Preparation and application of recombinant protein CFP-10 nanoparticles

A CFP-10, recombinant protein technology, applied in the directions of non-active ingredients medical preparations, bacterial antigen components, capsule delivery, etc., can solve the problems of missing RD1 region and limited protective efficacy in adults, to improve IgA levels, promote BCG immune effect, the effect of reducing tissue load

Active Publication Date: 2019-10-22
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Bacillus Calmette-Guerin (BCG) is the most widely used vaccine to prevent tuberculosis. It has a significant protective effect on children, but has limited protective effect on adults. Compare

Method used

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  • Preparation and application of recombinant protein CFP-10 nanoparticles
  • Preparation and application of recombinant protein CFP-10 nanoparticles
  • Preparation and application of recombinant protein CFP-10 nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the preparation method of recombinant protein CFP-10 nano vaccine

[0027] Preparation of nanoparticles by double emulsion method: according to the CFP10 gene sequence of M. The plasmid was transformed into E.coli BL21(DE3) competent cells for induced expression, and the expressed protein was purified and freeze-dried. Dissolve the purified and lyophilized protein in PBS solution to form an internal aqueous phase, inject the internal aqueous phase into the organic phase, and ultrasonically form colostrum, inject the colostrum into 1% PVA solution, ultrasonically form double emulsion, and Pour milk into 0.5% PVA solution, stir for 3-4 hours, and volatile oil phase. The recombinant protein CFP-10 nanoparticles can be obtained by centrifugal washing and freeze-drying. The specific operation steps are as follows:

[0028] (1) configuration of polylactic acid-glycolic acid copolymer PLGA solution;

[0029] Dissolve polylactic acid-glycolic acid copolymer PL...

Embodiment 2

[0038] Example 2: Physical Characterization and Morphological Observation of Nanoparticles

[0039] The samples prepared with the ratio of internal water phase and oil phase at 1:9 were characterized, and the particle size and potential were measured using a Malvern Zetasizer nanometer particle size potentiometer. The prepared CFP10 nanoparticles have an average particle size of about 247nm and a potential of -28.8mV. After being measured by an ultraviolet spectrophotometer, the encapsulation efficiency of the nanoparticles is calculated to be 80.53%. The results of scanning electron microscopy showed that the size of CFP10 nanoparticles was relatively uniform, and it was spherical with a smooth surface ( figure 1 ).

Embodiment 3

[0040] Example 3: Evaluation of Recombinant CFP-10 Protein Nanoparticles Enhanced BCG Immune Effect

[0041] The experimental animals were randomly divided into 5 groups, 9 mice in each group, divided into PBS (no immunization but challenge), BCG control group (BCG+PBS), BCG+rCFP-10, BCG+PBS-NPs (BCG+blank nano microparticles), BCG+rCFP-10-NPs (BCG+recombinant protein nanoparticles). Use BCG for the first free (10 6 CFU / monkey), and 4 weeks later, the first booster immunization with nanoparticles (rCFP-10 protein dose was 50 μg / bird) was carried out by nasal drops, with an interval of 2 weeks, a total of 3 times. Four weeks after the third immunization, 3 mice were randomly selected from each group to detect relevant immune indicators. The rest were challenged with the NTSE-2 strain (1000 CFU / bird) by nasal drops, and samples were taken for follow-up detection after 4 weeks.

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Abstract

The invention provides a preparation method of recombinant protein CFP-10 nanoparticles and the application thereof in preparing tuberculosis prevention vaccines. The preparation method includes the steps of (1) configuration of a polylactic acid-glycolic acid copolymer PLGA solution; (2) configuration of a recombinant protein CFP-10 solution; (3) preparation of primary emulsion; (4) preparation of compound emulsion; (5) formation of the nanoparticles. The recombinant protein CFP-10 nanoparticles prepared by the preparation method are used for enhancing the immunity of BCG-immunized mice through intranasal drip, and it is confirmed that the recombinant protein CFP 10 nanoparticles have the effects of improving mucosal immunity level and promoting BCG immunity.

Description

technical field [0001] The invention belongs to the field of new vaccine applications, and in particular relates to the preparation of recombinant protein CFP-10 nanoparticles and its application in the preparation of tuberculosis prevention vaccines. Background technique [0002] Bovine tuberculosis is an important zoonotic disease, which endangers the development of breeding industry and public health security. The causative bacterium of bovine tuberculosis is Mycobacterium bovis, which can break through the interspecies barrier and spread between other mammals and humans. It has a wide range of hosts and affects many mammal groups. The current investigation and research shows that bovine tuberculosis in my country has the characteristics of easy recurrence and national distribution, and it is transmitted to humans through food and contact. Bacillus Calmette-Guerin (BCG) is the most widely used vaccine to prevent tuberculosis. It has a significant protective effect on chi...

Claims

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Application Information

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IPC IPC(8): A61K9/51A61K47/34A61K39/04A61P31/06
CPCA61K9/5153A61K39/04A61P31/06
Inventor 周向梅李淼煊梁正敏
Owner CHINA AGRI UNIV
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