CN103463133A - Material including royal jelly-decomposing enzyme - Google Patents

Abstract

The invention relates to a material including royal jelly-decomposing enzyme, a royal jelly-decomposing enzyme, a royal jelly-decomposing material and a decomposing method of royal jelly. This material containing royal jelly-decomposing enzyme is prepared by a method including the following processes from the 2-3 day old queen bee larvae of Apis mellifera: (a) a process of centrifugal treatment of the suspension of body tissues of the queen bee larvae at <=6[deg.]C so as to separate to an upper layer of white solid form, a middle layer of solution and a lower layer of precipitate; and (b) a process of recovering the middle layer as the material containing royal jelly-decomposing enzyme. The royal jerry-decomposing enzyme is characterized by being contained in the above material containing royal jerry-decomposing enzyme. The royal jelly-decomposed products are prepared by using the material containing royal jelly-decomposing enzyme or the royal jelly-decomposing enzyme. The method for decomposing the royal jelly is characterized by using the material containing royal jelly-decomposing enzyme or the royal jelly-decomposing enzyme.

Description

Lac regis apis clastic enzyme contains thing

The application is to be that February 22, application number in 2008 are dividing an application of 200810007986.X, the denomination of invention application for a patent for invention that is " Lac regis apis clastic enzyme contains thing " for the applying date.

Technical field

The present invention relates to that Lac regis apis clastic enzyme prepared by 2～3 age in days queen bee nits by Apis mellifera (Apis mellifera) contains that thing, above-mentioned Lac regis apis clastic enzyme contain Lac regis apis clastic enzyme contained in thing and be prepared the decomposition method for the Lac regis apis analyte of feature and the Lac regis apis that uses above-mentioned Lac regis apis clastic enzyme to contain thing or above-mentioned Lac regis apis clastic enzyme so that contain thing or above-mentioned Lac regis apis clastic enzyme with above-mentioned Lac regis apis clastic enzyme.

Background technology

Lac regis apis is that worker bee is from the nutrient substance of the milky glue of hypopharyngeal gland and maxillary (mandible) glandular secretion in order to make the queen bee nit growth.Because queen bee and worker bee are homogeneities in heredity, therefore think in Lac regis apis that comprising some induces to the controlling elements of queen bee differentiation, but above-mentioned substance is not yet clear and definite.Above-mentioned controlling elements and to queen bee differentiation induce mechanism illustrate for the artificial production efficiency that improves queen bee etc. to be useful, therefore just to be studied by researcher in the world.

Lac regis apis changes according to the pick flowers kind etc. of flower of honey or pollen etc. of bee colony, kind, Apis, but usually fresh weight 2/3 be approximately moisture, and take protein, saccharide and fatty acid as main component.In addition, also contain a large amount of vitamin or mineral etc., nutritive value is very excellent.In addition, although be not confirmed, think a large amount of pharmacological actions such as thering is fatigue recovery effect, anti-allergic effects, antitumaous effect, immunological enhancement on science always.Therefore, be widely used in dietary supplement or pharmaceutical raw material.

In recent years, 40% the protein that accounts for the Lac regis apis dry weight is as the major physiological active substance of Lac regis apis and get most of the attention.Particularly, reported in a large number the invention of the physiologically active that utilizes the Lac regis apis protein decomposition product.For example, reported that (1) relates to the invention of infecting the defensive enginery reinforcing agent, it is characterized in that, containing as effective ingredient, molecular weight that obtain by the protein in the proteases for decomposing Lac regis apis is that peptide below 3000 is (for example,, with reference to patent documentation 1.）。Foregoing invention is based on following knowledge and the invention that proposes:, Lac regis apis protein decomposition product by protease-producing strain, with undecomposed Lac regis apis protein, compare, infection defensive enginery excellence, and viscosity is low and be water solublity, excellent in stability, so easily add in food and orally ingestible.In addition, also reported that (2) relate to the invention with the inhibiting albuminolysis thing of Angiotensin-Converting that obtains with trypsin treatment Lac regis apis raw material (for example,, with reference to patent documentation 2.）。

In addition, as the invention related to as the Lac regis apis analyte of food, also reported (3) low-allergen Lac regis apis, this low-allergen Lac regis apis is for having reduced hypersensitive low-allergen Lac regis apis, it is characterized in that, contain the 10-hydroxyl decanoic acid and, from the peptide composition of Lac regis apis, described peptide composition consists of (for example,, with reference to patent documentation 3 composition below molecular weight 4.5kDa.）。Foregoing invention is based on following knowledge and the invention that proposes:, process and use the carbohydrate-splitting enzyme of beta-Mannosidase to process by proteolytic enzyme, in the peptide composition from Lac regis apis, decomposition is removed and is easily caused that irritated molecular weight surpasses the composition of 4.5kDa, thereby easily reduces anaphylaxis.

So, for Lac regis apis analyte, the various physiologically actives that particularly the Lac regis apis protein decomposition product had, a lot of reports are arranged, but illustrate concrete biological active substances, so that the report of its concrete mechanism of action is considerably less.The illustrating of the differentiation mechanism that the illustrating of the specific and mechanism of action of biological active substances contributes to Apis, to the exploitation of the more effective medicine of the animals such as people or functional food etc., so strong expectation is further studied, studied the Lac regis apis analyte.And, for research of effectively carrying out the Lac regis apis analyte etc., importantly obtain the Lac regis apis analyte that contains the analyte that the abundant physiologically actives such as functional peptide are high.

