CN106994183A - Application of the trichosanthin in enhancing vaccine specific antibody - Google Patents

Abstract

The present invention discloses application of the trichosanthin in enhancing vaccine specific antibody.The present invention breaks through tradition, it was found that the trichosanthin in Chinese medicine source can significantly increase epidemic meningitis and influenza vaccines specific antibody level, this is to be clinically used for anti-AIDS after Trichosanthin, after Robust speaker feature, at home and abroad find that it is used to strengthen and improves existing infection prevention disease vaccine effect first, used dosage is again extremely low, natural traditional Chinese medicine vegetable protein of drawing materials, and is with Chinese characteristics, the efficient Chinese medicine preparation of low toxicity.

Description

Application of the trichosanthin in enhancing vaccine specific antibody

Technical field

The invention belongs to biomedical sector, more particularly, it is related to trichosanthin in enhancing vaccine specific antibody In application.

Background technology

Annual influenza virus prevalence, except higher fatal rate is triggered, is also a great prestige for pandemics disease The side of body, for children and the elderly, incidence and mortality is higher.Inoculation influenza vaccines are prevention and control the main of influenza generation One of measure, inoculation Seasonal Influenza Vaccine plays very important effect for control influenza.At present, go through The influenza vaccines used are divided into two major classes：Inactivated influenza vaccine and attenuated influenza live vaccine.Inactivated influenza vaccine be broadly divided into Under several classes：Inactivated virus vaccine, inactivation of viruses pyrolysis product vaccine and inactivation subunit vaccine.Relatively broad application at this stage Be trivalent inactivation split vaccine.And produced using chicken embryo, 6 months are at least needed to the vaccine of annual newfashioned strain.Though So in past 30 years, produce the speed of recombinant protein, the exploitation subunit vaccine that rises to of efficiency and provide new machine Meet.However, compared with traditional attenuation or inactivated vaccine, subunit vaccine immunogenicity lacks safely and effectively people than relatively low Such vaccine development is seriously constrained with adjuvant, the not enough problem of production capacity of vaccine shows lofty especially in influenza great outburst.Therefore, Influenza vaccines production capacity is improved, control influenza is pandemic to be to find the method that can improve influenza vaccines specific antibody at all, Correlative study is also one of focus of current vaccines research, than such as by the immunogenicity for improving vaccine antigen, so as to reduce Antigen consumption per vaccinating agent finally improves vaccine production capacity.

Meningococal meningitis (epidemic cerebrospinal meningitis) be most common infectious disease it One, with the characteristics of infectiousness is strong, crowd's subclinical infection is more, it is broadly divided into two kinds of plain edition and fulminant.Patients with clinical manifestations For the symptom such as severe headache, frequent vomiting, hyperpyrexia, the sequelae of brain parenchym infringement is often left.It is in a bad way and loses Mass formed by blood stasis and involve breathing and the circulatory system cause its MSOF, threat to life.

The hotspot of epidemic meningitis is Africa, Asia and South America, and the cases of 30-50 ten thousand occur every year on average, to children and always Year people's harm is particularly acute, and its case fatality rate of fulminant epidemic meningitis infected children may be up to 40%-60%, old age artificial 17.6%.I Once there are 5 national epidemic meningitis and was very popular in state, wherein the epidemic meningitis prevalence spring in 1967 creates China since the dawn of human civilization most Strong onset peak, the incidence of disease is up to 4,03/,100,000, case fatality rate 5.49%.

Therefore, vaccine of epidemic menigitis inoculation is the key measure that prevention and control epidemic meningitis occur, and China is formal batch from 1980 Quasi- A groups of epidemic meningitis polysaccharide vaccines of production, greatly control the whole nation outburst of epidemic meningitis.But according to the data over nearly 20 years, China Annual epidemic meningitis number of the infected can all reach thousands of people, and 1994, epidemic meningitis in 2003 there occurs the obvious incidence of disease twice Rebound phenomena, 2003 year whole nation reports 2490 rise 0.53%, dead 134, death toll increase with the same period last year 8.06%, statistics shows that its virulence has the outburst rule of 8-10 a cycles, it is contemplated that next number of the infected bounce-back is high Peak is at hand.Causing the main cause of this phenomenon mainly includes two aspects：

First, the A groups of popular preponderant genotypes of epidemic meningitis are periodically converted, and cause this disease cycle popular；

Secondly, it is immunized it was verified that polysaccharide antigen belongs to greatly T cell independent antigen, it is impossible to effectively activate auxiliary Helping property T cell (Th) and T memory cells, it is impossible to which induction produces immunological memory function, the immunoprotection time is of short duration, is immunized connects again Booster immunization reaction can not be produced by planting.And existing vaccine of epidemic menigitis is exactly polysaccharide vaccine, its immunogenicity is poor, stimulates body to produce The inefficiency of raw immune response even can produce exception response.

