CN104548081A - Fusion protein CTT3H contained tuberculosis subunit vaccine and vaccine adjuvants - Google Patents
Fusion protein CTT3H contained tuberculosis subunit vaccine and vaccine adjuvants Download PDFInfo
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Abstract
The invention discloses a fusion protein CTT3H contained tuberculosis subunit vaccine and vaccine adjuvants. The vaccine contains fusion protein CTT3H, and encoding genes of fusion protein CTT3H comprise CFP10, TB10.4, TB8.4, Rv3615c and HBHA (heparin-binding hemagglutinin adhesion) genes which are connected in series in the sequence of CFP10-TB10.4-TB8.4-Rv3615c-HBHA. The invention further provides three kinds of vaccine adjuvants. The vaccine adjuvants are matched with the fusion protein CTT3H contained tuberculosis subunit vaccine, so that a better immune response effect is provided. Fusion protein is prepared through selection of antigen genes and fusion sequences, the corresponding vaccine adjuvants are matched, the effect of prevention and treatment tuberculosis is good, and the safety is high.
Description
Technical field
The invention belongs to biomedicine field, more specifically, relate to a kind of tuberculosis subunit vaccine containing fusion rotein CTT3H and adjuvant.
Background technology
Vaccine lungy is uniquely prevented as current global range, bacillus calmette-guerin vaccine (BCG) can effectively prevent infant serious symptom tuberculosis as millet appearance tuberculosis and tuberculous meningitis, but very limited for the effect of the generation of prevention adult pulmonary tuberculosis disease, development.Therefore, be badly in need of the enhancing vaccine after exempting from the beginning of a kind of suitable BCG of development, to improve it to adult's preventive effect lungy.
With the exception of this, BCG is not suitable for the crowd of hypoimmunity, after the immunodeficiency crowds such as HIV inoculate BCG, causes the rising of mortality rate on the contrary; After exempting from baby at the beginning of BCG, the adult phase is immune BCG again, strengthens the limited efficiency of immunity, recommends only immunity inoculation BCG clinically.Therefore, adopting heterologous vaccine---subunit vaccine strengthens the effect of BCG immunity, the significant and more practical value of the prevention for adult pulmonary tuberculosis disease.Because definite ingredients, safety and stability, be convenient to produce, tuberculosis subunit vaccine is in widespread attention in tuberculosis vaccine development.But tuberculosis subunit vaccine known at present still exists the undesirable problem of immunne response effect, be mainly limited to alternative antigen type finite sum and lack the suitable adjuvant that can stimulate T cell immunne response.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of tuberculosis subunit vaccine containing fusion rotein CTT3H, its object is to by selecting antigen gene and fusion sequence to produce fusion rotein, coordinate vaccine adjuvant provided by the invention, for prevention and therapy tuberculosis, solve the generation of BCG for prevention adult pulmonary tuberculosis disease, the limited efficiency of development thus; Be not suitable for the crowd of hypoimmunity, after the immunodeficiency crowds such as HIV inoculate BCG, cause the rising of mortality rate on the contrary; Again inject after exempting from the beginning of BCG, strengthen the technical problems such as the limited efficiency of immunity.
For achieving the above object, according to one aspect of the present invention, provide a kind of tuberculosis subunit vaccine, containing fusion rotein CTT3H, its concentration is 0.1mg/ml to 1mg/ml, and described its encoding gene of fusion rotein CTT3H is the sequential series of CFP10, TB10.4, TB8.4, Rv3615c and HBHA gene according to CFP10-TB10.4-TB8.4-Rv3615c-HBHA.。
Preferably, described tuberculosis subunit vaccine, described in it, the sequence of fusion rotein CTT3H is:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.1; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.1 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.1 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more aminoacid.
Preferably, described tuberculosis subunit vaccine, itself or the Squalene oil containing 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A, the trehalose of 0.2mg/ml to 0.3mg/ml, volume fraction 1% to 4%, the sorbester p37 of volume fraction 1% to 4%, the tween 80 of volume fraction 0.1% to 0.4% and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml are Water-In-Oil sample solution.Described trehalose is 6,6 '-two mycolic acids preferably.
Preferably, described tuberculosis subunit vaccine, or it is containing 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml, is liposomal form.
Preferably, described tuberculosis subunit vaccine, or it is containing the trehalose of 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A and 0.2mg/ml to 0.3mg/ml, is liposomal form.Described trehalose is 6,6 '-two mycolic acids preferably.
According to another aspect of the present invention, provide a kind of vaccine adjuvant, the Squalene oil of the trehalose containing 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A, 0.4mg/ml to 0.6mg/ml, volume fraction 2% to 8%, the sorbester p37 of volume fraction 2% to 8%, the tween 80 of volume fraction 0.2% to 0.8% and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml are Water-In-Oil sample solution.
Preferably, described vaccine adjuvant, trehalose described in it is 6,6 '-two mycolic acids.
According to another aspect of the present invention, providing a kind of vaccine adjuvant, containing 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml, is liposomal form.
According to another aspect of the present invention, provide a kind of vaccine adjuvant, the trehalose containing 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A and 0.4mg/ml to 0.6mg/ml is liposomal form.
Preferably, described vaccine adjuvant, trehalose described in it is 6,6 '-two mycolic acids.In general, the above technical scheme conceived by the present invention compared with prior art, owing to screening specific antigen, and express in a certain order, the fusion rotein of preparation effectively can produce the TH1 type immunne response of antigen-specific, and cooperation the invention provides red adjuvant and can obtain better immunne response effect.Simultaneously relative to existing bacillus calmette-guerin vaccine, there is good safety.The immunoreactive beneficial effect of following enhancing body tuberculosis can be obtained.
