CN112972673B - PLGA-PEG-Poly I: preparation of C nano-particles and application thereof in tuberculosis subunit vaccine - Google Patents
PLGA-PEG-Poly I: preparation of C nano-particles and application thereof in tuberculosis subunit vaccine Download PDFInfo
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Abstract
The invention discloses PLGA-PEG-PolyI: the preparation method of the C nano-particles comprises the steps of firstly using a chemical synthesis method to make PLGA and NH 2 PEG forms PLGA-PEG copolymer, then two mycobacterium tuberculosis fusion proteins ESAT6-Rv2626C and Mtb10.4-HspX (abbreviated as M4) constructed in the experiment are wrapped in the nano-particles by a multiple emulsion solvent volatilization method (W/O/W), and then polydopamine and PolyI: C are used for modification to form PLGA-PEG-PolyI wrapping the mycobacterium tuberculosis fusion protein M4: c (NP for short) nanoparticle tuberculosis subunit vaccine NP/M4. After a mouse is immunized by adopting the nanoparticle tuberculosis subunit vaccine provided by the invention, the level of IFN-gamma and IL-2 secreted by spleen lymphocytes is obviously higher than that of a PBS group and a BCG group after the NP/M4 group is stimulated by HspX protein and Rv2626c protein, and the result shows that the NP/M4 group can generate IFN-gamma and IL-2 with higher levels; in addition, the HpsX and Rv2626c specific IgG and sIgA antibody levels were significantly higher in the NP/M4 group than in the PBS, BCG and fusion protein M4 group. PLGA-PEG-PolyI: the C nano-particles can assist antigens to induce cellular immunity and humoral immunity, and are ideal adjuvants for tuberculosis subunit vaccines.
Description
Technical Field
The invention relates to the field of biological engineering, in particular to a method for constructing a novel vaccine by applying chemical modification to construct a nanoparticle adjuvant.
Background
Tuberculosis is a serious long-term health hazard to human beingsOne of the diseases, nearly 1/3 of all people worldwide have been infected with Mycobacterium Tuberculosis (Mtb) [1] Some tuberculosis infected persons will develop into tuberculosis patients within about 2-5 years, and the rest latent infected persons are likely to form tuberculosis. China is one of the countries with high tuberculosis burden, and about 13 million people die each year due to tuberculosis.
Currently, vaccination with Bacillus Calmette-guerin (BCG) is an important measure for effective prevention of tuberculosis. However, different studies have shown that BCG is not protective in different populations, especially with protective titers in the range of 0-80% for adult tuberculosis. Therefore, the development and development of comprehensive and efficient antitubercular vaccines are important ways to control tuberculosis. At present, the immune protection mechanism of the anti-tuberculosis is not clear [2] Antigen-specific CD4 is generally considered + Th1 cells and CD8 + T cells play an important role in preventing Mtb infection. Th1 type cytokines such as IFN-. Gamma.and IL-2 are currently important indicators for assessing immunoprotection [3] . There is no doubt that the T cell mediated immune response is critical to eliminating Mtb infection. Furthermore, antigen-specific antibodies have recently received increased attention [4] . Busch M et al showed that IgA prevented the invasion of Mtb cells in vitro [5] . Kozakiewicz L et al also found that B cell depletion resulted in increased Mtb load in non-human primates infected with Mtb [6] . Therefore, an effective vaccine should elicit both cellular and humoral immunity.