Data is shown, though N.meningitidis polysaccharides can induce generation IgM and IgG antibody, the IgG produced by the mankind Mainly IgG2 subclass, and the Subclass Antibodies of human serum IgG 2 occur slower, typically can just rise to adult levels to 8-12 Sui, therefore And infant is inoculated with after the polysaccharide vaccine, the antibody of generation is based on the IgM antibody with of short duration effect, and which results in epidemic meningitis The problem of vaccine is especially weak to infant's protective capability of less than two years old, protection period is short.

Therefore, on the one hand inoculation vaccine of epidemic menigitis is promoted in reinforcement, and it is to strengthen it on the other hand to improve vaccine of epidemic menigitis immunogenicity Protecting effect is to preventing and controlling epidemic meningitis morbidity and prevalence significant.

In addition, being inoculated with the regulation of vaccine of epidemic menigitis immune programme for children according to country at present, 4 need to be inoculated with altogether by being completely inoculated with the vaccine Agent, the 1st, be immunized based on 2 doses, the 3rd, be booster immunization 4 doses times, children are inoculated with the 1st dose from 6 monthly ages, and 2 doses of minor ticks are no less than 3 months, the 3rd dose is inoculated with 3 years old, is no less than 1 year with the 2nd dose of interval time；The 4th dose is inoculated with 6 years old, is spaced with the 3rd dose of inoculation No less than 3 years.

It is not difficult to find out, existing immune programme for children is cumbersome compared with other vaccine inoculations and complexity, with more than inoculation times and flowing youngster The high shortcoming of virgin vaccine miss rate, directly influences the immune effect of vaccine.

For above vaccine of epidemic menigitis immunogenicity it is poor, the practical problem such as cumbersome is inoculated with, if we can develop more preferably Can strengthen the immunogenicity of existing vaccine, the strategy and technical method of the specificity protection antibody of induced high levels just have Wish that existing vaccine of epidemic menigitis can be solved especially weak to the low immunogenicity of infant, inducing immunological memory, immune response ability Only short-term protective effect the problems such as.

Latest research shows that polysaccharide is combined into combined vaccine with protein and can partly improve lacking for polysaccharide vaccine Fall into.Successful experience is researched and developed with reference to Type B haemophilus influenzae (Hib) combined vaccine, scholars are trying to explore with similar Mode improve vaccine of epidemic menigitis protecting effect, reduce inoculation adverse reaction.

Have in experience, adjuvant is non-specifically to strengthen specific immune response or change of the body to specific antigen Type of immune response, and the material of no antigen itself, can simultaneously be applied prior to or with antigen.Adjuvant should as immunopotentiator For influenza vaccines, it is of great significance for strengthening the immunogenicity of vaccine, the immune effect of raising vaccine.At present, Existing aluminium adjuvant is used in the trivalent inactivated influenza vaccine (TIV) of commercialization, and prove can with the immunogenicity of enhancement antigen, Reduce the antigen required for effective immune response.But aluminium adjuvant the disadvantage is that, it can not induce generation Th1 types reaction, do Cellular immunity is disturbed, and blocks CD8+CTL activation so that the immanoprotection action of vaccine is not comprehensive, not persistently, and also has one A little adverse reactions.But because the applicating history of aluminium adjuvant is longer, the relatively new adjuvant of security is more reliable, therefore can not also at present It is fully substituted.Although other many vaccine adjuvants were all once used to strengthen the research of influenza vaccines immunogenicity, only MF59 Oil in water emulsion such as (MF59 aqueous emulsions) shows stronger adjuvant effect and finally goes through to make in influenza vaccines in Europe With, but this kind of adjuvant is while immunogenicity is increased, also intensified response originality, in adult visible slight local pain, The increase of the symptoms such as heating, erythema, scleroma, myalgia.

In view of the shortcoming of above vaccine and adjuvant, find it is new, can specificity enhancing influenza and epidemic meningitis antiviral antibody level Adjuvant-type substances with the time are the task of top priority.