Accompanying drawing explanation
Fig. 1 is the structure of the fusion rotein CTT3H of embodiment 2, expression and purification and qualification result figure;
Fig. 2 is special IgG antibody, subclass and the IgG2a/IgG1 ratio result figure of the separate groups of mice change of serum C TT3H of embodiment 15;
Fig. 3 is the level result figure of cytokine IFN-γ and TNF-α in the special mouse boosting cell supernatant of embodiment 15, wherein-representing p<0.05, * represents p<0.05, vs.PBS control;
Fig. 4 is the special C57BL/6 mouse boosting cell total cytokine reaction result figure of flow cytometer detection fusion rotein CTT3H and PPD of embodiment 15, wherein-representing p<0.05, * represents p<0.05, vs.PBS control;
Fig. 5 is the special multi-functional T cell result figure of C57BL/6 mouse boosting cell of flow cytometer detection fusion rotein CTT3H and PPD of embodiment 15, wherein-representing p<0.05, * represents p<0.05, vs.PBS control;
Fig. 6 is the lymphocytic killing activity result figure of flow cytometer detection in vivo cytotoxicity T of embodiment 15;
Fig. 7 is the histopathological findings figure of spleen, lungs lotus bacterium amount and lungs after the aerosol challenge infection immunity C57BL/6 mice surrounding of embodiment 16; Wherein Fig. 7 A, Fig. 7 B are respectively lungs, spleen lotus bacterium amount after aerosol challenge infection immunity C57BL/6 mice surrounding; Fig. 7 C is the lungs pathological score of each group; Fig. 7 D is the histopathology microphotograph of each group of lungs, wherein HE-represent 100um, AFS-represent 50um, and arrow represents AFS positive bacteria.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.In addition, if below in described each embodiment of the present invention involved technical characteristic do not form conflict each other and just can mutually combine.
Tuberculosis subunit vaccine provided by the invention, containing fusion rotein CTT3H, its concentration is 0.1mg/ml to 1mg/ml, one group in also dividing containing following three compositions: (english abbreviation is MPL to (1) 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A, same afterwards), (english abbreviation is TDB to the trehalose of 0.2mg/ml to 0.3mg/ml, same afterwards), the Squalene oil of volume fraction 1% to 4%, the sorbester p37 of volume fraction 1% to 4%, the tween 80 of volume fraction 0.1% to 0.4%, and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml (english abbreviation is MV, same afterwards), for Water-In-Oil sample solution, (2) 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (english abbreviation is DDA, rear same) and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml, be liposomal form, (3) trehalose of 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A and 0.2mg/ml to 0.3mg/ml is liposomal form.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Described its encoding gene of fusion rotein CTT3H is the sequential series of CFP10, TB10.4, TB8.4, Rv3615c and HBHA gene according to CFP10-TB10.4-TB8.4-Rv3615c-HBHA.
The sequence of described fusion rotein CTT3H is:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.1; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.1 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.1 through increasing, really or replace the albumen derivative by (1) that one or more aminoacid has same isoreactivity.
Three kinds of vaccine adjuvants provided by the invention, respectively called after MTO/MV, DDA/MV and DMT.
MTO/MV vaccine adjuvant contains 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A, the trehalose of 0.4mg/ml to 0.6mg/ml, the Squalene oil of volume fraction 2% to 8%, the sorbester p37 of volume fraction 2% to 8%, the tween 80 of volume fraction 0.2% to 0.8% and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml, is Water-In-Oil sample solution.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
DDA/MV vaccine adjuvant contains 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml, is liposomal form.
DMT vaccine adjuvant contains the trehalose of 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A and 0.4mg/ml to 0.6mg/ml, is liposomal form.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Due to the popularization of new therapeutic strategy, global general report case load lungy is from the trend being in continuous decrease since 2005.But tuberculosis remains one of the most serious infectious disease.Still have 8,600,000 new cases every year and the population latent infection mycobacterium tuberculosis in the whole world 1/3.Although BCG is still prevent unique vaccine lungy clinically, and has used more than 90 year, in the different crowd of different regions, unstability is embodied to adult's protectiveness lungy, lack the ability of prevention mycobacterium tuberculosis latent infection.Thus need to develop new can vaccine to control tuberculosis.
Tuberculosis subunit protein candidate vaccine, can be used to alternative BCG or strengthen the effect after BCG immunity as strengthening vaccine, is one of control main vaccine classes lungy of new development.And it is safer relative to BCG that subunit vaccine is applied to immunodeficiency crowd.In addition, many tuberculosis antigens mix or are built into fusion rotein, can bring out strong antituberculotic protective effect.But, the Arrested Development of subunit vaccine in the antigenicity of albumen, lack effective candidate albumen and can inducing cell mediated immunity reaction adjuvant.The immunogenicity of many albumen of mycobacterium tuberculosis is weak, is used alone unprotect immunoreation, thus needs immunostimulation adjuvant to carry out the immunoreation of auxilin induction.Accept extensively the against mycobacterium tuberculosis infection of body and mainly rely on cellullar immunologic response, especially CD4
+the response of Th1 type is sent out to infect at antigen and is played an important role, CD8
+t cell is stoping the reactivation after latent infection, is namely grown up phthisically to have important function.BCG immunity mainly induces body to produce strong CD4
+th1 type is replied, and therefore, its former serious symptom tuberculosis caused after the primary infection of pre-child-resistant is comparatively effective.But BCG immunity body produces CD8
+t cell responses ability is more weak, so little to the value of prevention latent infection and adult pulmonary tuberculosis disease.Based on this immunologic mechanism, we, from the genome of mycobacterium tuberculosis, screen and have CD8 from some
+the important antigen of t cell epitope, comprises CFP10, TB10.4, TB8.4 and Rv3615c.In addition, HBHA is the adhesin of mycobacterium tuberculosis, with after m tuberculosis infection in vivo send out closely related, be also simultaneously an important T cell antigen.Industry also confirms, utilizes separately HBHA immunity, can strengthen the immune protective effect of BCG.Intend based on these antigens, set up subunit vaccine, utilize it at induction CD8
+t cell responses should have stronger potentiality, with the deficiency of supplementary BCG immunity.
Potential Th1 type cell effect effectively induced by vaccine, need in adjuvant, add extra composition, as TLR-4 agonist monophosphoryl lipid A (MPL), dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDA), trehalose (TDB), heat-inactivated Mycobacterium vaccae etc.Monophosphoryl lipid A is used for the AS01 of GSK company, AS02, AS02A and AS04.Wherein, AS04 is used for the vaccine of auxiliary HBV (Fendrix) and HPV (Cervarix) and has formally been applied to the prevention of corresponding disease clinically; AS02A be applied to tuberculosis candidate vaccine M72 carry out clinical 2 the phases test, dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and antigen combined all can inducing cell and humoral immune reaction at dissimilar animal model.Trehalose is the immunostimulating composition of the Mycobacterial cell wall of synthetic.The Mycobacterium vaccae of deactivation is a kind of tuberculosis immunotherapeutic agent, promotes the reaction of Th1 type, Th2 type can be suppressed to react and provide the cross reaction epi-position of mycobacteria to stimulate protective reaction widely simultaneously.And Mycobacterium vaccae can stimulate the CD8 relevant to antigen of mycobacterium tuberculosis
+the cytotoxic effect (CTL) of T cell.