Tuberculosis vaccines currently available for research are in the form of: subunit vaccines, recombinant BCG vaccines, attenuated live vaccines, and the like. The subunit vaccine also comprises protein vaccine, DNA vaccine and vaccine using virus as carrier, wherein the protein vaccine has the advantages of definite components, safe use and the like, and is easy to be accepted by people [7] . The protein subunit vaccine combined with the adjuvant has wide application prospect. However, the currently used adjuvant in clinical use is an aluminum adjuvant, which mainly induces a Th 2-type humoral immune response [8] . In order to improve the protective effect of tuberculosis vaccines, it is important to research novel adjuvants which can induce Th1 type cellular immunity. At present, some newThe liposomal adjuvants AS01 and CAF01 that induce cellular immunity have entered clinical trials. The core component of CAF01 is cationic liposome dimethyl dioctadecyl ammonium bromide (DDA), which can not only improve antigen presenting efficiency and protect antigen from being degraded in vivo, but also induce dendritic cell maturation, thereby enhancing immune effect [9] 。
DP adjuvant consisting of DDA and Poly I: C was previously prepared in our laboratory and proved to have good adjuvant activation and protective effects by animal experiments [10] . A large number of studies show that mucosal vaccination has a preventive effect on tuberculosis [11 , 12] . However, DDA is difficult to use for mucosal immunization [13,14] Therefore, there is an urgent need to develop an adjuvant with high biosecurity to stimulate mucosal immune response.
Polylactic-co-glycolic acid (PLGA) is a polymer with high biocompatibility and biodegradability. PLGA has been officially accepted and recorded by the FDA as a pharmaceutical adjuvant due to its excellent safety [15] . It has been demonstrated that PLGA-based nanoparticles exhibit sustained slow release properties. However, PLGA nanoparticles have limited applications due to their strong hydrophobicity and poor stability [16] . Polyethylene Glycol (PEG) is an FDA-approved hydrophilic polymer, and can be stably modified on PLGA by chemical bonds to increase the hydrophilicity of PLGA [17] . In addition, some researches show that the PEG modified PLGA nano-particles can stimulate antigens to generate cross reaction and improve the immune effect of the antigens [18] . Toll-like receptor 3 (Toll-like-receptor 3, tlrr 3) is one of the TLR family members and plays an important role in pathogen recognition and innate immune activation. Poly I: c is a synthetic double-stranded RNA, can be used as agonist of TLR3 receptor, and can induce natural immunity and adaptive immunity [19] . Poly I was observed: the C combined adjuvant can promote rhesus monkey to generate stronger protective Th1 immune response and high-titer antigen-specific IgG antibody response to plasmodium falciparum circumsporozoite protein (CSP). DDA and Poly I: binding of CThe immune protection effect of the tuberculosis subunit vaccine is also improved. The research prepares a novel tuberculosis nanoparticle fusion protein subunit vaccine, researches the specific immunogenicity of a nasal cavity immunized mouse, and compares the specific immunogenicity with a BCG group, a fusion protein M4 group and a DP/M4 group.
Reference to the literature
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[2]PaiM,BehrMA,DowdyD,DhedaK,Divangahi M,Boehme CC,GinsbergA,Swaminathan S,SpigelmanM,Getahun H,Menzies D,Raviglione M.Tuberculosis.Nat Rev Dis Primers.2016Oct 27；2:16076.doi:10.1038/nrdp.2016.76.PMID:27784885.
[3]Ryndak MB,Laal S.Mycobacterium tuberculosis Primary Infection and Dissemination:A Critical Role for AlveolarEpithelial Cells.Front Cell InfectMicrobiol.2019Aug 21；9:299.doi:10.3389/fcimb.2019.00299.PMID:31497538；PMCID:PMC6712944.
[4]Sia JK,Rengarajan J.Immunology of Mycobacterium tuberculosis Infections.Microbiol Spectr.2019Jul；7(4):10.1128/microbiolspec.GPP3-0022-2018.doi:10.1128/microbiolspec.GPP3-0022-2018.PMID:31298204；PMCID:PMC6636855.
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C,Mayer D,Zenk SF,Muche R,Lange C,Bloom BR,Modlin RL,Stenger S；TBornotTB Network.Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.Am J Respir Crit Care Med.2016Aug 1；194(3):345-55.doi:10.1164/rccm.201509-1746OC.PMID:26882070；PMCID:PMC5441105.