In this respect, the valuable medium-height grass Drug Storage of China has natural advantage.Trichosanthin (Trichosanthin, TCS) it is a kind of basic protein for being extracted from the root block of Curcurbitaceae Trichosanthes snakegourd, bimillennial go through of being used as medicine History.TCS clinics are mainly used in the treatment of Robust speaker feature and ectopic pregnancy, vesicular mole, suede cancer etc., while it is also uniquely to be clinically used for The Chinese medicine of anti-AIDS, there is obvious inhibiting effect to inhibition of HIV, and this inhibitory action and TCS concentration are proportionate.Smallpox Amyloid proteins as a kind of vegetable protein, have it is cheap, easy obtain, the features such as biological agent is extensive, make it have good research Prospect and practical value.

From the eighties, domestic and international many scholars have started from multi-angle observation shadow of the trichosanthin to immune system Ring.The result of study of different research groups finds that there are trichosanthin two kinds of immunological regulations of immunosupress and Immune-enhancing effect to make With：TCS can induce the immunosupress of human T cell's mediation, i.e. the T that TCS is induced soluble antigen and polyclonal activator is thin Born of the same parents' propagation has strong inhibitory action.Result to its mechanism of action shows that TCS induction immunosupress needs antigen submission thin Born of the same parents participate in, and are related to the functional expression of antigen processing submission and HLA-DQ molecules, the TCS of low dosage can also direct interference T lymphs The normal function of cells play.These results all illustrate that TCS may have depression effect to allogeneic immune response.But one Determine the TCS of concentration can also promote phytohemagglutin phytolectin stimulate lower PBMC of healthy people (PBMC) secretion IL-2 with IL-6, because IL-2 may act on Tc cells, produces killing and cellulotoxic effect, participates in cellullar immunologic response；IL-6 then stimulates B Cell propagation produces antibody, mediates the humoral immune response of body.Therefore TCS is played by promoting IL-2 and IL-6 secretion Strengthen the effect of immune response.TCS can promote IgG secretions in mouse salivary glands to increase, and promote mouse spleen immunocyte Formation and differentiation, make bone-marrow-derived lymphocyte and thick liquid cell quantity showed increased, strengthen humoral immunity.Certain density root of Chinese trichosanthes egg White enhancing immunoregulation effect prompting root of Chinese trichosanthes may have the effect of similar adjuvant.

The content of the invention

It is an object of the invention to provide application of the trichosanthin in enhancing vaccine specific antibody.

To realize object above, the present invention discloses following technical scheme：Trichosanthin is in enhancing vaccine specific antibody In application.

As a preferred scheme, the usage amount of the trichosanthin is 0.004-0.04mg/Kg.

As a preferred scheme, the vaccine refers to influenza vaccines and meningococal meningitis vaccine.

The advantage of the invention is that：Very low dose Trichosanthin can significantly increase vaccine specific antibody mediated immunity should Level is answered, provides strong to develop the enhanced new adjuvant influenza vaccines of immune effect and solving the problem of influenza vaccines production capacity is not enough Solution.The amount of original vaccine 1/10th only need to be used, you can reach protective effect, the titre of antibody and held time aobvious Increase is write, the usage amount of 90% vaccine is reduced.Research there is no to report both at home and abroad, trichosanthin is derived from Chinese medicine, great China is special Color.

Brief description of the drawings

The trichosanthin protein induced OVA specific humoral immune responses of Fig. 1, Land use models antigen-oralbumin (OVA) with Trichosanthin mixed immunity mouse, 1,2, the 3 weeks blood sampling after immune is coated with 96 orifice plates, general with OVA with 1ug/ml concentration After 100 times of the serum-dilution just exempted from, then 10000 times of serum-dilution after exempting from, OVA specific immunity ball eggs in serum are determined White IgG content, while setting up different control groups, determines that trichosanthin can induce OVA specific humoral immune responses.

Anti ova specific IgG 1 and IgG2a expresses general layout in Fig. 2 serum, and 96 holes are coated with 1ug/ml concentration with OVA Plate, by 100 times of the serum-dilution just exempted from, then after 10000 times of serum-dilution after exempting from, determines antigen in the serum after dilution special The IgG1 and IgG2a of the opposite sex, show the specific humoral immune response that TCS is dominated induction of Th2.