Combine MPL in the present invention, the active ingredient of TDB, MF59 as Squalene oil, sorbester p37 and tween 80, Mycobacterium vaccae (MV), and DDA.These adjuvant compositions are applied all clinically, possess safety.Based on this, make three kinds of different adjuvant MTO/MV, DDA/MV and DMT, auxiliary same PROTEIN C TT3H, builds subunit vaccine MTO/MV/CTT3H, DDA/MV/CTT3H and DMT/CTT3H, respectively to induce strong CD4
+the reaction of Th1 type and CD8
+t ctl response.
Through mouse model, after immune 6 weeks, confirm that three kinds of vaccines are all induced and produce high-caliber CTT3H Specific IgG antibody and subclass thereof, trend towards Th1 type immunne response, wherein the Th1 type immunne response of CTT3H/DMT induction is the most obvious.Compared with PBS, BCG group and three kinds of CTT3H vaccine group are all induced and are produced the specific IFN-γ of high-caliber PPD or CTT3H and TNF-α (P<0.05).Wherein, the IFN-γ level that CTT3H/DMT group induction PPD is special and CTT3H/MTO/MV are without statistics difference.The specific TNF-alpha levels of CTT3H/DDA/MV and CTT3H/DMT group PPD or CTT3H is without statistics difference.Compared with PBS, BCG and three kind of vaccine group produces the special total IFN-γ of more PPD or CTT3H
+or total IL-2
+cD4
+and CD8
+t cell, and total TNF-α
+cD4
+t cell (p<0.05).Particularly, compared with CTT3H/DDA/MV, total IFN-γ that CTT3H/DMT and CTT3H/MTO/MV provides more CTT3H special
+cD4
+and CD8
+t cell (p<0.05). total IL-2 of the antigen-specific between different CTT3H vaccine group
+cD8
+t cell number no difference of science of statistics.And CTT3H/DMT is induction of the IL-2 of top level
+cD4
+t cell.In addition, compare with PBS, total TNF-α that PPD or CTT3H that the induction of CTT3H/DMT and CTT3H/DDA/MV group produces is special
+cD8
+t cell number the highest (p<0.05). compare with PBS group, the single IFN-γ that the PPD that BCG and different adjuvant CTT3H vaccine group immune mouses produce is special
+, single IL-2
+, TNF-α
+iFN-γ
+, and IL-2
+iFN-γ
+cD4
+or CD8
+t cell number all significantly increases (p<0.05). in different adjuvant CTT3H vaccines, CTT3H/DMT and CTT3H/MTO/MV induces these special cell type quantity of CTT3H significantly to increase (p<0.05). the more important thing is, the lymphocytic killing activity of CTT3H/MTO/MV and CTT3H/DMT group in vivo cytotoxicity T is all the highest, is significantly higher than other each group (P<0.05).Finally; utilize Mycobacterium tuberculosis H37Rv strain 60CFU; through respiratory tract aerosol infection immune mouse; confirm that these three kinds of subunit vaccine protectiveness are all better than PBS matched group; CTT3H/MTO/MV and CTT3H/DMT protectiveness is on close level, and is all better than CTT3H/DDA/MV group (P<0.05).
Be below embodiment:
The design of embodiment 1 fusion gene and primer and synthesis
Obtained the sequence of CFP10, TB10.4, TB8.4, Rv3615c and HBHA gene by NCBI website, use each sequence restriction enzyme site of DNAman software analysis, homeotic mutation is carried out for specific site, to ensure that protein transcribes the correctness of translation process.Sequence assembly principle comprises: 1) if having a signal peptide to remove signal peptide, comprise original start codon, remove termination codon simultaneously, after splicing last complete gene order, add TAA; 2) gene order of no signal peptide then retains initiator sequences.If start codon is GTG's, because its original expression product is Met but not Val, GTG also must should be expressed as Met as during non-initial code, therefore GTG need be corrected to ATG.Sequence series connection also called after CTT3H sequence comparison completed according to the order of CFP10-TB10.4-TB8.4-Rv3615c-HBHA.
This fusion sequence is synthesized by the handsome biotech firm in Shanghai and is implemented in pDC316 plasmid, errorless through order-checking comparison.
The structure of embodiment 2 fusion protein prokaryotic expression carrier and protein purification
1, genes of interest obtains:
According to the multiple clone site design primer on the complete sequence of fusion gene and prokaryotic expression carrier pET30b (+), primer sequence is synthesized by Shanghai Sheng Gong biotech firm, and according to explanation, primer is diluted to 100pmol/ μ l, and-20 DEG C save backup.
CTT3H-Fwd：(Nde I)-TTCCATATGGCAGAGATGAAGACCGATG
CTT3H-Rev:(Xho I)-ATCTCGAGCTTCTGGGTGACCTTCTTGG take synthetic gene as template, sets up PCR reaction system, performing PCR of going forward side by side increases.PCR reaction system is as follows:
PCR reaction condition: 98 DEG C of 3min; Then 98 DEG C of 10s, 69 DEG C of 20s, 72 DEG C of 45s totally 36 circulations; 72 DEG C of 10min.1% agarose gel electrophoresis is observed and is reclaimed PCR primer ,-20 DEG C of preservations.
2, the structure of recombiant plasmid pET30b-CTT3H:
The PCR primer reclaimed through electrophoresis and carrier pET30b (+) are carried out double digestion respectively, and enzyme action system is:
The genes of interest CTT3H fragment reclaimed through enzyme action and carrier pET30b (+) are carried out coupled reaction according to following condition:
3, the qualification of conversion and recombinant bacterium
By recombiant plasmid through CaCl
2proceed to E.coli DH5 α and E.coli BL21 (DE3) respectively with heat shock, after resistance screening, obtain positive colony.
4, the purification of fusion rotein, qualification:
The E.coli BL21 (DE3) that pET30b-CTT3H transforms, induce 4 hours through 1mM IPTG, induced product is identified through solubility, and CTT3H exists with inclusion bodies.Therefore, with Denaturing purifying protein, and through correction and activity that Western-blotting verifies albumen, the results are shown in Figure shown in 1.According to the fusion rotein that GE Health company Ni-NTA affinity column operation instruction step purification obtains, Endotoxin Removal Solution test kit is used to remove remaining endotoxin in prokaryotic expression protein product also degerming with the frit of 0.22 μm, i.e. obtained described fusion rotein CTT3H.