[6]Kozakiewicz L,Phuah J,Flynn J,Chan J.The role of B cells and humoral immunity in Mycobacteriumtuberculosis infection.Adv Exp Med Biol.2013；783:225-50.doi:10.1007/978-1-4614-6111-1_12.PMID:23468112；PMCID:PMC4184189.
[7]Bertholet S,Ireton GC,Ordway DJ,Windish HP,Pine SO,Kahn M,Phan T,Orme IM,Vedvick TS,BaldwinSL,Coler RN,Reed SG.A defined tuberculosis vaccine candidate boosts BCG and protects againstmultidrug-resistant Mycobacterium tuberculosis.Sci Transl Med.2010 Oct 13；2(53):53ra74.doi:10.1126/scitranslmed.3001094.PMID:20944089；PMCID:PMC3110937
[8]Principi N,Esposito S.Aluminum in vaccines:Does it create a safety problemVaccine.2018 Sep18；36(39):5825-5831.doi:10.1016/j.vaccine.2018.08.036.Epub 2018 Aug 20.PMID:30139653.
[9]Nordly P,Agger EM,Andersen P,Nielsen HM,Foged C.Incorporation of the TLR4 agonist monophosphoryllipid A into the bilayer ofDDA/TDB liposomes:physico-chemical characterization and induction of CD8+T-cellresponses in vivo.Pharm Res.2011 Mar；28(3):553-62.doi:10.1007/s11095-010-0301-9.Epub 2010 Nov 2.PMID:21042837.
[10]X.Liu,Z.Da,Y.Wang,H.Niu,R.Li,H.Yu,S.He,M.Guo,Y.Wang,Y.Luo,X.Ma,B.Zhu,A novelliposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediatedimmunity and high protective efficacy in mice,Vaccine 34(11)(2016)1370-8.
[11]A.Rodriguez,A.Tjarnlund,J.Ivanji,M.Singh,I.Garcia,A.Williams,P.D.Marsh,M.Troye-Blomberg,C.Fernandez,Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increasedsusceptibility to intranasal infection with Mycobacterium bovis BCG,Vaccine 23(20)(2005)2565-72.
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[15]Allahyari M,Mohit E.Peptide/protein vaccine delivery system based on PLGA particles.Hum VaccinImmunother.2016 Mar 3；12(3):806-28.doi:10.1080/21645515.2015.1102804.PMID:26513024；PMCID:PMC4964737.
[16]Kole S,Qadiri SSN,Shin SM,Kim WS,Lee J,Jung SJ.PLGA encapsulated inactivated-viral vaccine:Formulation and evaluation of its protective efficacy against viral haemorrhagic septicaemia virus(VHSV)infection in olive flounder(Paralichthys olivaceus)vaccinated by mucosal delivery routes.Vaccine.2019 Feb8；37(7):973-983.doi:10.1016/j.vaccine.2018.12.063.Epub 2019 Jan 17.PMID:30661835.
[17]Cruz LJ,Tacken PJ,Fokkink R,Figdor CG.The influence of PEG chain length and targeting moiety onantibody-mediated delivery of nanoparticle vaccines to human dendritic cells.Biomaterials.2011Oct；32(28):6791-803.doi:10.1016/j.biomaterials.2011.04.082.Epub 2011 Jul 2.PMID:21724247.
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Disclosure of Invention
The invention provides a method for preparing PLGA-PEG-Poly I by combining PLGA and Poly I: c methods of novel nanoparticle adjuvants. The novel adjuvant provided by the invention and the M4 fusion protein prepared in the laboratory are combined to construct the tuberculosis subunit vaccine, and the vaccine can effectively induce the specific cell and humoral immune response reaction of tuberculosis antigen in a mouse body. The nano-particle is expected to become a novel tuberculosis vaccine adjuvant.
