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Method for preparing thermal stability vaccine based on calcium phosphate mineralization

A calcium phosphate mineralization and thermal stability technology, applied in the field of medicine and biotechnology, can solve the problem of low thermal stability of Streptococcus pneumoniae protein, stimulate humoral immunity and cellular immunity, improve thermal stability, improve subsidence effect

Inactive Publication Date: 2020-03-06
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above-mentioned shortcoming of prior art, the object of the present invention is to provide a kind of method based on calcium phosphate mineralization to prepare thermostable vaccine, be used to solve the thermal stability of Streptococcus pneumoniae protein in the prior art during transportation and cold storage. low stability issues

Method used

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  • Method for preparing thermal stability vaccine based on calcium phosphate mineralization
  • Method for preparing thermal stability vaccine based on calcium phosphate mineralization
  • Method for preparing thermal stability vaccine based on calcium phosphate mineralization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Expression, identification and purification of prokaryotic expression recombinant plasmids pET28a(+)-WT-PLY, pET28a(+)-ΔA146PLY and pET28a(+)-DnaJ in Escherichia coli

[0049] 1. The recombinant plasmids pET28a(+)-WT-PLY, pET28a(+)-ΔA146PLY and pET28a(+)-DnaJ were transformed into host bacteria BL21(DE3):

[0050] Add 10 μL of the recombinant plasmid to 200 μL of competent bacteria BL21(DE3), mix well, ice-water bath for 30 min; heat shock at 42°C for 90 s, ice-water bath for 2 min; add 800 μL LB medium, culture at 37°C×180rpm for 60min recovery; 3000rpm Centrifuge for ×5min, leave 200 μL of supernatant mixed with bacterial liquid, and spread it on LK plate; after incubation at 37°C for 13h, single clone colonies are picked.

[0051] 2. IPTG induces the massive expression of pET28a(+)-WT-PLY, pET28a(+)-ΔA146PLY and pET28a(+)-DnaJ:

[0052] E.coli BL21-WT-PLY and E.coli BL21-ΔA146PLY strains were activated in 5 mL of LB liquid medium containing 50 ng / mL kanamycin (kana)...

Embodiment 2

[0063] In situ calcium phosphate mineralization and biological characteristics analysis of recombinant proteins WT-PLY, ΔA146PLY and DnaJ

[0064] 1. In situ calcium phosphate mineralization of recombinant proteins WT-PLY, ΔA146PLY and DnaJ

[0065] 360 μg proteins WT-PLY, ΔA146PLY and DnaJ were mixed with 8.8 μmol CaCl, respectively. 2 , 8.8 μmol MgCl 2 and 5.28 μmol Na 2 HPO 4 / NaH 2 PO 4 (pH=7.5) buffers were mixed together and the remaining volume was ddH 2 O (pH=7.5) was made up to 1 mL. The mineralization system and the magnetic beads treated with DEPC were placed in a beaker treated with DEPC, and stirred at 4 °C for 4 h under the action of a magnetic stirrer before use. Take 40 μL of the mineralized suspension respectively, centrifuge at 4°C*3000rpm*5min, separate the supernatant and the precipitate, add 10 μL of 5× loading buffer to 40 μL of supernatant, add 50 μL of 1× loading buffer to the precipitate, boil in boiling water for 10 min, and take 20 μL of sampl...

Embodiment 3

[0080] Analysis of proteinase K degradation resistance of recombinant proteins WT-PLY, ΔA146PLY and DnaJ and their mineralized nanoparticles

[0081] Take 25 μL of WT-PLY protein and mineralized WT-PLY@CaP suspension, and treat them with 15 μL of 40 μg / mL proteinase K for 0, 1, 5, 10, and 15 min respectively; take 25 μL of ΔA146PLY protein and ΔA146PLY@CaP with 15 μg / mL 15 μL of proteinase K was treated for 0, 1, 5, 10, and 15 min, respectively; 25 μL of DnaJ protein and DnaJ@CaP were treated with 15 μL of proteinase K at 10 μg / mL, respectively, for 0, 1, 5, 10, and 15 min. Add 1 μL PMSF to stop the enzymatic hydrolysis reaction, add 10 μL 5×loading buffer, boil in boiling water for 10 min, take 20 μL samples into the sample wells of SDS-PAGE gel, run the gel at 80 V for 40 min, run the gel at 120 V for 80 min; use Coomassie brilliant blue Dyeing with dye solution for 2min (heating on high heat in microwave oven for 2min), use ddH 2 O microwave heating for 10 min (repeated tw...

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Abstract

The invention belongs to the technical field of medical biology, and particularly discloses a method for preparing a thermal stability vaccine based on calcium phosphate mineralization. The preparation method comprises the following steps: sequentially adding a Ca <2+> ion-containing compound, a Mg <2+> ion-containing compound and a PO4 <3-> ion-containing compound into a protein solution to prepare an initial system, and then stirring the materials for reaction to prepare calcium phosphate coated protein which is the thermal stability vaccine. The protein nanoparticle with the calcium phosphate shell is successfully prepared through in-situ mineralization, and the thermal stability of the protein nanoparticle is improved on the basis of not changing the original spatial conformation and immunocompetence of protein; when the mineralized protein is used as the vaccine, the protein can still effectively resist the degradation effect of protease, and still has immunocompetence after heattreatment. The method has a great application prospect in the preparation of the heat-stable protein vaccine.

Description

technical field [0001] The invention relates to the technical field of medicine and biotechnology, in particular to a method for preparing a thermostable vaccine based on calcium phosphate mineralization, in particular to the improvement of biomineralization in improving pneumolysin WT-PLY and pneumolysin mutant ΔA146PLY And the application of Streptococcus pneumoniae heat shock protein DnaJ stability, especially the application of the mineralized nanoparticles of vaccine protein ΔA146PLY@CaP as a vaccine in Streptococcus pneumoniae infection after treatment at higher temperature. Background technique [0002] Streptococcus pneumoniae (S.pn) can cause severe invasive diseases such as pneumonia, meningitis, and sepsis, and can lead to serious sequelae such as deafness, paralysis, and mental retardation. It is also the primary pathogenic bacteria for lethal bacterial infection secondary to influenza. . Vaccines are the most economical and effective means of preventing infecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/51A61K47/02A61K39/09A61P31/04
CPCA61K9/5115A61K39/092A61P31/04
Inventor 张雪梅王婧瑶闵娅君胥文春
Owner CHONGQING MEDICAL UNIVERSITY
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