CN105079801A - Application of cistanche deserticola polysaccharides as vaccine adjuvant - Google Patents
Application of cistanche deserticola polysaccharides as vaccine adjuvant Download PDFInfo
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Abstract
The invention provides application of cistanche deserticola polysaccharides as a vaccine adjuvant. Through tests such as detecting OVA (ovalbumin) specific antibody titer and cellular immunological activity in mice sera by animal grouping and immunizing methods and ELISA (enzyme linked immunosorbent assay), results show that the cistanche deserticola polysaccharides can serve as the adjuvant of a protein vaccine, a nucleic acid vaccine, a polypeptide vaccine, an inactivated vaccine or a live attenuated vaccine, and are obviously superior to an aluminum adjuvant in immunoenhancement effect, little in side effect, wide in source and easy to produce, and a preparation process of the cistanche deserticola polysaccharides is simple, low in cost, high in yield and beneficial to large-scale production.
Description
Technical field
The present invention relates to a kind of immunological adjuvant, particularly relate to the application of Herba Cistanches polysaccharides in vaccine adjuvant, belong to biological technical field.
Background technology
Herba Cistanches (HerbaCistanche) is Orobanchaceae plant, it is parasitic herbaceous plant, growth is in desert Environment, in the desert that main product is with in Xinjiang of China, Gansu, the Inner Mongol, Ningxia etc. and the Central Asia, West Asia one, there is the good reputation of desert " Radix Ginseng ", clinical extensive with Popular Utilization, " Chinese Pharmacopoeia " records Desert Herba Cistanches and Cistanche Tubulosa is Herba Cistanches.Xinjiang is the main producing region of Herba Cistanches, main product Desert Herba Cistanches and Cistanche Tubulosa.Nearly more than ten years, researcheres had conducted intensive studies its aspect such as chemical composition and pharmacological action; find that Herba Cistanches has immunomodulating, neuroprotective, antioxidation, anti-apoptotic, anti-inflammatory, resisting fatigue, anti-ageing multiple pharmacological effect of waiting for a long time; these pharmacological actions and the plurality of active ingredients contained by Herba Cistanches, as closely related in polysaccharide, phenethanol glycoside, flavone etc.
Vaccine adjuvant significantly can strengthen the consumption of the immunogenicity of vaccine and safety, saving antigen, serves very important effect for the control mankind and the popular of the great epidemic disease of animal.Adjuvant generally can use in multiple vaccine.Although many raw materials can be applied to vaccine adjuvant, mostly can only react or produce some side effect, because which limit its practical application by induce antibody.At present, can be used in clinical immunological adjuvant kind little, unique ratified by FDA the adjuvant that can be used for people----aluminium glue adjuvant can only excite humoral immunization, can not inducing cell mediation immunoreation, and invalid to antigens such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, so the research of new vaccine adjuvant that is safe, efficient, that have no side effect that screening can be used in human body and animal comes into one's own day by day.
Desirable immunologic adjuvant should be the cell of animal body or humoral immunization are all had to effective excitation, persistent are stable, immune time reduces, side effect is little, is convenient to produce and be easy to injection simultaneously.
Large quantifier elimination shows, the polysaccharide of plant origin plays many-sided important regulative to immune system, becomes again the candidate resource of safety, new high-efficiency vaccine adjuvant without serious toxicity.The immunologic active material of plant origin, has aboundresources, cheap, toxic and side effects is little, the advantage such as noresidue, therefore vegetable polysaccharides as adjuvant and vaccine with the use of being the important channel of adjuvant research and development and new trend.
Summary of the invention
The object of the present invention is to provide the application of Herba Cistanches polysaccharides (AqueousextractsofCistanchedeserticola, AECD) in vaccine adjuvant.
This research finds, Herba Cistanches polysaccharides can obvious stimulation Serum Antibody IgG, IgG
1and IgG
2ageneration, to ConA and LPS stimulate lymphproliferation response obviously increase, can excite lymphocytic emiocytosis produce cytokine IL-4 and IFN-γ.Show that Herba Cistanches polysaccharides not only induces stronger humoral immune reaction, and induction of stronger cell immune response, may be used for vaccine adjuvant.
For achieving the above object, the technical solution adopted in the present invention is: the present invention relates to the application of described Herba Cistanches polysaccharides in vaccine adjuvant.
Vaccine of the present invention is protein vaccine, gene vaccine, polypeptide vaccine, inactivated vaccine or attenuated live vaccine.