The technical scheme of the invention is as follows:
preparation of np nanoparticles:
(1) Dissolving 0.02mmol of PLGA in 8ml of Dichloromethane (DCM), adding 0.2mmol of 1-methyl- (3-dimethylaminopropyl) carbodiimide (N- (3-dimethylamino propyl) -N-ethylsuccinimide hydrochloride, EDC) and 0.2mmol of N-hydroxysuccinimide (N-Hydroxyscimide, NHS) after complete dissolution, introducing nitrogen, and slowly stirring for reaction for 24h; after the reaction is finished, precipitating the reactant by using glacial ethyl ether, and then repeatedly washing the product by using a methanol solution; redissolving with DCM and finally re-precipitating with glacial ethyl ether to give the product, and freeze-drying at low temperature.
(2)NH 2 -synthesis of PEG and activated PLGA:
activated 0.01mmol PLGA was dissolved in 3ml DCM, 0.03mmol NH 2 PEG dissolved in 1ml DCM; after complete dissolution, NH is added 2 PEG was added dropwise to the PLGA solution, 0.05mmol of EDC was added, 0.05mmol of 4-lutidine (DMAP) was added, and 50. Mu.l of triethylamine was added; slowly introducing nitrogen into the system, sealing, slowly stirring at a constant speed, and reacting for 24h; after the reaction is finished, precipitating the reactant by using glacial ethyl ether, and repeatedly washing the product by using a methanol solution; redissolving with DCM, and precipitating with glacial ethyl ether to obtain the product, and freeze drying at low temperature.
(3) The preparation process of the protein-nanosphere comprises the following steps:
250mg of PLGA-PEG were dissolved in 5mlIn DCM, 4mg of the fusion protein was dissolved in 1ml of PBS; slowly pouring PLGA-PEG solution into the fusion protein solution, and preparing colostrum (colostrum 600W,2min, start 3s, stop 3 s) with ultrasonication instrument; after the preparation of colostrum, mixing with 15 volumes of 2% polyvinyl alcohol (PVA) solution, and re-emulsifying in a homogenizer (300bar, 4 min); evaporate DCM with overnight stirring, then with ddH 2 And washing for three times.
(4) Modification process of nanospheres:
the freshly prepared nanoparticles were dissolved in 5ml of Tris buffer (salt concentration: 10mM, pH 8.5); at the same time, dopamine hydrochloride (pD) and Poly I: C were dissolved in Tris buffer solution to give final concentrations of 0.5mg/ml and 0.2mg/ml, respectively. Closing the cover at room temperature, stirring for 2.5h, centrifuging at 14000rpm, collecting the precipitate, and obtaining the polydopamine and Poly I: C modified PLGA nano-particles.
Application of NP nano-particles in tuberculosis subunit vaccine
The mycobacterium tuberculosis fusion protein-based subunit vaccine requires adjuvant to assist in inducing cellular immune response; the PLGA nano-particles are a novel vaccine adjuvant system, have the advantages of better biocompatibility, high safety and the like, and the stability and the adjuvant effect of the PLGA nano-particles need to be further improved. The invention aims to develop a novel PLGA nano-carrier to improve the efficacy of a tuberculosis subunit vaccine. The invention firstly chemically synthesizes PLGA-NH 2 And (2) copolymer, then coating two mycobacterium tuberculosis fusion proteins ESAT6-Rv2626C and Mtb10.4-HspX (expressed by M4) constructed in the experiment in nanoparticles by a double emulsion solvent volatilization method, and further modifying by using polydopamine and Poly I: C to form PLGA-PEG-Poly I coating the mycobacterium tuberculosis fusion protein antigen (M4): c nanoparticles, abbreviated M4/NP. The nasal drop immunization is carried out on C57BL/6 female mice with the age of 6-8 weeks, the priming interval is 8 weeks, and the immunization is carried out for 2 times. The immunogenicity of M4/NP was assessed 6 weeks after the last immunization by measuring the secretion levels of IFN-. Gamma.and IL-2 from spleen lymphocytes and measuring the titers of sIgA, serum IgG, igG1 and IgG2c in alveolar lavage fluid.