Fig. 3 TCS significantly increase cell factor IFN-r and IL-5 and significantly raised, and A figures, TCS significantly increases anti ova specificity IgG1 antibody levels；B figures TCS significantly increases the antiviral cell factor IFN-r and IL-5 secretion level relevant with antibody tormation, Pass through the measure of subtype-specific antibody in serum, it was demonstrated that TCS can significantly increase the leading humoral immune responses of Th2, while with ELISA checking TCS experimental groups in cell factor general layout change, as a result find with the antiviral cell relevant with antibody tormation because Sub- IFN-r and IL-5 are significantly raised.

Fig. 4 TCS significantly increase Specific anti-Flu virus IgG level in serum, and dosage ranging experiments determine TCS's Dose,optimum is 1 μ g/ mouse, the μ l of influenza vaccines Fluarix (GSK) 100 that GlaxoSmithKline PLC is produced and 1 μ g trichosanthins Mixed immunity mouse, while setting up blank control and aluminium adjuvant control, it is found that 1 μ g trichosanthins significantly increase mice serum Moderate resistance Flu specific antibody levels, are compared statistically significant (P ＜ 0.01) with control group.

Fig. 5 TCS significantly reduce influenza vaccines Fluarix consumption, still reach equivalent antibody level.By influenza Vaccine Fluarix is small with 1 μ g trichosanthin mixed immunities respectively according to (5 μ l, 10 μ l, 50 μ l, 100 μ l) after doubling dilution Mouse, while setting up control group, is detected by EILSA and found, if adding trichosanthin, even if only taking vaccine commonly uses agent 1/10th, 10 μ l of amount, still can reach the antibody level in 100 μ l vaccine dose equivalents, and both are compared without system Meter learns meaning.

Fig. 6 mice serums moderate resistance cerebrospinal meningitis antibody titre is with the change of immunization time, and TCS can significantly increase anti-current in serum The specific antibody of encephalovirus, improves specificity in vaccine of epidemic menigitis MCCV immune response, experimental group (MCCV+TCS) serum anti- Body titre is significantly higher than control group (MCCV) (P ＜ 0.01), Continuous Observation 7 weeks, and group difference started from the 2nd week, from the 4th week Start, the most notable in the 5th week difference, antibody titer reaches peak, shows that trichosanthin can significantly increase A groups of epidemic meningitis epidemic diseases The titre of seedling specific antibody.

Fig. 7 mice serums moderate resistance cerebrospinal meningitis antibody titre is with the change of immunization time, and it is anti-that TCS can significantly extend MC specificity Body time in vivo, thus strengthen guard time of the vaccine of epidemic menigitis to mouse.Protected after usual polysaccharide vaccine of epidemic menigitis traditional vaccination The shield time is about 3 months.It was found that compared with the control, after the 13rd week, specificity has been can't detect in control group serum anti- Body, and specific antibody still maintain higher level at 30 weeks in experimental group serum, still can reach highest titre When half.Illustrate that TCS make it that mouse is more lasting to the immune response of polysaccharide vaccine of epidemic menigitis, effectively prolongs by certain mechanism The guard time of vaccine is grown.

Fig. 8 A show that control group mice spleen B2 cells (B220+CD11b-) ratio is that 38.9%, Fig. 8 B show experimental group Mouse spleen B2 cell proportions are 57.5%, and experimental group B2 cells add 18.6% than control group, point out TCS to be obviously improved B2 cell percentages in Mice Body.

Fig. 9 A show control group mice marrow B2 progenitor cells (B220⁺CD11b^-) ratio be 12.9%, figure Fig. 9 B display experiment Group mouse bone marrow cells B2 progenitor cells ratio is 29.6%, and experimental group B2 cells add 16.7% than control group, points out TCS can be notable Lift B2 progenitor cells percentages in Mice Body.

Figure 10 A are the streaming result figure of control group mice cavum peritoneale B1 cells, and abscissa is CD11b, and ordinate is SSC, Control group mice cavum peritoneale CD11b+ ratios are 54.8%；Figure 10 B abscissas are B220, and ordinate is IgM, is set with CD11b+ Door, display control group mice B220lowIgMhigh ratios are that 84.3%, Figure 10 A, Figure 10 B show control group mice B1 cells (CD11b+B220lowIgMhigh) ratio is 54.8%*84.3%=46.2%.

