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

Inactive Publication Date: 2010-12-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the carrier protein used is an immune inert carrier, when used to prepare vaccines for immunizing animals, it cannot induce animals to produce ideal immune responses.

Method used

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  • Staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof
  • Staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof
  • Staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof

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preparation example Construction

[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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Abstract

The invention discloses a staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof. Salmonella typhimurium flagellin or polylysine salmonella typhimurium flagellin is taken as a carrier, and staphylococcus aureus capsular polysaccharide is subject to chemical modification, and then chemical coupling is carried out on the staphylococcus aureus capsular polysaccharide and the carrier protein. As the salmonella typhimurium flagellin and polylysine salmonella typhimurium flagellin have immunologic adjuvant function, the new formed conjugate can cause staphylococcus aureus capsular polysaccharide with extremely low immunogenicity to have higher immunogenicity, and the carrier protein plays the immunologic adjuvant function when in inoculation, so that specific immune response of immune animal to the staphylococcus aureus capsular polysaccharide is enhanced. The invention has wide application in preparation of vaccine resisting staphylococcus aureus infection.

Description

technical field [0001] The invention relates to a staphylococcus aureus capsular polysaccharide and protein conjugate, a preparation method and application. Background technique [0002] Mastitis is an inflammation of mammary gland tissue, usually caused by microbial infection, and is one of the important diseases of dairy cows. According to the statistics of National Mastitis Council (NMC) in 1996, the annual economic loss caused by mastitis is about 1.7 billion US dollars. In my country, the incidence of dairy cow mastitis is high, up to 85% in some places [1], causing huge economic losses to cattle farmers every year. Since antibiotic treatment is likely to cause drug residues in dairy products and endanger the health of consumers, vaccination of dairy cows is a safe precautionary measure. [0003] Staphylococcus aureus is the main pathogen of cow mastitis. The bacteria can be isolated from the milk of many mastitis cows [2,3]. 94% to 100% of these isolated strains co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/255C07K1/14C12N15/31C12N15/63A61K39/39A61K39/085A61P31/04
Inventor 胡松华袁林王治才
Owner ZHEJIANG UNIV
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