The novel nanoparticle tuberculosis subunit vaccine provided by the invention is adopted to immunize C57BL/6 female mice. The results of five groups of results of PBS, BCG, M4, NP/M4 and DP/M4 are compared, and the results are displayed; after the NP/M4 group is stimulated by HspX protein, rv2626c protein, the level of IFN-gamma and IL-2 secreted by spleen lymphocytes is obviously higher than that of PBS, BCG and M4 groups, the statistical difference is obvious (p is less than 0.001), and the NP/M4 group can generate higher level of IFN-gamma and IL-2. The levels of specific antibodies IgG and sIgA generated against the antigens HpsX and Rv2626c protein were significantly higher than those of PBS, BCG and M4 groups.
Drawings
FIG. 1 shows NMR of PLGA (a) and PLGA-PEG copolymer (b) 1 And H, spectrum.
FIG. 2 is an infrared spectrum of PLGA (a) and PLGA-PEG copolymer (b).
FIG. 3 is a graph of PLGA-PEG nanoparticle size and Zeta potential simulation curve.
(A) The particle size of PLGA-PEG nanoparticles; and (B) a Zeta potential simulation curve.
Fig. 4 is a Zeta potential detection graph of nanoparticles at different stages.
FIG. 5 is a scanning electron micrograph of PLGA-PEG-poly (I: C) nanoparticles.
FIG. 6 is a graph of the effect of NP nanoparticles on THP-1 cells.
After incubating the THP-1 cells with different concentrations of NP for 24 hours and 48 hours, the cell viability of the THP-1 cells was determined by the MTT method.
FIG. 7 is a graph showing the expression detection of antigen-specific IL-2 and IFN-. Gamma.at week 6 after vaccine immunization.
Detection of CD 46 weeks after last vaccine immunization + T cells and CD8 + The ability of T cells to produce IL-2 and IFN- γ (A-D). (E) IFN-. Gamma.content in lymphocyte culture supernatant. means + -SD,. P<0.05，**p<0.01，***p<0.001。
FIG. 8 is a graph of antigen-specific sIgA detection in alveolar lavage fluid at week 6 after the final immunization of the vaccine.
Antigen-specific sIgA antibody levels in alveolar lavage fluid at week 6 after the last immunization of the vaccine. (A) Amount of antigen-specific sIgA detected in alveolar lavage fluid, 1:1 and 1:3 are dilutions of alveolar lavage fluid, the level of Rv2626 c-specific sIgA in NP/M4 group is significantly higher than that in PBS, BCG and M4 groups. (ii) a (B) Reactivity of different dilutions of alveolar lavage (1:4 to 1, 128, 100 liters/well) to v2626 c. means ± SD, # p <0.05, # p <0.01, # p <0.001.
Detailed Description
Example 1PLGA-PEG-Poly I: preparation of C nanoparticles
1. Dissolving 250mg PLGA-PEG copolymer in 5ml DCM, dissolving 4mg fusion protein in 1ml PBS, colostrum 600W,2min, starting for 3s, stopping for 3s, and multiple emulsion in a homogenizer for 300Bar,4min;
2. DCM was evaporated overnight (magnetic stirrer, 400 rpm) before using ddH 2 And O, washing to remove the unencapsulated protein and the redundant PVA.
3. The freshly prepared nanoparticles were dissolved in 5ml of Tris buffer (salt concentration: 10mM, pH 8.5); at the same time, pD and Poly I: C were dissolved in Tris buffer solutions to give final concentrations of 0.5mg/ml and 0.2mg/ml, respectively. Closing the cover at room temperature, stirring for 2.5h, centrifuging at 14000rpm, collecting the precipitate, and obtaining the polydopamine and Poly I: C modified PLGA nano-particles.