From experimental result: the expression cassette of fusion rotein CTT3H as shown in Figure 1A, enzyme action qualification confirms as figure B., because pET30b (+)-CTT3H is confirming the successful expression (before figure C lane 1 induction, after lane 2 induces) of destination protein at E.coli through IPTG induction.Confirm after protein purification procedures is carried out SDS-PAGE electrophoresis, CTT3H protein expression and size is about 61kDa.C end carry 6 histidine-tagged, and confirm that CTT3H protokaryon albumen exists with inclusion bodies in E.coli body by solubility qualification, therefore thalline ultrasonication liquid is combined with Ni affinity column after urea-denatured, and protein purification is completed under Denaturing, eluted product is shown in lane 4-6.Final purified product is shown in lane 7.The anti-CTT3H specific serum anti-CTT3H sera combining the acquisition of incomplete Freund's adjuvant injection mice with anti-His Tag mouse monoclonal antibody or CTT3H carries out Western-blotting, confirms specificity and the biological activity (figure D) of fusion rotein CTT3H respectively.
The preparation of embodiment 3 vaccine adjuvant MTO/MV
MTO/MV vaccine adjuvant contains 0.4mg/ml monophosphoryl lipid A, the trehalose of 0.4mg/ml, the Squalene oil of volume fraction 2%, the sorbester p37 of volume fraction 2%, the tween 80 of volume fraction 0.2% and the heat-inactivated Mycobacterium vaccae of 10mg/ml, is Water-In-Oil sample solution.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows:
Accurately take the trehalose 0.4mg of monophosphoryl lipid A (MPL) 0.4mg, synthetic, add in the aseptic PBS of 1ml.Separately add 2% Squalene oil (v/v), the sorbester p37 (v/v) of volume fraction 2% and the Tween-80 (v/v) of 0.2%, machinery or manually vibration or stirring, after abundant mixing, be rendered as uniform Water-In-Oil sample solution, in addition heat-inactivated mycobacterium vaccae 10mg is joined in this Water-In-Oil sample solution, vibration or stirring, fully after mixing, be namely prepared into described MTO/MV vaccine adjuvant.
The preparation of embodiment 4 vaccine adjuvant MTO/MV
MTO/MV vaccine adjuvant contains 0.5mg/ml monophosphoryl lipid A, the trehalose of 0.5mg/ml, the Squalene oil of volume fraction 5%, the sorbester p37 of volume fraction 5%, the tween 80 of volume fraction 0.5% and the heat-inactivated Mycobacterium vaccae of 20mg/ml, is Water-In-Oil sample solution.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows:
Accurately take the trehalose 0.5mg of monophosphoryl lipid A (MPL) 0.5mg, synthetic, add in the aseptic PBS of 1ml.Separately add 5% Squalene oil (v/v), the sorbester p37 (v/v) of volume fraction 5% and the Tween-80 (v/v) of 0.5%, machinery or manually vibration or stirring, after abundant mixing, be rendered as uniform Water-In-Oil sample solution, in addition heat-inactivated mycobacterium vaccae 20mg is joined in this Water-In-Oil sample solution, vibration or stirring, fully after mixing, be namely prepared into described MTO/MV vaccine adjuvant.
The preparation of embodiment 5 vaccine adjuvant MTO/MV
MTO/MV vaccine adjuvant contains 0.6mg/ml monophosphoryl lipid A, the trehalose of 0.6mg/ml, the Squalene oil of volume fraction 8%, the sorbester p37 of volume fraction 8%, the tween 80 of volume fraction 0.8% and the heat-inactivated Mycobacterium vaccae of 40mg/ml, is Water-In-Oil sample solution.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows:
Accurately take the trehalose 0.6mg of monophosphoryl lipid A (MPL) 0.6mg, synthetic, add in the aseptic PBS of 1ml.Separately add 8% Squalene oil (v/v), the sorbester p37 (v/v) of volume fraction 8% and the Tween-80 (v/v) of 0.8%, machinery or manually vibration or stirring, uniform Water-In-Oil sample solution is rendered as after abundant mixing, in addition heat-inactivated mycobacterium vaccae 40mg is joined in this Water-In-Oil sample solution, vibration or stirring, after abundant mixing, be namely prepared into described MTO/MV vaccine adjuvant.
The preparation of embodiment 6 vaccine adjuvant DDA/MV
A kind of DDA/MV vaccine adjuvant contains 2mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDA) and the heat-inactivated Mycobacterium vaccae of 10mg/ml, is liposomal form.
Its preparation method is as follows:
Accurately take DDA 2mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Add the aseptic PBS of 1ml to dissolve, 60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve) are liposomal form after cooling.In addition heat-inactivated Mycobacterium vaccae 10mg is joined in liposome solutions, vibration or stirring, be fully namely prepared into described vaccine adjuvant after mixing.
The preparation of embodiment 7 vaccine adjuvant DDA/MV
A kind of DDA/MV vaccine adjuvant contains 5mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 20mg/ml, is liposomal form.
Its preparation method is as follows:
Accurately take DDA 5mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Add the aseptic PBS of 1ml to dissolve, 60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve) are liposomal form after cooling.In addition heat-inactivated mycobacterium vaccae 20mg is joined in liposome solutions, vibration or stirring, be fully namely prepared into described vaccine adjuvant after mixing.
The preparation of embodiment 8 vaccine adjuvant DDA/MV
A kind of DDA/MV vaccine adjuvant contains 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 40mg/ml, is liposomal form.
Its preparation method is as follows:
Accurately take DDA 8mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Add the aseptic PBS of 1ml to dissolve, 60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve) are liposomal form after cooling.In addition heat-inactivated mycobacterium vaccae 40mg is joined in liposome solutions, vibration or stirring, be fully namely prepared into described vaccine adjuvant after mixing.
The preparation of embodiment 9 vaccine adjuvant DMT
A kind of DMT vaccine adjuvant contains the trehalose of 2mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDA), 0.4mg/ml monophosphoryl lipid A and 0.4mg/ml, is liposomal form.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows
Accurately take DDA 2mg, MPL 0.4mg, TDB 0.4mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Dissolve with 1mlPBS, 60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve), being namely prepared into described DMT vaccine adjuvant after cooling, is liposomal form.