Herba Cistanches polysaccharides of the present invention adopts ultrasonic wave added decoction and alcohol sedimentation technique to extract.The present invention by fresh or dry Xinjiang Herba Cistanches powder, defat with petroleum ether, it is ultrasonic to add distilled water, and add absolute ethyl alcohol and stirring precipitation after filtrate is concentrated, precipitate is through centrifugal, and vacuum drying, obtains the semifinished product of Herba Cistanches polysaccharides;
Again to Herba Cistanches crude polysaccharides Sevage reagent (chloroform: n-butyl alcohol=4:1) removing protein, centrifugal, merge supernatant, repeat aforesaid operations repeatedly, collect supernatant, add ethanol, hold over night, precipitate is through centrifugal, and vacuum drying, obtains Herba Cistanches polysaccharides and can be used as vaccine adjuvant.
Using method of the present invention: Herba Cistanches crude polysaccharides and antigen adopt together with subcutaneous route and injects body.Described vaccine is ejection preparation, and the dosage of Herba Cistanches polysaccharides in described ejection preparation is 1.5mg/kg ~ 50mg/kg.
The application of Herba Cistanches crude polysaccharides of the present invention in vaccine adjuvant, divided into groups and immunization method by animal, ELISA detects the experiment such as anti ova Specific antibody titre and cellular immunologic competence in mice serum, result proves that Herba Cistanches polysaccharides can as protein vaccine, gene vaccine, polypeptide vaccine, the adjuvant of inactivated vaccine or attenuated live vaccine, its immunoenhancement result is obviously better than aluminium adjuvant, it is little that it has side effect, wide material sources, be easy to produce, and the preparation process of Herba Cistanches polysaccharides of the present invention is simple, cost is lower, and yield is higher, be conducive to large-scale production.
Accompanying drawing explanation
Fig. 1 is mouse immune OVA specific antibody IgG titre testing result.
Fig. 2 is mouse immune OVA specific antibody IgG
1and IgG
2atesting result.
Fig. 3 is the situation of mouse lymphocyte breeder reaction after ConA and LPS stimulates.
Fig. 4 is the impact that Herba Cistanches polysaccharides adjuvant produces the cytokine IL-4 that mouse lymphocyte is bred.
Fig. 5 is the impact that Herba Cistanches polysaccharides adjuvant produces the cytokine IFN-γ that mouse lymphocyte is bred.
Fig. 6 is the impact that Herba Cistanches polysaccharides adjuvant produces influenza vaccines IgG antibody level.
Fig. 7 is that Herba Cistanches polysaccharides adjuvant is to influenza vaccines IgG
1and IgG
2athe impact that antibody horizontal produces.
Fig. 8 is the impact that Herba Cistanches polysaccharides adjuvant produces foot-and-mouth disease vaccine IgG antibody level.
Fig. 9 is that Herba Cistanches polysaccharides adjuvant is to foot-and-mouth disease vaccine IgG
1and IgG
2athe impact that antibody horizontal produces.
Figure 10 is the impact that Herba Cistanches polysaccharides adjuvant produces mice OVA gene vaccine IgG antibody level.
Detailed description of the invention
Below in conjunction with the specific embodiment of the invention, the application of Herba Cistanches polysaccharides of the present invention at vaccine adjuvant is described in detail.
Embodiment 1: various dose Herba Cistanches polysaccharides is to the zoopery of mouse immune model antigen oralbumin (OVA)
The preparation method of the present embodiment Herba Cistanches polysaccharides is:
Step 1: fresh or dry Xinjiang Herba Cistanches powder, defat with petroleum ether, it is ultrasonic to add distilled water, and add absolute ethyl alcohol and stirring precipitation after filtrate is concentrated, precipitate is through centrifugal, and vacuum drying, obtains the semifinished product of Herba Cistanches polysaccharides;
Step 2: Herba Cistanches crude polysaccharides Sevage reagent (chloroform: n-butyl alcohol is 4:1) removing protein, centrifugal, merge supernatant, repeat aforesaid operations repeatedly, collect supernatant, add ethanol, hold over night, precipitate is through centrifugal, and vacuum drying, obtains Herba Cistanches polysaccharides.
Implementation method:
Test material: Herba Cistanches polysaccharides mother solution configures: the Herba Cistanches polysaccharides of 0.1g is dissolved in 10ml normal saline, is configured to the mother solution of 10mg/ml.Configuration OVA mother solution: 0.01gOVA is dissolved in the normal saline of 10ml, is configured to the mother solution of 1mg/ml.