Example 2 Properties and characterization of nanoparticles
Nuclear magnetic hydrogen spectrum determination of PLGA-PEG copolymer
And (3) verifying by using a 600MHz liquid superconducting nuclear magnetic resonance spectrometer. As a result, it was found that PLGA and NH were present 2 Characteristic absorption peaks of PEG, demonstrating the success of the coupling of the copolymer, the nuclear magnetic hydrogen spectrum is shown in FIG. 1. Typical absorption peaks for PLGA were detected at 1.53,4.83 and 5.23ppm, and we detected NH in the copolymer at 3.54 and 3.42ppm 2 Characteristic absorption peak of-PEG corresponding to-OCH 3 and-CH 2 -a group. These observations flank successful coupling of PLGA and PEG. (FIG. 1)
Determination of Infrared Spectrum of PLGA-PEG copolymer
The formation of amide bonds in the polymer was also confirmed using fourier transform infrared spectroscopy, as shown in fig. 2. Respectively at 1561.3cm -1 (ν C＝O ) And 1646.0cm -1 (β NH ) The absorption peak confirms PLGA and NH 2 Coupling of PEG. And at 1093.9cm -1 V can be observed C-N Absorption peak, description of NH 2 -PEG is present. The absorption peak of PLGA is 2950.3cm -1 And 2995.7cm -1 To (3). (FIG. 2)
Particle size distribution of PLGA-PEG nanoparticles
The particle size distribution of the protein-encapsulated PLGA-PEG nanoparticles was determined using a Brookhaven Instrument. The average diameter of the nano particles is 200.21 +/-1.07 nm (figure 3A), and the detection result well simulates a sine function (figure 3B), so that the detection result is true and reliable.
4. Potential detection of nanoparticles at different stages
And respectively measuring the zeta potential of PLGA-PEG nano-particles, the nano-particles after PLGA-PEG modified dopamine and Poly I: C coated by the PLGA-PEG modified dopamine by adopting a Brookhaven Instrument. The zeta potential of the nanoparticles was found to decrease from 20mV to about-24 mV after dopamine modification, and to decrease to about-33 mV after the last modification of Poly I: C, indicating that Poly I: C was successfully modified. (FIG. 4)
5. Nanoparticle drug loading and encapsulation efficiency
The amount of protein contained in the supernatant is detected after centrifugation, and the amount of unencapsulated protein is subtracted from the total amount of protein, so that the actual protein encapsulation rate and drug loading rate of the nanoparticles are calculated. Finally obtaining; the protein encapsulation efficiency is 73.25 percent, and the drug loading rate is 6.81 percent.
6. Scanning electron microscope
The surface morphology of the nanoparticles is observed by adopting a scanning electron microscope SEM (figure 5), and the prepared nanoparticles are circular and have good uniformity.
Example 3 in vitro toxicity testing of PLGA-PEG-Poly (I: C) nanoparticles
The cytotoxicity of nanoparticle vaccines was tested by MTT assay using THP-1 cells. First 100. Mu.L of serially diluted nanoparticle samples (from 6.25. Mu.g/mL to 800. Mu.g/mL) were added to a 96-well plate, followed by 1X 10 density differentiation into macrophages with phorbol esters 4 THP-1 cells from cells/well, plates were incubated for 24h to 48h, then 20. Mu.l addedLMTT (5 mg/mL). After 4 hours of incubation, 100 μ L DMSO was added and absorbance was measured at 490nm using a microplate reader. (FIG. 6)
Example 4 vaccine Immunity assay
1. Experimental materials: fusion protein (M4), hspX protein, rv2626C protein, poly (I: C), BCG, phosphate buffered saline PBS.
2. Experimental animals: c57BL/6 mice
3. Experimental animals were grouped (total five groups):
A:PBS
B:BCG
C:M4
D:NP/M4
E:DP/M4
4. immunizing animals:
BCG group was injected once at week 0 (5X 10) 6 CFU,100 μ L). All 3M 4 vaccine groups (M4, DP/M4 and NP/M4) were 100ul by nasal drip vaccination with a M4 content of 10ug. PBS control group (PBS only) and M4 group were immunized twice by nasal drip at week 0 and 8, respectively.