The preparation of embodiment 10 vaccine adjuvant DMT
A kind of DMT/MV vaccine adjuvant contains the trehalose of 5mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDA), 0.5mg/ml monophosphoryl lipid A and 0.5mg/ml, is liposomal form.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows
Accurately take DDA 5mg, MPL 0.5mg, TDB 0.5mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Dissolve with 1mlPBS.60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve), being namely prepared into described DMT vaccine adjuvant after cooling, is liposomal form.
The preparation of embodiment 11 vaccine adjuvant DMT
A kind of DMT/MV vaccine adjuvant contains the trehalose of 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDA), 0.6mg/ml monophosphoryl lipid A and 0.6mg/ml, is liposomal form.Described trehalose is 6 of synthetic, 6 '-two mycolic acids.
Its preparation method is as follows
Accurately take DDA 8mg, MPL 0.6mg, TDB 0.6mg, be dissolved in chloroform and methyl alcohol mixed liquor (9:1, v/v), slowly pass into nitrogen subsequently and organic solvent is dried up, until form one deck white film.Lipid membrane is at room temperature spent the night and dries, to remove residual organic solvent.Dissolve with 1mL PBS.60 DEG C of heating in water bath 1h (every 10min vortex makes it fully dissolve), being namely prepared into described DMT vaccine adjuvant after cooling, is liposomal form.
The preparation of embodiment 12 tuberculosis subunit vaccine CTT3HMTO/MV
A kind of tuberculosis subunit vaccine, containing fusion rotein CTT3H prepared by embodiment 2, its concentration is for being followed successively by 0.1mg/ml, 0.5mg/ml, 1mg/ml, mix with the MTO/MV adjuvant equal-volume prepared containing embodiment 3,4,5 respectively, conventional mechanical vibration or stirring, be mixed into homogeneous suspension.
The preparation method of described tuberculosis subunit vaccine is as follows:
Draw the MTO/MV adjuvant of preparation in the fusion rotein solution and 100 μ l embodiments 3,4,5 that 100 μ l working concentrations are 0.2mg/ml, 1mg/ml, 2mg/ml respectively in aseptic EP pipe, after covering tightly pipe lid, interval concussion 2 to 3 minutes in vortex oscillator, the uniform emulsion of final formation, is namely prepared into described tuberculosis subunit vaccine CTT3H/MTO/MV.
The preparation of embodiment 13 tuberculosis subunit vaccine CTT3HDDA/MV
A kind of tuberculosis subunit vaccine, containing fusion rotein CTT3H prepared by embodiment 2, its concentration is respectively 0.1mg/ml, 0.5mg/ml, 1mg/ml, mixes respectively with the DDA/MV adjuvant equal-volume prepared containing embodiment 6,7,8, and conventional mechanical vibrates or is uniformly mixed becomes homogeneous suspension.
The preparation method of described tuberculosis subunit vaccine is as follows:
Draw the DDA/MV adjuvant of preparation in the fusion rotein solution and 100 μ l embodiments 6,7,8 that 100 μ l working concentrations are 0.2mg/ml, 1mg/ml, 2mg/ml respectively in aseptic EP pipe, after covering tightly pipe lid, interval concussion 2 to 3 minutes in vortex oscillator, the uniform emulsion of final formation, is namely prepared into described tuberculosis subunit vaccine DDA/MV/CTT3H.
The preparation of embodiment 14 tuberculosis subunit vaccine DMT/CTT3H
A kind of tuberculosis subunit vaccine, it is characterized in that, containing fusion rotein CTT3H prepared by embodiment 2, its concentration is respectively 0.1mg/ml, 0.5mg/ml, 1mg/ml, respectively with the DMT adjuvant prepared containing embodiment 9,10,11, equal-volume mixes, and conventional mechanical vibration or stirring, be mixed into homogeneous suspension.
The preparation method of described tuberculosis subunit vaccine is as follows:
Draw the DMT adjuvant of preparation in the fusion rotein solution and 100 μ l embodiments 9,10,11 that 100 μ l working concentrations are 0.2mg/ml, 1mg/ml, 2mg/ml respectively in aseptic EP pipe, after covering tightly pipe lid, interval concussion 2 to 3 minutes in vortex oscillator, the uniform emulsion of final formation, is namely prepared into described tuberculosis subunit vaccine DMT/CTT3H.
The immunological evaluation of embodiment 15 tuberculosis subunit vaccine
1. vaccine and adjuvant preparation
MTO/MV adjuvant adopts embodiment 4 to prepare, and DDA/MV adjuvant adopts embodiment 7 to prepare, and DMT adjuvant adopts embodiment 10 to prepare.
Tuberculosis subunit vaccine MTO/MV/CTT3H adopts embodiment 12 to prepare, and DDA/MV/CTT3H adopts embodiment 13 to prepare, and DMT/CTT3H adopts embodiment 14 to prepare.
1) laboratory animal and grouping
Select 6 week age, female C57BL/6 mice, SPF level, purchased from Wuhan University's Experimental Animal Center, lot number is No.4200593074.Commodity in use feedstuff and distilled water are fed.
Experimentally need, laboratory animal be divided into following eight groups: negative control group---PBS group; Positive controls---BCG group; MTO/MV group; DDA/MV group; DMT group; CTT3H/MTO/MV group; CTT3H/DDA/MV group; CTT3H/DMT group.Often organize 3.
2) animal immune
All adopt injected s. c to carry out immunity, volume injected is 100 μ l.Adjuvant group and vaccine group need repeat immune secondary, every minor tick 3 weeks.BCG group volume injected 100 μ l, the bacterium amount contained is 1 × 10
6cFU.PBS group and BCG group all immune 1 time,
2.ELISA detects immune serum specific antibody
1) mice after immune 6 weeks, gets after blood through eyeball and collects serum, and frozen in-20 DEG C after subpackage;
2) diluted according to working concentration 5 μ g/ml by fusion rotein CTT3H, routine bag is carried out by 96 hollow plates in 100 μ l/ holes;
3) the aseptic 1 × PBS after the filtration of each group mice serum sample doubly dilutes by 1:400,200 μ l/ holes add to often row first hole, often row second hole to octal adds 100 μ l PBS respectively, each sample does multiple hole, then carry out doubling dilution to 1:51200 from the first hole, blank control wells adds PBS 100 μ l;
4) the anti-dilution factor of HRP labelling sheep anti mouse two is respectively: IgG 1:5000, IgG11:10000, IgG2a 1:10000;
5) result treatment: after each each sample OD value is deducted blank control wells OD value, is averaged OD value with the multiple hole of a blood serum sample.Wherein with the OD value of PBS negative control group for negative control (N), immune group is sample (P), when blood-serum P/N value >=2.1 can be judged as the positive.Antibody titer represents with the inverse of the most highly diluted multiple occurring positive findings;
6) result calculates: each sample result is expressed as log10 (antibody titer), and averaging between the multiple hole of each sample is the actual value of this mice, and calculating mean value and standard error in same group, carry out statistical analysis; IgG2a:IgG1 compares with the antibody titer of every mice, and in same group, result represents with meansigma methods ± standard error.
