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Biological preparation method of typhoid glycoprotein and application thereof

A protein and pili protein technology, applied in the field of biological preparation of typhoid glycoprotein, can solve the problem of low specificity of polysaccharides, achieve the effects of improving uniformity, reducing costs, and wide application prospects

Active Publication Date: 2016-08-17
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, with the development of sequencing technology and bioinformatics, natural protein glycosylation modification systems have been found in a variety of bacteria, such as the N-glycosylation modification system of Campylovibrio jejuni, Neisseria and green The O-glycosylation modification system of Pseudomonas, in these glycosylation modification systems, the polysaccharide synthesis pathway is very similar to the lipopolysaccharide and capsular polysaccharide synthesis pathway of bacteria, the polysaccharide structure depends on the glycosyl synthesis gene cluster, and its Oligosaccharyltransferases are less specific for the polysaccharides they transfer

Method used

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  • Biological preparation method of typhoid glycoprotein and application thereof
  • Biological preparation method of typhoid glycoprotein and application thereof
  • Biological preparation method of typhoid glycoprotein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Example 1. Preparation of Salmonella typhi O-polysaccharide-recombinant EPA fusion protein and vaccine thereof by one-step biological cross-linking method

[0074] 1. Preparation of Salmonella typhi deficient in O-antigen ligase gene waaL

[0075] (1) Preparation of linear targeting DNA fragments

[0076] 1. Design of PCR primers

[0077] According to the O-antigen ligase gene waaL (Genbank No. 3925131-3926345 of GI: 29139723) listed in the complete genome sequence of Salmonella typhi on NCBI (NC_004631.1) and its upstream and downstream sequences, the upstream of the waaL gene ( 5' end) and downstream (3' end) respectively design a pair of primers, namely 96waaLu1 / 96waaLu2 and 96waaLd1 / 96waaLd2. For the convenience of operation, restriction enzyme cutting sites BamH Ⅰ and Sal Ⅰ will be added to the end of the primer of the upstream homology arm up, and restriction enzyme cutting sites Hind III and Xho I will be added to the end of the primer of the downstream homolog...

Embodiment 2

[0157] Example 2, One-step biological cross-linking method to prepare Salmonella typhi O-polysaccharide-PilE protein

[0158] 1. Preparation of Salmonella typhi deficient in O-antigen ligase gene waaL

[0159] The steps are the same as Step 1 in Example 1.

[0160] 2. Molecular and phenotypic identification of Salmonella typhi defective in lipopolysaccharide synthesis

[0161] The steps are the same as Step 2 in Example 1.

[0162] 3. Preparation of Salmonella typhi O-polysaccharide-PilE protein by one-step biological cross-linking method

[0163] (1) Construction of glycosyl-engineered Salmonella typhi

[0164] The vectors pMMB66EH-pilE and pETtac28-pglL were electroporated to transform the host bacteria 50096ΔwaaL successively, and the double-antibody LB plate containing the final concentration of 50 μg / mL kanamycin and 100 μg / mL ampicillin was coated, and the grown clones were Typhoid glycosylated Engineering bacteria 50096ΔwaaL / pMMB66EH-pilE / pETtac28-pglL.

[0165] Th...

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Abstract

The invention discloses a biological preparation method of typhoid glycoprotein and an application thereof. The invention discloses a method for preparing bacteria polysaccharide-modified protein, a substrate protein of neisseria meningitidis O-oligosaccharyltransferase PglL and the neisseria meningitidis O-oligosaccharyltransferase PgIL are co-expressed in a bacteria defective in O-antigen ligase genes, and the bacteria polysaccharide-modified protein is obtained. The biological preparation method has wide application prospect for increasing uniformity of vaccine, increasing the production efficiency of the vaccine, and reducing cost; and the vaccine can be used for preventing the diseases caused by typhoid pathogen.

Description

technical field [0001] The invention relates to a biological preparation method and application of typhoid glycoprotein, belonging to the field of biomedicine. Background technique [0002] Salmonella (Salmonella spp.) is a highly contagious Gram-negative enteropathogenic bacterium with strong endotoxin and invasiveness. Fever, gastroenteritis, and sepsis, etc., can lead to intestinal bleeding or perforation in severe cases. Salmonella mediates initial bacterial invasion of the intestinal mucosa and subsequent systemic disease through two type III secretion systems. [0003] For most serotypes of Salmonella, half the infection rate is between 10 5 -10 8 between, but in outbreaks, the number of infections is generally less than 10 3 cells, sometimes even fewer than 100 bacteria. In 2012, the Ministry of Health of my country announced the annual statutory report of infectious disease epidemics, among the intestinal infectious diseases, the incidence of typhoid and paratyp...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/62C12N15/54C12N15/31C12N1/21C12P21/00A61K39/112A61P31/04
CPCY02A50/30
Inventor 王恒樑朱力彭哲慧潘超冯尔玲刘先凯吴军孙鹏曾明王斌
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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