5. Method for measuring immunological index
(1) Cellular immunoassay
(1) ELISA method for detecting expression of mouse spleen lymphocyte IFN-gamma
And (3) performing aseptic separation on spleen lymphocytes after the mice are immunized for 6 weeks for the last time, co-incubating HspX protein, rv2626c protein and spleen lymphocytes in a 24-hole cell culture plate for 68 hours, collecting cell culture supernatant, and detecting the expression of IFN-gamma of the spleen lymphocytes after stimulation of the HspX protein and the Rv2626c protein by an ELISA method.
The method comprises the following specific steps: the spleen was removed aseptically, ground, filtered through a 200 mesh nylon mesh, and the lymphocytes were separated with a lymphocyte separation medium. The isolated lymphocytes were added to a 24-well cell culture plate to a final concentration of 5X 10 6 /well, stimulation with HspX protein (5 ug/ml) and Rv2626c protein (5 ug/ml), respectively. At 37 ℃ 5% CO 2 After co-incubation under conditions for 68 hours, cell culture supernatant was collected. Adding cell culture supernatant into 96-well ELISA plate (100 u 1/well), sequentially adding reagents such as detection antibody according to ELISA specification, washing plate, washing,Developing, terminating the reaction, reading the plate by a microplate reader, and determining the content of IFN-gamma (pg/ml) according to a standard curve.
The results show that: comparing the results of the PBS, BCG, M4, NP/M4 and DP/M4 groups, the level of IFN-gamma secretion of spleen lymphocytes after the stimulation of HspX protein and Rv2626c protein in the NP/M4 group is obviously higher than that of the PBS, BCG and M4 group, which indicates that the NP/M4 group can produce higher level of IFN-gamma. (the results are shown in FIG. 7E)
(2) Intracellular cytokine staining method for detecting expression of IFN-gamma and IL-2 of spleen lymphocytes of mice
Performing aseptic separation on spleen lymphocytes after mice are immunized for 6 weeks for the last time, performing in-vitro stimulation on Rv2626c antigen (5 ug/ml) and HspX antigen (5 ug/ml) for 4h, adding 1ul protein transport inhibitor, incubating for 8h, and detecting CD4 + T cells and CD8 + Secretion levels of IFN-gamma and IL-2 by T cells.
The results show that: PBS, BCG, M4, NP/M4, DP/M4 groups were compared. NP/M4 group CD4 after stimulation by HspX protein, rv2626c protein + T and CD8 + The IL-2 secretion level of T cells is higher than that of the other four groups, CD4 + T and CD8 + T cell IFN-gamma secretion levels were all higher than in PBS, BCG and M4 groups. The result proves that the nanoparticle tuberculosis subunit protein vaccine can cause stronger cellular immune response. (the results are shown in FIGS. 7A-D)
(2) Humoral immune response
(1) The expression level of mouse serum IgG antibody was measured by ELISA.
HspX and Rv2626c (5 ug/ml) were coated onto 96-well plates separately and left overnight at 4 ℃; wash the plate 5 times with PBST solution 300u1/we 11; sealing with 5% skimmed milk powder, and washing. 100u of a 1-fold diluted serum sample was added and left at 37 ℃ for 1 hour. After washing the plates, 100u1/we11 of 1: a5000 dilution of goat anti-mouse IgG was left at 37 ℃ for lh. After washing the plate, 100u1/we11 TMB developing solution was added, and after development of color by reaction for 15 minutes in the dark at room temperature, 50u1/we11 stop solution (2M H) was added 2 SO 4 ) Terminating the reaction; OD was measured at 450 nm.
(2) And detecting the expression level of the sIgA antibody in the mouse alveolar lavage fluid by an ELISA method.