Experimental result is shown in Fig. 2.
From experimental result: compared with BCG, the IgG of the CTT3H that three kinds of vaccine-induced high-level CTT3H are special, IgG1 and IgG2a antibody, other three kinds of vaccine group IgG2a/IgG1 ratios are all greater than 1 and higher than BCG group (P<0.05), point out three kinds of vaccine-induced immunoreation to tend to the immunoreation of TH1 type.The strongest with the Th1 type immunne response of CTT3H/DMT group induction.
3.ELISA detects immune mouse spleen cell culture supernatant antigen specific cytokine
1) extracting spleen cell suspension 100 μ l is inoculated in (cell number 2.5 × 10 in 96 orifice plates
6);
2) stimulus object is as follows: CTT3H 20 μ l, final concentration 10 μ g/ hole; Positive control PPD working solution 20 μ l, final concentration 10 μ g/ hole; Negative control RPMI1640 complete medium 20 μ l;
3) cultivate after 72 hours, liquid carry out labelling, 2000rpm, centrifugal 10min in collection hole, collects supernatant and is also sub-packed in EP pipe respectively, frozen stand-by in-80 DEG C after labelling;
4) content of cytokine TNF-α and IFN-γ in supernatant is detected according to Mouse ELISA kit workbook:
5) after cessation reaction in 10min, with blank control wells zeroing, microplate reader is used to read double UV check light absorption value: 450nm is determined wavelength, and 630nm is reference wavelength;
6) establishing criteria sample wells Plotting data standard curve, surveys OD value per sample and records corresponding cytokine concentrations.Every mice multiple hole results averaged, after deducting RPMI1640 values of control groups, in same group, difference calculating mean value and standard deviation, carry out statistical analysis.
Experimental result is shown in Fig. 3.
As shown in the figure: after immune 6 weeks, PBS group PPD and the special IFN-γ of CTT3H albumen and TNF-alpha levels all lower.Compared with PBS, the IFN-γ that PPD and the CTT3H albumen of BCG group induced high levels is special and TNF-α (P<0.05), three kinds of CTT3H vaccine group also all induction produce the specific IFN-γ of high-caliber PPD or CTT3H and TNF-α (P<0.05).Wherein, the IFN-γ level that CTT3H/DMT group induction PPD is special and CTT3H/MTO/MV are without statistics difference.The specific TNF-alpha levels of CTT3H/DDA/MV and CTT3H/DMT group PPD or CTT3H is without statistics difference.
4. the T lymphocyte of intracellular cytokine dyeing and Flow cytometry immune mouse spleen cell antigenic specificity secrete cytokines
1) draw the cell suspension 100 μ l adjusting concentration, be inoculated in 24 orifice plates, make spleens cell number in every hole be 2.5 × 10
6;
2) the concrete sample loading alternative of different stimulated thing is as follows:
Experimental port: every hole adds 20 μ l fusion rotein CTT3H, final concentration 10 μ g/ hole; Separately add anti-CD28mcAb20 μ l;
Positive control wells: every hole adds 20 μ l PPD working solutions, i.e. 10 μ l PPD stock solution+10 μ l RPMI1640 complete mediums, PPD final concentration 10 μ g/ hole; Separately add anti-CD28mcAb 20 μ l;
Negative control hole: every hole adds RPMI1640 complete medium 20 μ l; Separately add anti-CD28mcAb 20 μ l;
Blank control wells: add Cocktail working solution 40 μ l;
Positive test hole: add Cocktail working solution 40 μ l;
Singly mark pipe: add Cocktail working solution 40 μ l;
3) every hole adds 840 μ l RPMI1640 complete mediums, is placed in 37 DEG C and cultivates 16h;
4), after adding stimulus object cultivation 4h, every hole adds blocker Brefeldin A+Monensin mixed liquor 20 μ l;
5) cultivate and terminate rear collection pipettor and move in streaming pipe by liquid in hole and cell, 500 × g, centrifugal 5min, abandons supernatant for subsequent use.
6) pre-configured dyeing liquor is added, specific as follows:
Experimental port, Positive control wells, negative control hole and positive test hole: every hole adds 0.5 μ l Anti-Mouse CD3 FITC, 1.25 μ l APC/Cy7anti-mouse CD4Antibody, 1.25 μ l Anti-Mouse CD8a PE and 97 μ lPBS;
Blank control wells: do not add any antibody;
Singly mark pipe: the antibody that surface marker list mark pipe is corresponding according to often pipe adds corresponding antibodies respectively, cytokine list mark pipe does not add any antibody;
After mix homogeneously, 4 DEG C of lucifuges hatch 30min;
7) often pipe adds 1 × PBS 2ml to wash away antibody staining liquid, 4 DEG C, and the centrifugal 5min of 500 × g, abandons supernatant;
8) step 2 is repeated;
9) often pipe adds the Intracellular Fixation 100 μ l of pre-cooling, and piping and druming mixing is so that fully fixing, and room temperature lucifuge hatches 30min;
10) often pipe adds 1ml 1 × Permeabilization Buffer, and the centrifugal 5min of 500 × g, abandons supernatant;
11) step 5 is repeated;
12) add corresponding cytokine antibodies respectively through fixing and after wearing film cell, concrete mode is as follows:
Experimental port, Positive control wells, negative control hole and positive test hole: every hole adds 1.25 μ l Anti-Mouse IFN-gamma PerCP-Cy5.5,1.25 μ l PE-Cy7Rat Anti-Mouse TNF, 1.25 μ l Anti-Mouse IL-2APC and 96.25 μ l 1 × Permeabilization Buffer.
