Method for improving S-adenosyl-L-cysteine hydrolase immunogenicity and hexasaccharide modified ahcy protein

A cysteine, immunogenic technology, applied in the direction of hydrolase, pharmaceutical formulations, bacterial antigen components, etc., to achieve the effect of improving the level of humoral immunity

Inactive Publication Date: 2016-06-15
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the defects of some attenuated vaccines and inactivated vaccines and the development of some new vaccines are in the laboratory stage, there is no more ideal Brucella vaccine for the control of human and animal brucellosis.

Method used

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  • Method for improving S-adenosyl-L-cysteine hydrolase immunogenicity and hexasaccharide modified ahcy protein
  • Method for improving S-adenosyl-L-cysteine hydrolase immunogenicity and hexasaccharide modified ahcy protein
  • Method for improving S-adenosyl-L-cysteine hydrolase immunogenicity and hexasaccharide modified ahcy protein

Examples

Experimental program
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Effect test

Embodiment 1a

[0042] Preparation and purification of embodiment 1ahcy protein

[0043] 1. Preparation of ahcy protein

[0044] (1) After the E. coli expression host strain BL21 (DE3) was prepared into competent cells, the recombinant plasmid pET28a-ahcy was transformed to construct the pET-28a-ahcy / BL21 expression strain, and the final concentration of pET-28a-ahcy / BL21 expression strain was 1 mmol / L of IPTG induces 6h expression of protein;

[0045] (2) Collect E.coliBL21 cells by centrifugation, wash twice with PBS, centrifuge at 5000rmp / min for 15min, and collect the bacterial liquid;

[0046] (3) Add ice-precooled 50mmol / L Tris-HCl (pH8.0) and 2mmol / L EDTA, wash the bacteria solution once, the total volume of added Tris-HCl and EDTA is 1 / of the bacteria solution volume 5;

[0047] (4) Collect the bacterial liquid by centrifugation, resuspend in ice-precooled 10mmol / L Tris-HCl (pH8.0), the volume of Tris-HCl is 1 / 10 of the bacterial liquid volume, add lysozyme to 0.1mg / L ml, then a...

Embodiment 2 6

[0060] Embodiment 2 Preparation of hexasaccharide-modified ahcy protein (ahcymhe)

[0061] Step 1. Mix hexasaccharide and sodium periodate at a molar ratio of 1:1 at 4°C, and react in PBS with pH 6.0 for 1 hour;

[0062] Step 2. At room temperature, add EDC (water-soluble carbodiimide) and NHS (N-hydroxysuccinimide ) reaction 6h;

[0063] Step 3. At room temperature, according to the molar ratio of ahcy protein to hexasaccharide of 1:1000, add ahcy protein to the reaction system obtained in step 2 to react overnight;

[0064] Step 4. Dialyze in a carbonate solution of 50 mmol / L and pH 8.0 to 9.6 at 4°C for 5 hours;

[0065] Step 5. Dialyze in 50 mmol / L PBS with pH 7.0-8.0 for one day at 4° C. to obtain hexasaccharide-modified ahcy protein (ahcymhe).

[0066] The ratio of the reaction between hexasaccharide and ahcy protein is confirmed by the following method: use the BCA kit to measure the content of ahcy protein in the reaction product and determine the amount of substanc...

Embodiment 3 6

[0067] Embodiment 3 Preparation of six kinds of oligosaccharide modified proteins

[0068] According to the steps of Example 2, six kinds of oligosaccharide modified proteins were prepared, i.e. monosaccharide modified ahcy protein (ahcym), disaccharide modified ahcy protein (ahcymbi), trisaccharide modified ahcy protein (ahcymtr), tetrasaccharide modified ahcy protein (ahcymte ), pentasaccharide modified ahcy protein (ahcympe), hexasaccharide modified ahcy protein (ahcymhe).

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Abstract

The invention provides a method for improving S-adenosyl-L-cysteine hydrolase immunogenicity and hexasaccharide modified ahcy protein, relates to the field of protein immunogenicity, aiming at providing better vaccine candidate antigens for Brucella. The method comprises the steps of mixing hexasaccharide and sodium periodate at 4 DEG C in a PBS of which the pH is 6.0 according to the molar ratio of 1 to 1, and reacting for 1h; adding EDC and NHS at normal temperature into a system according to the molar ratio of 1 to 1, and reacting for 6h; adding ahcy protein into the system at the normal temperature according to the molar ratio of 1 to 10000 of the ahcy protein to the hexasaccharide, and reacting overnight; dialyzing at 4 DEG C for 5h in a carbonate solution of which the volume is 50mmol/L and the pH is 8.0 to 9.6, and then dialyzing for one day in the PBS of which the volume is 50mmol/L and the pH is 7.0 to 8.0. A stimulation proliferation index, an antibody titer, the amount of IFN-gamma and IL-2 generated by stimulating mice spleen lymphocyte of the obtained hexasaccharide modified ahcy protein are greatly improved.

Description

technical field [0001] The invention relates to the technical field of protein immunogenicity, in particular to a method for improving the immunogenicity of S-adenosyl-L-cysteine ​​hydrolase and hexasaccharide modified ahcy protein. Background technique [0002] S-adenosyl-L-cysteinehydrolase (S-adenosyl-L-homocysteinehydrolase, ahcy) is an immunogenic protein of Brucella and a candidate antigen for subunit vaccines against Brucella. Rutella has better immune protection. [0003] Brucella is an intracellular parasite. At present, there is no ideal treatment method and drug. Vaccine prevention is the only effective way to prevent and control the disease. The immune mechanism of Brucella is not yet fully understood, but both humoral immune response and cellular immune response play a role in the elimination of Brucella, but the cellular immune response is the most important immunity to eliminate intracellular parasites mechanism. Brucella live attenuated vaccines and some p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14A61K39/10A61P31/04
CPCA61K39/00C12N9/14
Inventor 王秀然许春晓卢天成王晓龙王浩王岩李海燕李校堃孙喆李晓薇孙天旭王法微杨晶王艳芳刘秀明付玉芹
Owner JILIN AGRICULTURAL UNIV
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