Animal immune: by 6-8 ICR mice in age in week, random assortment becomes 7 groups, often organizes 6, and experiment grouping is as shown in table 1,
Table 1 mouse immune divides into groups
| Group number | Kind (female) | Number of elements | Injectable composition |
| 1 | ICR | 6 | 0.9%NaCl |
| 2 | ICR | 6 | AECD 400 μg |
| 3 | 6 | OVA 10 μg | |
| 4 | ICR | 6 | Alum200 μg+OVA 10 μg |
| 5 | ICR | 6 | AECD20 μ g+OVA 10 μ g(low dosage) |
| 6 | ICR | 6 | Dosage in AECD 100 μ g+OVA 10 μ g() |
| 7 | ICR | 6 | AECD 400 μ g+OVA 10 μ g(high dose) |
Often organizing vaccine is dissolved in normal saline, every injected in mice 100 microlitre, the 0th day subcutaneous injection, and after first time injection, interval is injected for 14 days again, injects twice altogether.
In order to detect mouse humoral immune level, take a blood sample after subcutaneous injection, separating mouse serum, indirect elisa method detects IgG, IgG respectively
1and IgG
2aantibody horizontal.With in the antigen coated 96 hole ELISA Plate of 2 μ g/mlOVA, 4 DEG C are spent the night, the closed 2h of 3% bovine serum albumin 37 DEG C; PBST (Tween20 is dissolved in PBS, makes the final concentration of Tween20 be 0.05% solution obtained) washs 4 times; Add the mice serum by different multiples dilution, hatch 1h for 37 DEG C; PBST washs 4 times, each 5 minutes; Add sheep anti-Mouse horseradish peroxidase-labeled IgG, IgG
1and IgG
2aantibody (Southernbiotech) (all by 1:5000 dilution), hatches 1h for 37 DEG C.Every hole adds substrate TMB, and 37 DEG C of lucifuge colour developing 5-15min, add stop buffer 2mol/LH
2sO
4, every hole 50 μ l, color development stopping, is placed in 450nm/655nm and measures every hole optical density value, and the terminal of positive titers is OD value higher than waiting 2.1 times of dilution factor negative serum (normal saline group) meansigma methods.
Result is (numerical value in table 2 is the titre of 6 mice pooled serums) as shown in Table 2 and Figure 1: saline control group, holosaccharide group without antigen, IgG antibody level is lower, shows that above combination does not possess immune effect.OVA antigen group without adjuvant creates low-level IgG, and this shows that OVA antigen has certain immune effect.Compare above combination, aluminium adjuvant group, all high-caliber IgG can be produced containing the low dose group of adjuvant, middle dosage group, high dose group, and the effect of AECD100 μ g group is the most remarkable, compared with antigen control group, antibody titer enhances 30 times, and a little more than aluminium adjuvant group, and the persistent period of antibody is longer.The above results shows, adjuvant Herba Cistanches polysaccharides can make OVA protein vaccine induce body to produce very high humoral immune reaction.
Antibody titre results after table 2 immune mouse
| Group | 14d | 21d | 35d | 49d |
| 0.9%NaCl | 100 | 100 | 100 | 100 |
| AECD 400 μg | 100 | 100 | 100 | 100 |
| OVA 10 μg | 1000 | 8000 | 6000 | 9000 |
| Alum200 μg+OVA 10 μg | 4000 | 240000 | 280000 | 300000 |
| AECD 20 μg+OVA 10 μg | 2000 | 150000 | 150000 | 200000 |
| AECD 100 μg+OVA 10 μg | 4000 | 250000 | 300000 | 320000 |
| AECD 400 μg+OVA 10 μg | 3000 | 230000 | 250000 | 250000 |
Antibody subtype testing result is as table 3 and Fig. 2 (numerical value in table 3 is the mean+SD of 6 mices, different letter representation P < 0.01): saline control group, holosaccharide group without antigen, IgG
1and IgG
2aantibody horizontal lower, show that above combination does not possess immune effect.OVA antigen group without adjuvant creates the IgG of certain level
1and IgG
2a, this shows that OVA antigen has certain immune effect.Compare above combination, aluminium adjuvant group can produce high-caliber IgG
1but, the IgG produced
2alevel lower, all can produce the IgG of higher level containing the low dose group of adjuvant, middle dosage group, high dose group
1, and the effect of AECD100 μ g group is the most remarkable, significant difference (P<0.05) compared with antigen control group, suitable with aluminium adjuvant, and all can produce the IgG of higher level containing each group of adjuvant
2athe effect of AECD100 μ g group is the most remarkable, difference is compared extremely significantly (P<0.01) with aluminium adjuvant with antigen control group, the result that antibody subtype detects shows, adjuvant Herba Cistanches polysaccharides can not only make OVA protein vaccine induce body to produce very high humoral immune reaction, also can produce the cell immune response of high level.
