CN110368488B - Preparation and application of recombinant argF protein nanoparticles - Google Patents
Preparation and application of recombinant argF protein nanoparticles Download PDFInfo
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- CN110368488B CN110368488B CN201910665776.8A CN201910665776A CN110368488B CN 110368488 B CN110368488 B CN 110368488B CN 201910665776 A CN201910665776 A CN 201910665776A CN 110368488 B CN110368488 B CN 110368488B
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Abstract
The invention relates to a preparation method of recombinant argF protein nanoparticles, the prepared recombinant argF protein nanoparticles and application of the recombinant argF protein nanoparticles in preparation of a tubercle bacillus vaccine, wherein the preparation method comprises the following steps: dissolving the lyophilized recombinant protein argF powder in a PBS solution as an internal aqueous phase; weighing a proper amount of PLGA, dissolving the PLGA in ethyl acetate with a certain volume, and taking the PLGA as an oil phase for later use; preparing 1% PVA aqueous solution as an external water phase and 0.5% PVA aqueous solution as a diffusion phase; slowly dripping the inner water phase into the oil phase, and performing ultrasonic treatment under ice bath for a certain time to form primary emulsion; then, dropwise adding the formed colostrum into a certain volume of external water phase solution, and performing ultrasonic treatment for a certain time under ice bath to form multiple emulsion; dripping the formed multiple emulsion system into the diffusion phase, and placing the multiple emulsion system on a magnetic stirrer to ensure that the organic solvent is fully diffused into the diffusion phase, thereby solidifying the polymer emulsion droplets; and adding sterilized distilled water into the formed polymer emulsion droplets, and washing to obtain the recombinant argF protein nanoparticle emulsion. Compared with the traditional BCG immune control, the vaccine obtained by the recombinant argF protein nanoparticles prepared by the method can obviously improve the IgA (immunoglobulin A) level of mucosal immunity, reduce the tissue bacterium carrying amount and promote the BCG immune effect by using the argF nanoparticle vaccine.
Description
Technical Field
The invention belongs to the field of novel vaccine application, and particularly relates to application of argF nanoparticles in preparation of a tuberculosis prevention vaccine.
Background
Tuberculosis (TB) is a chronic, consumable zoonosis caused by bacteria of the Mycobacterium Tuberculosis Complex (MTBC). Bovine Tuberculosis (BTB) is mainly caused by mycobacterium bovis (m.bovis), but may also cause Tuberculosis in humans, and thus BTB also threatens human health. There is currently no effective vaccine to prevent BTB-induced tuberculosis, and there is an urgent need to develop new effective vaccines for preventing or as booster vaccines for BCG to control this disease which poses a global health threat.
argF has an indispensable role in the survival of m.bovis, one of the key factors in m.bovis survival, and argF also has an inconstant regulatory function in the innate immune response against m.bovis infection. Polylactic-co-glycolic acid (PLGA), PLGA is a degradable high molecular organic compound formed by random polymerization of lactic acid and glycolic acid. PLGA has been approved by the Food and Drug Administration (FDA) as a delivery vehicle for vaccines and drugs with non-toxic, good biocompatibility, and encapsulation and film-forming properties, and its vaccine formulation has the ability to deliver both antigen and immunopotentiator to the same dendritic cells or macrophages.
The argF mutant strain of m.bovis is an attenuated L-Arg auxotrophic mycobacterium strain that is impaired in growth within somatic macrophages, is unable to activate and proliferate, but retains the ability to start the immune system, is immunogenic, and is a suitable antigen for m.bovis vaccines. However, subunit vaccines do not have the inherent ability to deliver themselves to the appropriate site within the infected organism for optimal immune stimulation. Therefore, in order to ensure that vaccines are better able to exhibit both innate and adaptive immune responses, related studies have developed a number of alternative forms of vaccine delivery systems. The invention selects PLGA high molecular material to encapsulate argF protein to prepare the subunit nano-particles for preventing and treating M.bovis infection, and proves that the recombinant protein argF nano-vaccine has obvious enhanced BCG immunocompetence.
Disclosure of Invention
The invention aims to provide a novel tuberculosis nano vaccine, and particularly relates to an application of the novel tuberculosis nano vaccine in preparation of a tuberculosis vaccine. The invention strengthens immunity through BCG priming and argF nano vaccine, and then establishes a mouse infection model by using mycobacterium bovis to prove the immune activity of the vaccine.