Figure 11 A are the streaming result figure of experimental mice cavum peritoneale B1 cells, and abscissa is CD11b, and ordinate is SSC, experiment Group mouse peritoneum chamber CD11b+ ratios are 70.0%；Figure 11 B abscissas are B220, and ordinate is IgM, with CD11b+ gatings, are shown Show that control group mice B220lowIgMhigh ratios show experimental mice B1 cells (CD11b+ for 80.2%, Figure 11 A, Figure 11 B B220lowIgMhigh) ratio is 70.0%*80.2%=56.1%.

Figure 12 is foot-pad immunization and blood sampling flow and explanation.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.Experimental method used in following embodiments for example without Specified otherwise, is conventional method.Material, reagent used etc. in following embodiments, unless otherwise specified, can be from business way Footpath is obtained.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.

Present invention firstly discovers that Trichosanthin can significantly increase vaccine (subunit's influenza and polysaccharide epidemic meningitis epidemic disease Seedling) specific antibody level：

(1) providing trichosanthin strengthens the valid density of vaccine specific antibody；

(2) its enhanced application effect and Partial Mechanism are provided.

TCS strengthens the art technique method of subunit influenza vaccine specific antibody：

(1) Land use models antigen-oralbumin (OVA) and trichosanthin mixed immunity mouse, after immune 1, 2nd, take a blood sample within 3 weeks, determine the content of OVA specific immunoglobulins IgG in serum, while setting up different control groups, determine smallpox Amyloid proteins can induce OVA specific humoral immune responses (Fig. 1).

(2) dose,optimum that trichosanthin plays a role is determined, under the conditions of optimal dose, trichosanthin can be same When induction body fluid and cell immune response.First, by OVA and various concentrations trichosanthin mixed immunity mouse, ELISA is surveyed It is 1 μ g/ mouse come the dose,optimum for determining trichosanthin to determine OVA-specific IgG antibody in mice serum, It is scaled 0.004-0.04mg/Kg (Fig. 2).

(3) by dose,optimum TCS and OVA mixed immunity mouse, while different control groups are set up, in initial immunity and again Different time points after immune, with OVA stimulated in vitro mouse boosting cells, ELISA detects the secretion situation of Th1 cytokines； Simultaneously with MHC-I tetramer stainings method detection CD8 T cell number changes, and carry out cell inner dyeing, flow cytometry analysis The immune response of CD8 T cells.

(4) checking trichosanthin enhancing influenza antigen specific immune response.Using intramuscular injection immunization route, Detect the humoral and cellular immune response reaction of trichosanthin auxiliary influenza vaccines inducing mouse antigentic specificity.It is immune with reference to OVA Response intensity determines TCS dose,optimum (Fig. 3), and ELISA determines the specific total IgG of influenza antigen and IgA and IgG Subclass antibodies (Fig. 4)；Hirst's hemagglutination Inhibition test (HIA) evaluates the protecting effect of humoral immunity；Plaque assay determines blood The neutralizing antibody titers of resisiting influenza virus in clear, further evaluate humoral immunity protecting effect；Exempted from using ELISPOT analyses measure The level of the CD4 and CD8 T cells secretion of gamma-IFN of epidemic disease mouse influenza virus antigentic specificity；Influenza antigens are pierced in vitro Swash immune mouse spleen cell, ELISA determine cell supernatant in cell factor, such as IL-2, IFN-gamma, IL-4, IL-5 and IL-13 etc., determines immune response general layout.

(5) influenza vaccines are with after trichosanthin mixed immunity mouse, passing through challenge test, checking trichosanthin enhancing Influenza vaccines induce the protective effect of immune response.Meanwhile, mixed respectively with influenza vaccines or by influenza vaccines with trichosanthin Immune mouse is closed, the serum adoptive transfer of booster immunization mouse is fed back in normal mouse body, then to feeding back the small of serum Mouse carries out challenge test, the protective effect of adopting of the enhanced specific antibodies of checking TCS.

TCS strengthens the art technique method of polysaccharide vaccine of epidemic menigitis specific antibody：

(1) the C57BL/6 female mices of 8-10 week old strengthen stream by being randomly divided into 2 groups (separately setting one group of blank control) with reference to TCS The dosage of influenza vaccine specific antibody, strengthens A groups of vaccine of epidemic menigitis (A-MCCV) special according to below scheme (table 1) checking TCS Heterogenetic antibody titre.It was found that, control group mice serum moderate resistance cerebrospinal meningitis antibody titre is significantly lower than the experimental group that TCS is added in vaccine (Fig. 5).