35d antibody subtype testing result after table 3 immune mouse
| Group | IgG | IgG 1 | IgG 2a |
| 0.9%NaCl | 0.024±0.008 a | 0.014±0.004 a | 0.023±0.007 a |
| AECD 400 μg | 0.023±0.009 a | 0.015±0.003 a | 0.024±0.006 a |
| OVA 10 μg | 0.324±0.079 b | 0.509±0.203 b | 0.566±0.455 b |
| Alum200 μg+OVA 10 μg | 0.794±0.255 c | 0.730±0.167 c | 0.494±0.279 b |
| AECD 20 μg+OVA 10 μg | 0.675±0.159 c | 0.592±0.302 b | 0.819±0.393 c |
| AECD 100 μg+OVA 10 μg | 0.973±0.241 c | 0.824±0.318 c | 1.368±0.276 d |
| AECD 400 μg+OVA 10 μg | 0.456±0.102 b | 0.3518±0.05 b | 0.685±0.432 b |
The cellular immune level after mouse immune is detected with mtt assay, specific as follows: within after second time subcutaneous inoculation 7 days, to put to death mice, aseptically, get mouse spleen, grind, remove erythrocyte with erythrocyte cracked liquid, and nylon column removing B cell makes single cell suspension excessively, PBS liquid washes three times, centrifugal and carry out cell counting, and adjustment cell concentration is to 1 × 10
6individual/ml, often organizing cell suspension divides 4 parts to add in 96 porocyte culture plates: a copy of it adds OVA antigen to final concentration 10 μ g/ml, portion adds ConA(concanavalin A, Con A) or LPS(lipopolysaccharide) be that 5 μ g/ml or 10 μ g/ml are as positive control to final concentration, portion adds BSA to final concentration 2 μ g/ml as negative control, cultivate 48 hours, every hole adds the MTT(tetramethyl azo azoles salt of 20 μ l), cultivate after 4 hours, 2000 leave the heart 5 minutes, abandon cell conditioned medium, add the DMSO(dimethyl sulfoxide of 100 μ l), after 37 DEG C of incubators place 15 minutes, microplate reader reads the OD value at 570nm place, calculate stimulation index (stimulationIndex, SI), SI=(experimental group OD-culture medium OD)/(cell OD-culture medium OD).Wherein, experimental group OD refers to the OD value that the cell of antigenic stimulus reads, and cell OD refers to the OD value that the cell without antigenic stimulus reads.
Result (is the mean+SD of 3 mices in table 4 as shown in table 4 and Fig. 3, different letter representation P < 0.05): blank group, saline control group, substantially stimulate the effect of T cell very low without adjuvant OVA antigen group, aluminium adjuvant group, Herba Cistanches polysaccharides has obvious potentiation, significant difference (P < 0.05) compared with other each group to the mice spleen lymphocytes proliferation reaction that ConA and LPS induces.
After table 4 immune mouse, 21d detects Spleen cell proliferation
| Group | ConA 5μg/mL | LPS 10μg/mL |
| Blank | 1.435±0.400 a | 1.328±0.312 a |
| 0.9%NaCl | 1.371±0.330 a | 1.422±0.172 a |
| OVA10 μg | 1.656±0.319 a | 1.576±0.227 a |
| Alum200 μg | 2.378±0.633 ab | 2.007±0.646 a |
| AECD100 μg +OVA10 μg | 3.768±0.176 c | 3.411±0.466 b |
Vaccine is dissolved in normal saline, every injected in mice 100 microlitre, and the 0th day subcutaneous first, and after first time injection, interval is injected for 14 days again, injects twice altogether.
In order to detect the expression of cytokine after mouse immune, with the expression of CD4+ cell IL-4 and IFN-γ after Flow cytometry immunity.Specific as follows: within after second time subcutaneous inoculation 7 days, put to death mice, obtain T lymphocyte as stated above, adjustment cell concentration is to 5 × 10
6individual/ml, every hole adds 100 μ l cell suspension on 96 porocyte culture plates, adding OVA every hole final concentration is 10 μ g/ml, stimulate 12-14 hour, add after monensin cultivates 2h again, centrifugal collecting cell, anti-Fc gamma antibodies (BD company, the U.S.) 4 DEG C of closed 15min, PBS washes 3 times, anti-CD4-FITC antibody (BD company, the U.S.) 4 DEG C of dyeing 30min, PBS washes 3 times, the fixing 10min of 4% paraformaldehyde 4 DEG C, PBS washes 3 times, 0.1% Saponin rupture of membranes 10min, PBS washes 3 times, anti-IL-4-PE or IFN-γ-PE antibody (BD company, the U.S.) dye 15min, PBS washes 3 times, flow cytomery.