Blank control wells: do not add any antibody;
Singly mark pipe: the antibody that the corresponding single mark pipe of cytokine is corresponding according to often pipe adds corresponding antibodies respectively, and surface marker list mark pipe does not add any antibody;
After mix homogeneously, room temperature lucifuge hatches 30min;
13) often pipe adds 1ml 1 × Permeabilization Buffer to wash away antibody staining liquid, and the centrifugal 5min of 500 × g, abandons supernatant;
14) often pipe adds 1ml 1 × PBS to wash away antibody staining liquid and to wear film liquid, and the centrifugal 5min of 500 × g, abandons supernatant;
15) often pipe adds 300 μ l 1 × PBS re-suspended cells, and 4 DEG C keep in Dark Place to be checked;
16) first go up machine-readable each list of getting and mark pipe data, compensate to complete fluorescence associated according to result regulating parameter; Each test all needs preparation blank control wells and positive test hole to get rid of reagent difference;
17) sorting strategy is: take lymphocyte populations as P1 door, then with CD3+CD4+ and CD3+CD8+ sorting CD4+ and CD8+ cell, analyzes the percentage of lymphocyte that Secretion answers cytokine respectively;
18) according to dissimilar T lymphocyte proportion and mouse boosting cell count results in total cell number of upper machine testing, every lymphocytic quantity of Mouse spleen cells cytokine secretion type T is calculated respectively.Often organize data calculating mean value and standard deviation respectively, use one factor analysis of variance to carry out statistical evaluation between difference group.
Experimental result is shown in Fig. 4.
Immunity is after 6 weeks, and compared with PBS, BCG and three kind of vaccine group produces the special total IFN-γ of more PPD or CTT3H
+or total IL-2
+cD4
+and CD8
+t cell, and total TNF-α
+cD4
+t cell (p<0.05).Particularly, compared with CTT3H/DDA/MV, total IFN-γ that CTT3H/DMT and CTT3H/MTO/MV provides more CTT3H special
+cD4
+and CD8
+t cell (p<0.05). total IL-2 of the antigen-specific between different CTT3H vaccine group
+cD8
+t cell number no difference of science of statistics.And CTT3H/DMT is induction of the IL-2 of top level
+cD4
+t cell.In addition, compare with PBS, total TNF-α that PPD or CTT3H that the induction of CTT3H/DMT and CTT3H/DDA/MV group produces is special
+cD8
+t cell number the highest (p<0.05). compare with PBS group, the single IFN-γ that the PPD that BCG and different adjuvant CTT3H vaccine group immune mouses produce is special
+, single IL-2
+, TNF-α
+iFN-γ
+, and IL-2
+iFN-γ
+cD4
+or CD8
+t cell number all significantly increases (p<0.05). and in different adjuvant CTT3H vaccines, CTT3H/DMT and CTT3H/MTO/MV induces these special cell type quantity of CTT3H significantly to increase (p<0.05).
CTL specific killing activity in 5.CFSE labelling method detection bodies
(1) target cell preparation:
1. the splenocyte of non-immune C57BL/6 mice (with same batch of experimental group) is collected, with 1ml 1640 complete medium re-suspended cell, counting.By plating cells.
2. the target cell of load peptide: get 500 μ l cell suspension (5 × 10
7individual/ml cell) add the CD8 peptide (1mg/ml) of 100 μ l TB10.44 – 11 (IMYNYPAM), put into 15ml cell centrifugation pipe after mixing, mend 400 μ l complete mediums to 1ml.
3. the target cell of unsupported peptide: get 500 μ l cell suspension (5 × 10
7individual/ml cell), add in 15ml cell centrifugation pipe, mend 500 μ l complete mediums.37 DEG C, in 5%CO2 incubator, cultivate 6h.The centrifugal 10min of 1500rpm/min, abandons supernatant.
(2) target cell labelling CFSE:
1. CFSE (the CFSE of the target cell labelling high concentration of load peptide
high): the target cell (final concentration is 5 μMs) of the resuspended load peptide of CFSE dyestuff of high concentration.
2. CFSE (the CFSE of the target cell labelling low concentration of non-load peptide
low): the target cell (final concentration is 0.5 μM) of the resuspended non-load peptide of CFSE dyestuff of low concentration.
3. hatch 15min for 37 DEG C, period mixes twice (use have gentle hands bullet, must guard against piping and druming) gently.
4. add 1ml containing 10%FCS RPMI1640 liquid, mix 1min gently, hatch 30min for 37 DEG C.The centrifugal 10min of 1500rpm/min.Abandon supernatant.
5. add the 10ml PBS of pre-cooling containing 10%FCS, mix 1min gently, the centrifugal 10min of 1500rpm/min.Wash 3 times.
(3) tail venous re-transfusion: the target cell of collecting two group echos, the resuspended rear 1:1 mixed in equal amounts of PBS.Experimental group and matched group every mice be tail vein injection cell mixing 200 μ l respectively.
(4) FCM detects: after 14h, puts to death mice.Prepare splenocyte suspension, need splitting erythrocyte, counting, gets 100 ten thousand cells, upper machine analysis.488nm excites, and collects CFSE fluorescence signal.The CFSE positive cell sum collected is more than 2 ~ 5 × 104 cells.
(5) result calculates:
Cell killing rate=(CFSE
lowpositive cell number ÷ CFSE
highpositive cell number) × 100%.
CTL specific killing activity in body=[1-(non-sensitized animal cell killing rate ÷ sensitized animal kill rate)] × 100%
Calculate according to formula the CTL specific killing activity often organizing every mice respectively, finally calculate each group of mean and standard error, carry out statistical analysis.
Experimental result is shown in Fig. 6.
Three kinds of lymphocytic killing activities of CTT3H vaccine group group in vivo cytotoxicity T are all higher than BCG group (P<0.05), and CTT3H/MTO/MV and CTT3H/DMT is higher than CTT3H/DDA/MV group (P<0.05).
Embodiment 16 subunit vaccine immune mouse mithridatism Mycobacterium tuberculosis H37Rv is through the protectiveness of aerosol infection
Vaccine adjuvant adopts the vaccine adjuvant of preparation in embodiment 15.
Tuberculosis subunit vaccine adopts the tuberculosis subunit vaccine of preparation in embodiment 15.
1, the grouping of laboratory animal and immunity
The female C57BL/6 mice of SPF level in 6 week age, quality certification lot number is No.4200592074, purchased from Wuhan University ABSL-3 animal experimental center.Raise the Ventirack animal breeding cabinet in Wuhan University animal experimental center ABSL-3 laboratory, all operations all strictly observes Wuhan University's zoopery workbook.
