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
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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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