IFN-γ and IL-4 expression of results as shown in table 5 and Fig. 4, Fig. 5 (in table 5 average ± the standard deviation of 3 mices, different letter representation P<0.01).
The expression of blank group, saline control group IFN-γ and IL-4 is very low, shows that above combination does not possess immune effect substantially; All produce low-level IFN-γ and IL-4 without the OVA antigen group of adjuvant and aluminium adjuvant immune group to express, this shows that immunizing antigen group has certain immune effect; Compared to above combination, the immunizing antigen group containing adjuvant AECD100 μ g can produce high-caliber IFN-γ and IL-4, and compared with only immune OVA antigen group, the level of IFN-γ and IL-4 has had significant raising (P<0.01).The above results shows, AECD100 μ g can be good at induction OVA vaccine and produces IFN-γ and IL-4, and these results illustrate that Herba Cistanches polysaccharides obviously can promote CD4 as the adjuvant of protein vaccine
+t cell secretion IL-4 and IFN-γ, promotes Th
1and Th
2the activation of cell, thus strengthen body fluid and cellular immune level.
Table 5IFN-γ and IL-4 expression of results
| Group | INF-γ/CD4+Tcell(%) | IL-4/CD4+Tcell(%) |
| Blank | 5.186±1.778 a | 6.180±4.061 a |
| 0.9%NaCl | 5.710±3.859 a | 6.646±2.178 a |
| OVA10 μg | 8.596±0.951 a | 6.667±3.141 a |
| Alum200 μg+OVA 10 μg | 8.237±4.033 a | 13.3433±0.963 b |
| AECD100 μg +OVA10 μg | 14.656±0.478 b | 14.530±2.0403 b |
Above experimental result shows, meat desert polysaccharide, as the adjuvant of protein vaccine, significantly can strengthen humoral immunity level, cellular immune level, and effect is better than aluminium adjuvant.
Embodiment 2: Herba Cistanches polysaccharides adjuvant infected by influenza inactivated vaccine (Flu) Immune pattern zoopery
The preparation of the present embodiment Herba Cistanches polysaccharides obtains by method described in the embodiment of the present invention 1.
Test material: the many pools of Herba Cistanches such as embodiment 1 is prepared, and influenza vaccines are commercially available.
Animal immune: 6-8 Female ICR mice in age in week, divide 6 groups, often organize 6, experiment grouping is as shown in table 6,
Table 6 mouse immune divides into groups
| Group number | Kind (female) | Number of elements | Injectable composition |
| 1 | ICR | 6 | 0.9%NaCl |
| 2 | ICR | 6 | AECD800μg |
| 3 | ICR | 6 | Influenza vaccines 0.5 μ g |
| 4 | ICR | 6 | Influenza vaccines 0.5 μ g+AECD100 μ g |
| 5 | ICR | 6 | Influenza vaccines 0.5 μ g+AECD400 μ g |
| 6 | ICR | 6 | Influenza vaccines 0.5 μ g+AECD800 μ g |
Vaccine is dissolved in normal saline, every injected in mice 100 microlitre, the 0th day subcutaneous injection, and after first time injection, interval is injected for 14 days again, injects twice altogether.
Second time subcutaneous injection after 7 days, blood sampling separation of serum, indirect elisa method detects IgG respectively, IgG
1and IgG
2aantibody horizontal.Detection method such as the embodiment of the present invention 1 is carried out.
As shown in table 7 and table 8, Fig. 6 and Fig. 7, (numerical value is the mean+SD of 6 mices to result, different letter representation p<0.05): blank group and holosaccharide group antibody horizontal minimum, show that combinations thereof does not possess immune effect substantially; Without the IgG that the influenza vaccines group of adjuvant and the Herba Cistanches polysaccharides group of low dosage produce, IgG
1and IgG
2athere is no significant difference, show that Herba Cistanches polysaccharides low dosage adjuvant group does not produce substantially and significantly promote immune effect; Compared to combinations thereof, produce high-caliber IgG, IgG containing dosage in adjuvant and high dose immune function
1and IgG
2a, and in Herba Cistanches polysaccharides, the effect of dosage 400 μ g immune group is the most remarkable, with influenza vaccines group difference extremely remarkable (p<0.01).The above results shows, adjuvant Herba Cistanches polysaccharides can make vaccinum influenzae inactivatum induce body to produce very high humoral immune reaction and cell immune response.