Experiment sets group such as table 1：

Foot-pad immunization and blood sampling flow and explanation such as Figure 12.

Foot-pad immunization

1. g/ bottles of 90 μ A meningococcal polysaccharides vaccine (A-MCCV) is divided into two groups, every bottle plus 1.5ml buffered physiologics A-MCCV of the saline into 60 μ g/mL.One of which is standby, and another group of concentration according to 1 μ g/50 μ l is added after TCS mixings It is standby.

2. the immunoreagent configured is subjected to foot pad initial immunity, left and right foot pad to female mice respectively with insulin injection syringe Each 50 μ l, every mouse co-injection 0.1ml reagent.At the 24th day according to initial immunity dosage booster immunization.Booster immunization agent Amount is identical with initial immunity dosage.

3. two experimental groups can be set again according to result, one, in the case where other conditions are constant, suitably reduces injection The dosage of vaccine；Another cancels the booster immunization of the 21st day.Observation and the difference of other experimental groups and control group.

Blood sampling：

1. after being immunized 6 days, the eye socket blood sampling of first week is carried out.Estimate and obtain every μ l of mice serum 100.Each of which part Serum takes 5-10 μ l to carry out ELISA experiments (10 μ l then do secondary orifices), observes dramatically increasing for antibody.Remaining serum is preserved in time In -80 DEG C of refrigerators.

2. it is immune for the first time latter 13rd day, carry out second week eye socket blood sampling.

3. it is immune for the first time latter 20th day, carry out eye socket blood sampling in the 3rd week.

4. the 23rd day after being immunized for the first time, enter to be about to be immunized by force, immunizing dose is immune with first time.

5. booster immunization relief mouse has three days off.

6. it is immune for the first time latter 26th day, carry out 4th week eye socket blood sampling.

7. it is immune for the first time latter 33rd day, carry out eye socket blood sampling in the 5th week.

8. it is immune for the first time latter 40th day, eyeball of mouse is extractd, blood is collected as much as possible, mouse is put to death.

(2) unitary variant experiment is designed, checking addition TCS vaccine of epidemic menigitis can reduce immunogenic dose, reduction and be immunized The conditions such as number of times are issued to equivalent or more preferable protecting effect.

(3) mouse infection Neisseria meningitidis model is built, the weight data of mouse is monitored weekly since vaccine inoculation, Verify that TCS strengthens the protective effect of vaccine simultaneously.

After trichosanthin is mixed with vaccine of epidemic menigitis (MPV), foot-pad immunization mouse, respectively at the 2nd, 6,7,9,10 days periorbits Veniplex takes blood, continues to collect serum in many weeks, 96 orifice plates is coated with vaccine of epidemic menigitis with 20ug/ml concentration, by serum with 1:10 Dilution, IgM ELIAS secondary antibodies are with 1：1000 dilutions.Antigen-specific antibodies in the serum after dilution, analysis day are determined with ELISA method Change (Fig. 8-Figure 11) of the pollen protein to the ratio, the ratio of cavum peritoneale B1 cells of marrow B2 progenitor cells and spleen B2 cells.

Fig. 8-Figure 11 shows that TCS can dramatically increase individual spleen B2 cells, marrow B2 progenitor cells, cavum peritoneale B1 cells Percentage, point out growth and the B cell percentage of specific antibody to occur simultaneously.Take Mouse spleen cells, bone marrow cell, abdomen Membrane cavity cell, flow cytometry detects mouse spleen B2 cells respectively, marrow B2 progenitor cells, the percentage of cavum peritoneale B1 cells, Trichosanthin can improve the ratio of marrow B2 progenitor cells and spleen B2 cells, while the ratio of cavum peritoneale B1 cells also increases, The possibility that prompting B2 is transformed by B1 is consistent with ELISA results than relatively low.

For immunology angle, influenza and vaccine of epidemic menigitis can represent whole vaccines substantially, from vaccine antigen classification For be albumen or polysaccharide, and both of the above is albumen and polysaccharide respectively, thus above effect can be summarized into it is corresponding anti- All vaccines of original classification.

