., Ltd.
[0075] 2. Cloning of the Flic gene
[0076] 2.1 Cloning of flagellin (Flic)
[0077] The whole gene of Salmonella typhimurium flagellin (Flic) was amplified with reference to the Genbank (AY353373.1) sequence, and the primers were designed using P...
Embodiment 2
[0146] The preparation of embodiment 2 poly-lysine Salmonella typhimurium flagellin (6×Lys-Flic)
[0147] 1. Materials and methods
[0148] The poly-lysine Salmonella typhimurium flagellin (Lys-Flic) primer introduces 6 lysine codons (AAA or AAG) at the N-terminal of the flagellin, namely
[0149] Lys-Flic-f: 5'----CGCGGATCCAAAAAGAAAAAGAAAAAAGGCACAAGTCATTAATACAAAC AGC C----3'
[0150] Lys-Flic-r: 3'----CGTTTTGCAGGAGAGAAATGACGCAATTCGATCGATC----5'
[0151] The preparation process of the polylysine Salmonella typhimurium flagellin is the same as the cloning, expression and purification process of the Flic gene in Example 1.
[0152] 2. Results
[0153] 2.1 Enzyme digestion and identification analysis of recombinant expression vector
[0154] image 3 The plasmid pET-his-6Lys-Flic, which is a recombinant expression vector and its products before and after induction, was analyzed by BamHI single-enzyme digestion and EcoR / NheI double-enzyme digestion electrophoresis, which prov...
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