IgG antibody testing result after table 7 immune mouse
| Group | 14d | 28d | 42d |
| 0.9%NaCl | 0.040±0.006 a | 0.035±0.007 a | 0.038±0.006 a |
| AECD800μg | 0.038±0.010 a | 0.032±0.011 a | 0.031±0.012 a |
| Flu0.5μg | 0.077±0.011 a | 1.215±0.243 b | 1.358±0.229 b |
| AECD100μg+ Flu0.5μg | 0.171±0.005 a | 1.194±0.203 b | 1.318±0.235 b |
| AECD400μg +Flu0.5μg | 0.405±0.067 b | 1.746±0.367 c | 1.816±0.229 c |
| AECD800μg +Flu0.5μg | 0.223±0.040 a | 1.631±0.419 c | 1.675±0.337 c |
35 days antibody subtype testing results after table 8 immune mouse
| Group | IgG | IgG1 | IgG2a |
| 0.9%NaCl | 0.009±0.002 a | 0.008±0.002 a | 0.024±0.005 a |
| AECD800μg | 0.008±0.001 a | 0.008±0.002 a | 0.024±0.004 a |
| Flu0.5μg | 0.874±0.170 b | 0.963±0.026 b | 1.329±0.249 b |
| AECD100μg+ Flu0.5μg | 1.127±0.264 c | 1.445±0.271 c | 0.555±0.173 c |
| AECD400μg+ Flu0.5μg | 1.433±0.281 d | 1.627±0.358 c | 1.844±0.226 d |
| AECD800μg+ Flu0.5μg | 1.371±0.305 d | 1.604±0.311 c | 1.832±0.261 d |
Above experimental result shows, Herba Cistanches polysaccharides can the humoral immune reaction of enhanced flow influenza vaccine and cell immune response effectively as the adjuvant of vaccinum influenzae inactivatum.
Embodiment 3: Herba Cistanches polysaccharides adjuvant is to the zoopery of inactivated foot-and-mouth disease vaccine Immune pattern
The preparation of the present embodiment Herba Cistanches polysaccharides obtains by method described in the embodiment of the present invention 1.
Experiment material: the many pools of Herba Cistanches are prepared by the embodiment of the present invention 1, foot and mouth disease virus (FootandMouthvirusdisease, FMDV) inactivated vaccine is Tian Kang drugmaker product.
Animal immune: 6-8 Female ICR mice in age in week, be divided into 6 groups at random, often organize 6, experiment grouping is as shown in table 9,
Table 9 mouse immune divides into groups
| Group number | Kind (female) | Number of elements | Injectable composition |
| 1 | ICR | 6 | 0.9%NaCl |
| 2 | ICR | 6 | AECD800μg |
| 3 | ICR | 6 | FMDV 0.5μg |
| 4 | ICR | 6 | FMDV+ commodity adjuvant |
| 5 | ICR | 6 | FMDV+AECD100μg |
| 6 | ICR | 6 | FMDV+AECD400μg |
| 7 | ICR | 6 | FMDV+AECD800μg |
Vaccine is dissolved in normal saline, every injected in mice 100 microlitre, the 0th day subcutaneous injection, and after first time injection, interval is injected for 14 days again, injects twice altogether.
Second time subcutaneous injection after 7 days, blood sampling separation of serum, indirect elisa method detects IgG respectively, IgG
1and IgG
2aantibody horizontal.Detection method such as the embodiment of the present invention 1 is carried out.
(numerical value in table is the mean+SD of 6 mices to result such as table 10, different letter representation p<0.05) and table 11(show in numerical value be the OD value of 6 mice pooled serums), shown in Fig. 8 and Fig. 9: blank group and holosaccharide group antibody horizontal minimum, show that combinations thereof does not possess immune effect substantially; Without the IgG that the Herba Cistanches polysaccharides group of the inactivated foot-and-mouth disease vaccine group of adjuvant, inactivated foot-and-mouth disease vaccine group and low dosage produces, IgG
1and IgG
2athere is no significant difference, show that Herba Cistanches polysaccharides low dosage adjuvant group does not produce substantially and significantly promote immune effect; Compared to combinations thereof, produce high-caliber IgG, IgG containing dosage in adjuvant and high dose immune function
1and IgG
2a, and in Herba Cistanches polysaccharides, the effect of dosage 400 μ g immune group is the most remarkable, with inactivated foot-and-mouth disease vaccine group and inactivated foot-and-mouth disease vaccine commodity adjuvant group difference extremely remarkable (p<0.01).The above results shows, adjuvant Herba Cistanches polysaccharides can make inactivated foot-and-mouth disease vaccine induce body to produce very high humoral immune reaction and cell immune response, and effect is better than commodity adjuvant group.