Main agents of the present invention and equipment：

Main agents：Trichosanthin parenteral solution (Shanghai Jinshan pharmaceutical Co., Ltd.), A meningococcal polysaccharide vaccines (Beijing Tiantan Biological Products Co.ltd), ELISA mouse cytokines detection kit (eBioscience), alkalescence Phosphatase Substrate Tablets (SIGMA), lymphocyte separation medium, hyclone (Hyclone Lab, Inc), RPMI1640 nutrient solutions (Invitrogen Corp),Anti-Mouse IgG2a(gamma 2a chain)(Rabbit)Antibody Alkaline Phosphatase Conjugated(ROCKLAND),Anti-Mouse IgG(H&L)(Goat)Antibody Alkaline Phosphatase Conjugated(ROCKLAND),Anti-Mouse IgG1(Gamma 1 chain)(Rabbit) Antibody Alkaline Phosphatase Conjuated(ROCKLAND)。

The main well culture plate of equipment 96 and 24 well culture plates, CO2 cell culture incubators (Heraeus), Thermo Scientific SORVALL Legend R+ desk centrifuges, the automatic enzyme mark detector (Molecular of MAX250 all-waves Device)。

Influenza vaccines：Trade name：Fluarix (Fluarix), composition (contains the purifying of following Strain per 0.5ml vaccines Antigen：) A3 types influenza virus cracking haemagglutinin antigen 15ug (2011/2012 years：First/Perth/16/2009 (H3N2)-pleiston：First/Victoria/210/2009NYMC X-187) A1 types influenza virus cracking haemagglutinin antigen 15ug (2011/2012 years：First/California/7/2009 (H1N1)-pleiston：First/California/7/2009NYMC X-181) Type B influenza virus cracking haemagglutinin antigen 15ug (2011/2012 years：Second/Brisbane/60/2008).

Vaccine of epidemic menigitis：07052571 (MCCV), epidemic meningitis-actor playing a martial role in Chinese operas's-D-A polysaccharide, A group meningitis ash Neisser bacteria culture fluid, warp Extract and obtain capsular polysaccharide antigen, add that suitable stabilizer is lyophilized to be made after purification.

Advantage of the present invention is shown：(1) influenza and vaccine of epidemic menigitis are strengthened by Trichosanthin inducing T cell subpopulations Specific antibody level, embody Chinese Resources characteristic, the Chinese medicine of materials has been used as medicine more than 2,000 years, with safety, it is cheap, be easy to get Feature, has no relevant report both at home and abroad.

(2) influenza virus variation at present is more, and production capacity is not enough；Vaccine of epidemic menigitis spininess, the cumbersome vaccine program at long interval make " missing " phenomenon it is increasingly frequent, especially for Floating Children, this problem is extremely serious.This allows vaccine protecting effect significantly Reduction.The present invention can improve vaccine service efficiency, while making to be inoculated with the case where reducing vaccine inoculation number of times, dosage Crowd obtains identical and persistently protected.

(3) Tk has by changing the method that cell factor general layout and the skew of T cell subgroup improve humoral immune response level Help fully understand the characteristic of China's Chinese medicine preparation trichosanthin from molecule and cellular level, and its play various biological work Property mechanism, be that this vegetable protein is used to treating larger range of clinical disease and shows new prospect, and may be new for development Vaccine provide thinking.

The present invention is simple and easy to apply, need to be only simply mixed with vaccine, you can reach specific antibody and the increased effect of efficiency. Very low dose Trichosanthin has the effect of good enhancing vaccine efficacy, can be used as vaccine efficacy reinforcing agent.By pole Low dosage trichosanthin and Seasonal Influenza Vaccine use in conjunction, can accelerate induction of antibodies to exempt to answer, by low dosage root of Chinese trichosanthes egg In vain with vaccine of epidemic menigitis use in conjunction, specific antibody can be significantly increased and exempt to answer, improved vaccine and exempt from originality, and significantly reduce anti-consumption Promote cell and humoral response simultaneously, reach more preferable protecting effect, be development of new adjuvant influenza vaccines with solving production capacity not Sufficient problem provides new approaches, has opened up the development space of vaccine reinforcing agent research.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (3)

1. application of the trichosanthin in enhancing vaccine specific antibody.

2. application of the trichosanthin according to claim 1 in enhancing vaccine specific antibody, it is characterised in that institute The usage amount for stating trichosanthin is 0.004-0.04mg/Kg.

3. application of the trichosanthin according to claim 1 in enhancing vaccine specific antibody, it is characterised in that institute State vaccine and refer to influenza vaccines and meningococal meningitis vaccine.