Respectively using HspX protein and Rv2626c proteinCoating 96-well plate (5 ug/ml) and standing overnight at 4 deg.C; wash the plate 5 times with PBST solution 300u1/we 11; sealing with 5% skimmed milk powder, and washing plate. HspX-coated alveolar lavage fluid is diluted by 1:1; the alveolar lavage fluid coated with the Rv2626c protein was diluted with 1:3 and left at 37 ℃ for 1h. After washing, 100u1/we11 of 1. After washing the plate, 100u1/we11 TMB developing solution was added, and after development of color by light-shielding reaction at room temperature for 15 minutes, 50u1/we11 stop solution (2M H) was added 2 SO 4 And (b) terminating the reaction; OD was measured at 450 nm.
The results show that: the results of PBS, BCG, M4, NP/M4 and DP/M4 groups are compared, and the antibody levels of IgG and sIgA specific to the HpsX and Rv2626c proteins of the NP/M4 group and the DP/M4 group are obviously higher than those of the PBS, BCG and M4 group.
The result shows that the nanoparticle tuberculosis subunit vaccine provided by the invention can stimulate an organism to generate specific antibodies of HspX and Rv2626c proteins, has stronger humoral immune response, and shows that after the fusion protein and the nanoparticles are combined for application, mucosal and whole body humoral and cellular immune response of the protein are obviously enhanced after nasal drip immunization. (the results are shown in FIGS. 8 and
watch 1)
TABLE 1 detection of specific antibody expression levels after vaccine immunization
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. Polydopamine, PLGA-PEG-Poly I: the preparation method of the C nano-particles comprises the following steps:
(1) Activated PLGA
(2) Synthesis of PLGA-PEG
(3) Preparation of protein-nanoparticles
(4) Modification of nanoparticles;
the step (1) is specifically as follows:
dissolving 0.02mmol of PLGA in 8ml of DCM, adding 0.2mmol of EDC and 0.2mmol of NHS after complete dissolution, introducing nitrogen, and slowly stirring for reaction for 24h; after the reaction is finished, precipitating the reactant by using ethyl acetate, and then repeatedly washing the product by using a methanol solution; redissolving with DCM, precipitating with glacial ethyl ether again to obtain product, and freeze drying at low temperature;
the step (2) is specifically as follows:
activated 0.01mmol PLGA was dissolved in 3ml DCM, 0.03mmol NH 2 PEG dissolved in 1ml DCM; after complete dissolution, NH is added 2 PEG is added into PLGA solution drop by drop, 0.05mmol of EDC is added, 0.05mmol of DMAP is added, and 50 mu l of triethylamine is added; slowly introducing nitrogen into the system, sealing, slowly stirring at a constant speed, and reacting for 24h; after the reaction is finished, precipitating the reactant by using glacial ethyl ether, and repeatedly washing the product by using a methanol solution; redissolving with DCM, precipitating with ethyl acetate again to obtain product, and freeze drying at low temperature;
the step (3) is specifically as follows:
250mg PLGA-PEG was dissolved in 5ml DCM and 4mg fusion protein was dissolved in 1ml PBS; slowly pouring PLGA-PEG solution into the fusion protein solution, and preparing colostrum with an ultrasonication instrument, wherein the colostrum is 600W,2min, 3s on and 3s off; mixing the prepared primary emulsion with 15 times of 2% PVA solution, and preparing multiple emulsion in a homogenizer, 300Bar,4min; evaporate DCM with overnight stirring, then with ddH 2 Washing for three times;
the step (4) is specifically as follows:
dissolving the newly prepared nanoparticles in 5ml of Tris buffer, 10mM of salt concentration and 8.5 pH; simultaneously, pD and Poly I: c are respectively dissolved in Tris buffer solution to lead the final concentration to be 0.5mg/ml and 0.2mg/ml respectively; closing the cover at room temperature, stirring the mixture for 2.5h, centrifuging the mixture at 14000rpm, collecting the precipitate to obtain polydopamine, poly I: c modified PLGA nanoparticles.
2. Polydopamine, PLGA-PEG-Poly I: and C, nano-particles.
3. The polydopamine, PLGA-PEG-Poly I of claim 2: the application of the C nano-particles in the preparation of tuberculosis subunit vaccines.
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