In order to achieve the above object, the present invention provides a method for preparing recombinant argF protein nanoparticles, which comprises the following steps:
(1) dissolving the lyophilized recombinant protein argF powder in a PBS solution as an internal aqueous phase;
(2) weighing a proper amount of PLGA, dissolving the PLGA in ethyl acetate with a certain volume, and taking the PLGA as an oil phase for later use;
(3) preparing 1% PVA aqueous solution as an external water phase and 0.5% PVA aqueous solution as a diffusion phase;
(4) slowly dripping the inner water phase into the oil phase, and carrying out 60W power ultrasonic treatment for a certain time under ice bath to form primary emulsion;
(5) then, dropwise adding the primary emulsion formed in the step (4) into a certain volume of external water phase solution, and carrying out 100W power ultrasonic treatment for a certain time under ice bath to form multiple emulsion;
(6) dropwise adding the multiple emulsion system formed in the step (5) into the diffusion phase, placing the multiple emulsion system on a magnetic stirrer, and continuously stirring at 700rpm for 4 hours at room temperature to fully diffuse the organic solvent into the diffusion phase, so as to solidify polymer emulsion droplets;
(7) and (4) adding 6-10 times of sterilized distilled water into the polymer emulsion drops formed in the step (6), centrifuging at 5000rpm and 4 ℃ for 3min, and washing twice to obtain the recombinant argF protein nanoparticle emulsion.
Further, the ratio of the internal water phase to the oil phase in step (4) is 1: 9.
further, the average particle size of the recombinant argF protein nanoparticles is about 186.6nm, and the potential is-28.8 mV.
Further, the encapsulation efficiency of the recombinant argF protein nanoparticles is 76%.
The invention also provides a recombinant argF protein nanoparticle prepared according to the method, which is characterized in that the nanoparticle is obtained by coating PLGA with a recombinant argF antigen.
Further, the average particle size of the recombinant argF protein nanoparticles is about 186.6nm, and the potential is-28.8 mV.
Further, the encapsulation efficiency of the recombinant argF protein nanoparticles is 76%.
The invention finally provides the application of the recombinant argF protein nanoparticles in the preparation of a vaccine for preventing mycobacterium tuberculosis.
Further, the average particle size of the recombinant argF protein nanoparticles is about 186.6nm, and the potential is-28.8 mV.
Further, the encapsulation efficiency of the recombinant argF protein nanoparticles is 76%.
Compared with the traditional BCG immune control, the vaccine obtained by the recombinant argF protein nanoparticles prepared by the method can obviously improve the IgA (immunoglobulin A) level of mucosal immunity, reduce the tissue bacterium carrying amount and promote the BCG immune effect by using the argF nanoparticle vaccine.
Drawings
FIG. 1: scanning electron microscope images of the recombined argF nanoparticles;
FIG. 2: the recombinant argF nanoparticles affect the IgG and IgA levels of BCG immunized mice;
FIG. 3: the argF nanoparticles have an effect on the lung tissue bacterial load of BCG-immunized mice.
In fig. 2 and 3, P < 0.05, P < 0.01, P < 0.001.
Detailed Description
The embodiments are described in detail below with reference to the accompanying drawings.
Example 1: preparation method of recombinant protein argF nanoparticles
The specific operation steps are as follows:
(1) amplifying MB1684 gene of M.bovis, constructing a recombinant expression plasmid pProEx-MB1684, transforming the recombinant plasmid into BL21 competent cells to obtain recombinant bacteria, inducing the recombinant bacteria to express argF by IPTG, purifying the expressed argF by adopting an affinity chromatography, dialyzing to remove salt, and freeze-drying to obtain recombinant protein argF;
(2) dissolving the lyophilized recombinant protein argF powder in a PBS solution as an internal aqueous phase;
(3) weighing a proper amount of PLGA, dissolving the PLGA in ethyl acetate with a certain volume, and taking the PLGA as an oil phase for later use;
(4) preparing 1% PVA aqueous solution as an external water phase and 0.5% PVA aqueous solution as a diffusion phase;
(5) slowly dripping the inner water phase into a PLGA organic solvent, and carrying out 60W power ultrasonic treatment for a certain time under ice bath to form primary emulsion;
(6) then, dropwise adding the primary emulsion into a 1% PVA aqueous solution with a certain volume, and carrying out 100W power ultrasonic treatment for a certain time under ice bath to form multiple emulsion;
(7) dripping the multiple emulsion system into the diffusion phase, placing the multiple emulsion system on a magnetic stirrer, and continuously stirring the multiple emulsion system for 4 hours at the room temperature of 700rpm to ensure that the organic solvent is fully diffused into the diffusion phase, thereby solidifying polymer emulsion droplets;
(8) and adding 6-10 times of sterilized distilled water into the argF nano-particle emulsion, centrifuging at 5000rpm and 4 ℃ for 3min, and washing twice to obtain the argF nano-particle emulsion.