IgG antibody testing result after table 10 immune mouse
| Group | 14d | 28d | 42d |
| 0.9%NaCl | 0.039 scholar 0.008 a | 0.045 scholar 0.008 a | 0.048 scholar 0.007 a |
| AECD800μg | 0.036 scholar 0.006 a | 0.047 scholar 0.009 a | 0.084 scholar 0.007 a |
| FMDV | 0.256 scholar 0.056 b | 0.263 scholar 0.087 a | 0.300 scholar 0.067 b |
| FMDV+ adjuvant | 0.279 scholar 0.079 b | 0.277 scholar 0.073 b | 0.277 scholar 0.127 b |
| FMDV+AECD 100μg | 0.139 scholar 0.040 b | 0.456 scholar 0.099 c | 0.460 scholar 0.179 b |
| FMDV+AECD 400μg | 0.669 scholar 0.141 d | 0.717 scholar 0.234 d | 0.801 scholar 0.195 d |
| FMDV+AECD 800μg | 0.499 scholar 0.099 c | 0.664 scholar 0.165 d | 0.655 scholar 0.188 c |
24 days antibody subtype testing results after table 11 immune mouse
| Group | IgG | IgG 1 | IgG 2a |
| 0.9%NaCl | 0.041 scholar 0.002 | 0.024 scholar 0.006 | 0.045 scholar 0.002 |
| AECD800μg | 0.050 scholar 0.001 | 0.034 scholar 0.002 | 0.033 scholar 0.003 |
| FMDV | 0.421 scholar 0.014 | 0.365 scholar 0.008 | 0.072 scholar 0.004 |
| FMDV+ adjuvant | 0.509 scholar 0.009 | 0.409 scholar 0.006 | 0.207 scholar 0.004 |
| FMDV+AECD 100μg | 0.573 scholar 0.007 | 0.521 scholar 0.027 | 0.065 scholar 0.003 |
| FMDV+AECD 400μg | 0.940 scholar 0.037 | 0.647 scholar 0.018 | 0.329 scholar 0.016 |
| FMDV+AECD 800μg | 0.725 scholar 0.038 | 0.544 scholar 0.029 | 0.169 scholar 0.009 |
This result shows, Herba Cistanches polysaccharides can strengthen humoral immune reaction effectively as the adjuvant of inactivated foot-and-mouth disease vaccine.
Embodiment 4: Herba Cistanches polysaccharides adjuvant is tested OVA DNA gene vaccine model animal
The preparation of the present embodiment Herba Cistanches polysaccharides obtains by method described in the embodiment of the present invention 1, and the many pools of Herba Cistanches are prepared by the embodiment of the present invention 1.
Experiment material: recombiant plasmid proVAX-OVA, proVAX alkaline lysis extracts in a small amount, after enzyme action qualification is correct, prepares plasmid DNA in a large number through alkaline lysis, then through PEG8000 purification, uses agarose gel electrophoresis quantitative method to carry out quantitatively for subsequent use.
Animal immune: the Female ICR mice getting 6-8 age in week respectively, is divided at random and often organizes 6, and experiment grouping is as shown in table 12.
Table 12 mouse immune grouped table
| Group number | Kind (female) | Number of elements | Injectable composition |
| 1 | ICR | 6 | 0.9%NaCl |
| 2 | ICR | 6 | proVAX100μg |
| 3 | ICR | 6 | AECD 800100μg |
| 4 | ICR | 6 | proVAX-OVA 100μg |
| 5 | ICR | 6 | proVAX-OVA 100μg+AECD 100μg |
| 6 | ICR | 6 | proVAX-OVA 100μg+AECD 400μg |
| 7 | ICR | 6 | proVAX-OVA 100μg +AECD 800μg |
Often organizing vaccine is dissolved in normal saline, every injected in mice 100 microlitre, intramuscular injection first in the 0th day, and after first time injection, interval is injected for 14 days again, injects three times altogether.
After third time intramuscular injection, blood sampling separation of serum, indirect elisa method detects IgG antibody level.Detection method such as the embodiment of the present invention 1 is carried out.
Result is (numerical value in table 13 is the titre of 6 mice pooled serums) as shown in table 13 and Figure 10: saline control group, minimum without the proVAX matched group of adjuvant without antigen, AECD vehicle control group IgG level without antigen, shows that above combination does not possess immune effect substantially; Without adjuvant proVAX-OVA immune group and have the low dosage immunizing antigen group of AECD adjuvant (proVAX-OVA+AECD100 μ g) all to produce low-level IgG, this shows that immunizing antigen group (proVAX-OVA) has certain immune effect, and AECD100 μ does not substantially produce and significantly promotes immune effect; Compared to above combination, the immunizing antigen group containing adjuvant proVAX-OVA+AECD400 μ g can produce high-caliber IgG, and effect is the most remarkable.