Experimentally need, by laboratory animal with embodiment 15, often organize 5.Animal immune method and dosage the same.
Zoogenetic infection approach: after immune 6 weeks, utilizes M.tb H37Rv standard strain through aerosol infection immune mouse (actual counteracting toxic substances bacterium amount is 60CFU/).
2, the lotus bacterium component analysis of lungs and spleen
Put to death mice after 4 weeks, with 75% alcohol-pickled sterilization 10min, aseptic taking-up lungs and spleen, add the aseptic 1 × PBST of 2ml respectively according to every internal organs, grind and rinse in mortar, transfer in 10ml sterile tube, agitator fully mixes.Get 100 μ l stock solutions to join in 900 μ l PBST and do doubling dilution, after agitator fully mixes, get 4 each 100 μ l coating 7H11-OADC culture dishs of dilution bacterium liquid and (during preparation, add cycloheximide, the growth of Antifungi, add TCH can Selective depression remain the growth of BCG).37 DEG C of incubators are cultivated after 3 ~ 4 weeks and are carried out colony counting.With Log
10(CFU) component analysis of each group of internal organs lotus bacterium is carried out.Calculate average and the standard error of each group.
Experimental result is shown in Fig. 7.
The lungs of PBS group mice and the lotus bacterium amount of spleen all the highest.The lungs of three kinds of vaccine group and BCG group and spleen lotus bacterium amount, all significantly reduce (P<0.05) respectively.Compared with PBS group, CTT3H/MTO/MV, CTT3H/DDA/MV and CTT3H/DMT group lungs decline-0.99log ,-0.69log and-1.00log respectively, and spleen declines-1.09log ,-0.51log and-1.06log respectively.In addition, CTT3H/MTO/MV and CTT3H/DMT group spleen, lungs lotus bacterium amount decline more than CTT3H/DDA/MV group (P<0.05).The antiacid positive bacteria of consistent with lotus bacterium amount is three kinds of vaccine group and BCG group is more less than PBS group.In addition, PBS group lungs granuloma sample changes the most serious.And three kinds of vaccine group and BCG group pathological change are relatively light, and pathological score all lower (Fig. 7).
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a tuberculosis subunit vaccine, it is characterized in that, containing fusion rotein CTT3H, its concentration is 0.1mg/ml to 1mg/ml, and described its encoding gene of fusion rotein CTT3H is the sequential series of CFP10, TB10.4, TB8.4, Rv3615c and HBHA gene according to CFP10-TB10.4-TB8.4-Rv3615c-HBHA.
2. tuberculosis subunit vaccine as claimed in claim 1, it is characterized in that, the sequence of described fusion rotein CTT3H is:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.1; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.1 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.1 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more aminoacid.
3. tuberculosis subunit vaccine as claimed in claim 1 or 2, it is characterized in that, or Squalene oil containing 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A, the trehalose of 0.2mg/ml to 0.3mg/ml, volume fraction 1% to 4%, the sorbester p37 of volume fraction 1% to 4%, the tween 80 of volume fraction 0.1% to 0.4% and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml, be Water-In-Oil sample solution.
4. tuberculosis subunit vaccine as claimed in claim 1 or 2, is characterized in that, or containing 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 5mg/ml to 20mg/ml, is liposomal form.
5. tuberculosis subunit vaccine as claimed in claim 1 or 2, it is characterized in that, or the trehalose containing 1mg/ml to 4mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.2mg/ml to 0.3mg/ml monophosphoryl lipid A and 0.2mg/ml to 0.3mg/ml, be liposomal form.
6. the tuberculosis subunit vaccine as described in claim 3 or 5, is characterized in that, described trehalose is 6,6 '-two mycolic acids.
7. a vaccine adjuvant, it is characterized in that, the Squalene oil of the trehalose containing 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A, 0.4mg/ml to 0.6mg/ml, volume fraction 2% to 8%, the sorbester p37 of volume fraction 2% to 8%, the tween 80 of volume fraction 0.2% to 0.8% and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml are Water-In-Oil sample solution.
8. a vaccine adjuvant, is characterized in that, containing 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium and the heat-inactivated Mycobacterium vaccae of 10mg/ml to 40mg/ml, is liposomal form.
9. a vaccine adjuvant, is characterized in that, the trehalose containing 2mg/ml to 8mg/ml dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium, 0.4mg/ml to 0.6mg/ml monophosphoryl lipid A and 0.4mg/ml to 0.6mg/ml, is liposomal form.
10. vaccine adjuvant as described in claim 7 or 9, is characterized in that, described trehalose is 6,6 '-two mycolic acids.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105944094A (en) * | 2016-06-14 | 2016-09-21 | 中国科学院过程工程研究所 | Inulin-chitosan modified mycobacterium tuberculosis subunit vaccine and preparation method thereof |
| CN109813576A (en) * | 2019-03-25 | 2019-05-28 | 中国动物卫生与流行病学中心 | Prepared by a kind of perlsucht stimulation supernatant uses hemostix |
-
2014
- 2014-12-26 CN CN201410836669.4A patent/CN104548081A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| LOXTON AG ET AL: "heparin-binding hemagglutinin induces ifn-γ+ il-2+ il-17+ multifunctional cd4+ t cells during latent but not active tuberculosis disease", 《CLIN VACCINE IMMUNOL》 * |
| SKJOT RLV ET AL: "epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the eat-6 gene family", 《INFECT IMMUN》 * |
| 滕新栋等: "不同佐剂辅助的CTT3H结核病亚单位疫苗的免疫效果评价", 《第九届全国免疫学学术大会论文集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105944094A (en) * | 2016-06-14 | 2016-09-21 | 中国科学院过程工程研究所 | Inulin-chitosan modified mycobacterium tuberculosis subunit vaccine and preparation method thereof |
| CN105944094B (en) * | 2016-06-14 | 2019-09-13 | 中国科学院过程工程研究所 | A kind of mycobacterium tuberculosis subunit vaccine and preparation method thereof chitosan-modified based on inulin- |
| CN109813576A (en) * | 2019-03-25 | 2019-05-28 | 中国动物卫生与流行病学中心 | Prepared by a kind of perlsucht stimulation supernatant uses hemostix |
| CN109813576B (en) * | 2019-03-25 | 2021-04-02 | 中国动物卫生与流行病学中心 | Blood collector for preparing bovine tuberculosis stimulating supernatant |
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Application publication date: 20150429 |