Example 2: nanoparticle characterization and morphology observation
The ratio of the internal water phase to the oil phase is 1: 9 characterization, particle size and potential were measured using a Malvern Zetasizer nano particle size potentiometer. The prepared argF nanoparticles had an average particle size of about 186.6nm and a potential of-28.8 mV. The calculated encapsulation efficiency of the nanoparticles was 76% after measurement using an ultraviolet spectrophotometer. The results of scanning electron microscopy showed that the argF nanoparticles were of uniform size and were spherical with a smooth surface (FIG. 1).
Example 3: evaluation of BCG immune effect enhanced by recombinant argF protein nanoparticles
The test animals were randomly divided into 5 groups of 9 mice each, and divided into a PBS group (not immunized but only challenged), a BCG control group (BCG + PBS), a BCG + argF group, a BCG + PBS-NP group (BCG + blank nanoparticles), and a BCG + argF-NP group (BCG + argF nanoparticles). Primary immunization with BCG (about 1X 10)6CFU/one), 4 weeks later, 1 st booster immunization with recombinant argF protein nanoparticles (argF protein nanoparticles dose 75 μ g/one) was performed by nasal drip, 2 weeks apart, for 3 times. 4 weeks after 3 rd immunization, 3 mice were randomly selected from each group for detection of relevant immune indexes. The remaining mice were treated by nasal drip using m.bovis (NTSE-2 strain) NTSE-2 strain for nasal drip challenge (about 1000 CFU/mouse), and sampled 4 weeks later for subsequent detection.
Example 4: effect of recombinant argF protein nanoparticles on IgA levels in BCG-immunized mice
After the BCG immunized mice, bronchoalveolar lavage fluid is taken to measure IgA content so as to evaluate the mucosal immunity level. As shown in FIG. 2, IgA levels in tracheal alveolar lavage fluid were significantly elevated after boost following BCG priming (P < 0.001). The argF nanoparticles can obviously promote the secretion of immunoglobulin IgA on the basis of BCG pre-immunization.
Example 5: effect of recombinant argF protein nanoparticles on lung tissue bacterial load of BCG-immunized mice
And (3) detecting the bacterial load of the lungs of the mice after the mice are infected by the bovis challenge for four weeks, wherein the results show that the bacterial load of the lungs of each treatment group is reduced to different degrees compared with a PBS group, and the bacterial load of the lungs of a PLGA no-load group (P is less than 0.05) and a nanoparticle group (P is less than 0.01) is obviously reduced compared with a BCG control group, so that the bacterial load of the lungs of the infected mice is obviously reduced by argF nanoparticles on the basis of BCG pre-immunization.
The present invention is not limited to the above embodiments, and any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. The application of the recombinant argF protein nanoparticles in preparing the tuberculosis prevention vaccine is characterized in that the preparation method of the recombinant argF protein nanoparticles specifically comprises the following operation steps:
(1) amplifying MB1684 gene of M.bovis, constructing a recombinant expression plasmid pProEx-MB1684, transforming the recombinant expression plasmid into BL21 competent cells to obtain recombinant bacteria, inducing the recombinant bacteria to express argF by IPTG, purifying the expressed argF by adopting an affinity chromatography, dialyzing to remove salt, and freeze-drying to obtain recombinant protein argF;
(2) dissolving the lyophilized powder of the recombinant protein argF in a PBS solution as an internal aqueous phase;
(3) weighing PLGA, dissolving in ethyl acetate, and taking the PLGA as an oil phase for later use;
(4) preparing 1% PVA aqueous solution as an external water phase and 0.5% PVA aqueous solution as a diffusion phase;
(5) slowly and dropwise adding the internal water phase into the oil phase, and ultrasonically forming colostrum under ice bath at 60W power, wherein the ratio of the internal water phase to the oil phase is 1: 9;
(6) then dropwise adding the colostrum formed in the step (5) into the external water phase solution, and carrying out 100W power ultrasonic treatment under ice bath to form multiple emulsion;
(7) dropwise adding the multiple emulsion system formed in the step (6) into the diffusion phase, placing the multiple emulsion system on a magnetic stirrer, and continuously stirring at 700rpm for 4 hours at room temperature to fully diffuse the organic solvent into the diffusion phase, so as to solidify polymer emulsion droplets;
(8) and (3) adding 6-10 times of sterilized distilled water into the emulsion formed in the step (7), centrifuging at 5000rpm and 4 ℃ for 3min, and washing twice to obtain the recombinant argF nano-particle emulsion, wherein the recombinant argF protein nano-particles are obtained by coating PLGA with recombinant argF antigen, the encapsulation rate is 76%, the average particle size is 186.6nm, the potential is-28.8 mV, and the recombinant argF protein nano-particles can enhance the immunocompetence of BCG.
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