IgG antibody titre testing result after table 13 mouse immune
| Group | 14d | 21d | 35d | 49d |
| 0.9%NaCl | 150 | 200 | 100 | 150 |
| proVAX | 200 | 300 | 200 | 400 |
| AECD800μg | 300 | 300 | 400 | 450 |
| proVAX-OVA | 300 | 350 | 500 | 600 |
| proVAX-OVA+AECD100μg | 300 | 400 | 600 | 800 |
| proVAX-OVA+AECD400μg | 900 | 3000 | 3000 | 2500 |
| proVAX-OVA+AECD800μg | 350 | 500 | 600 | 1000 |
The above results shows, adjuvant AECD can make OVA gene vaccine induce body to produce higher humoral immune reaction.
Claims (6)
1. Herba Cistanches polysaccharides is as the application of vaccine adjuvant.
2. Herba Cistanches polysaccharides, as the application of vaccine adjuvant, is characterized in that as claimed in claim 1: described vaccine is protein vaccine.
3. Herba Cistanches polysaccharides, as the application of vaccine adjuvant, is characterized in that as claimed in claim 1: described vaccine is gene vaccine.
4. Herba Cistanches polysaccharides, as the application of vaccine adjuvant, is characterized in that as claimed in claim 1: described vaccine is inactivated vaccine.
5. Herba Cistanches polysaccharides, as the application of vaccine adjuvant, is characterized in that as claimed in claim 1: described vaccine is attenuated live vaccine.
6. as described in claim 1,2,3,4 and 5, Herba Cistanches polysaccharides, as the application of vaccine adjuvant, is characterized in that: described vaccine assistant is ejection preparation, and injected dose is per kilogram of body weight 1.5mg ~ 50mg.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636321A (en) * | 2016-07-21 | 2017-05-10 | 湖州市农业科学研究院 | Method for studying reaction of cordyceps militaris polysacharide to humoral immune response and cellular immune response of organisms |
| CN106729699A (en) * | 2016-11-29 | 2017-05-31 | 浙江美保龙生物技术有限公司 | A kind of pseudorabies live vaccine dilution |
| CN107007830A (en) * | 2017-06-02 | 2017-08-04 | 中山大学 | A kind of purposes, vaccine and the preparation method of avirulent strain toxoplasm and herbal polysaccharide adjunvant composition |
| CN114306590A (en) * | 2021-12-31 | 2022-04-12 | 皖西学院 | Veterinary vaccine diluent with immunity enhancement effect and preparation method and application thereof |
| CN116549482A (en) * | 2023-05-24 | 2023-08-08 | 广东药科大学 | Application of ginseng polysaccharide in preparation of immunoregulation product |
-
2015
- 2015-08-14 CN CN201510498302.0A patent/CN105079801A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 曾群力等: "肉苁蓉多糖的免疫活性作用及机制", 《浙江大学学报(医学版)》 * |
| 李科: "肉苁蓉多糖脂质体制备及对鸡新城疫苗免疫效果影响的研究", 《中国优秀硕士学位论文全文数据库，农业科技辑，西南大学硕士学位论文》 * |
| 王翔岩: "肉苁蓉多糖的免疫调节活性及吸收特性研究", 《中国优秀硕士学位论文全文数据库，医药卫生科技辑，中国协和医科大学硕士学位论文》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636321A (en) * | 2016-07-21 | 2017-05-10 | 湖州市农业科学研究院 | Method for studying reaction of cordyceps militaris polysacharide to humoral immune response and cellular immune response of organisms |
| CN106729699A (en) * | 2016-11-29 | 2017-05-31 | 浙江美保龙生物技术有限公司 | A kind of pseudorabies live vaccine dilution |
| CN107007830A (en) * | 2017-06-02 | 2017-08-04 | 中山大学 | A kind of purposes, vaccine and the preparation method of avirulent strain toxoplasm and herbal polysaccharide adjunvant composition |
| CN107007830B (en) * | 2017-06-02 | 2020-07-14 | 中山大学 | Application of toxoplasma gondii and traditional Chinese medicine polysaccharide adjuvant composition, vaccine and preparation method |
| CN114306590A (en) * | 2021-12-31 | 2022-04-12 | 皖西学院 | Veterinary vaccine diluent with immunity enhancement effect and preparation method and application thereof |
| CN116549482A (en) * | 2023-05-24 | 2023-08-08 | 广东药科大学 | Application of ginseng polysaccharide in preparation of immunoregulation product |
| CN116549482B (en) * | 2023-05-24 | 2023-11-07 | 广东药科大学 | Application of ginseng polysaccharide in preparation of immunoregulation product |
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