Patent documentation 1: Japanese kokai publication hei 8-59499 communique

Patent documentation 2: TOHKEMY 2005-255670 communique

Patent documentation 3: TOHKEMY 2005-287411 communique

But the method for the present use that above-mentioned (1) method of take is representative is just suitably decomposed Lac regis apis protein with general protease etc. mostly.Above-mentioned general protease etc., owing to not usining Lac regis apis as original substrate, therefore decompose at unsuitable position, likely make the function of original useful peptide lose activity.Using Lac regis apis as the protease of substrate etc. by using, can address the above problem, but not yet find this kind of enzyme.

Summary of the invention

The object of the invention is to, provide best Lac regis apis clastic enzyme and above-mentioned Lac regis apis clastic enzyme to contain thing, for effectively obtain the high analytes of physiologically active such as functional peptide from Lac regis apis.

In addition, the object of the invention is to, provide so that contain thing etc. with above-mentioned Lac regis apis clastic enzyme and be prepared the decomposition method for the Lac regis apis analyte of feature and the Lac regis apis that uses above-mentioned Lac regis apis clastic enzyme to contain thing etc.

The inventor conducts in-depth research in order to solve above-mentioned problem, result is thought, the Lac regis apis of take is controlled to the queen bee nit of queen bee differentiation is congenital and is had a catabolic enzyme that is suitable for decomposing Lac regis apis most as staple food and by the Lac regis apis of ingesting, thereby, by the fraction that separates the enzyme that contains the degrading activity with Lac regis apis protein the larva body fluid extract from queen bee nit, complete the present invention.

That is, the invention provides Lac regis apis clastic enzyme and contain thing, the method by comprising following operation is by 2～3 age in days queen bee nits preparations of Apis mellifera (Apismellifera):

(a) carry out centrifugal treating in the soma's float to above-mentioned queen bee nit below 6 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, solution middle level, precipitation lower floor;

(b) reclaim the operation that thing is contained as Lac regis apis clastic enzyme in above-mentioned middle level.

In addition, the invention provides Lac regis apis clastic enzyme and contain thing, it is characterized in that, above-mentioned centrifugal treating is 8000～12000 * g, the centrifugal treating more than 5 minutes.

In addition, the invention provides Lac regis apis clastic enzyme and contain thing, the method by comprising following operation is by 2～3 age in days queen bee nits preparations of Apis mellifera:

(a ') by filtration after ice-cooled normal saline washing for above-mentioned queen bee nit, thus the operation of preparation soma float;

After (b ') used the 50mM phosphate buffer of pH7 to dilute above-mentioned soma float, carry out the centrifugal treating of 10 minutes under 10000 * g, 5 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, aaerosol solution middle level, precipitation lower floor.

(c ') reclaims the operation that thing is contained as Lac regis apis clastic enzyme in above-mentioned middle level.

In addition, the invention provides the Lac regis apis analyte, use the Lac regis apis clastic enzyme of above-mentioned any one record to contain the thing preparation.

In addition, the invention provides the Lac regis apis analyte, by the Lac regis apis clastic enzyme that uses above-mentioned any one record, contain thing, under pH9, Lac regis apis carried out to the enzyme processing and obtain, and it has following characteristics:

(1) to nonspecific cytopathy effect of neurocyte, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, compare reduction;

(2) to the inhibitory action of the nerve cell death of being induced by amyloid, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, equate;

(3) to the cel l proliferation of neurogliocyte, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, compare increase;

(4) infection inhibitory action Human reoviruslike agent infected, compare increase with the Lac regis apis water soluble fraction that does not carry out the enzyme processing.

In addition, the invention provides antioxidant, will contain thing by the Lac regis apis clastic enzyme that uses the record of above-mentioned any one, under pH9, Lac regis apis is carried out to the enzyme processing and the Lac regis apis analyte that obtains as effective ingredient.

In addition, the invention provides cell proliferating agent, will contain thing by the Lac regis apis clastic enzyme that uses the record of above-mentioned any one, under pH9, Lac regis apis is carried out to the enzyme processing and the Lac regis apis analyte that obtains as effective ingredient.

In addition, the invention provides infection inhibitor, will contain thing by the Lac regis apis clastic enzyme that uses the record of above-mentioned any one, under pH9, Lac regis apis is carried out to the enzyme processing and the Lac regis apis analyte that obtains as effective ingredient.

In addition, the invention provides nerve cell death and induce inhibitor, will contain thing by the Lac regis apis clastic enzyme that uses the record of above-mentioned any one, under pH9, Lac regis apis is carried out to the enzyme processing and the Lac regis apis analyte that obtains as effective ingredient.

In addition, the invention provides the decomposition method of Lac regis apis, it is characterized in that, use the Lac regis apis clastic enzyme of above-mentioned any one record to contain thing.

In addition, the invention provides Lac regis apis clastic enzyme, it is characterized in that, the Lac regis apis clastic enzyme that is included in above-mentioned any one record contains in thing.

In addition, the invention provides Lac regis apis clastic enzyme, it is characterized in that, the best pH of enzymatic activity is 7 or 9.

In addition, the invention provides the Lac regis apis analyte, use the Lac regis apis clastic enzyme preparation of above-mentioned any one record.

In addition, the invention provides the decomposition method of Lac regis apis, it is characterized in that, use the Lac regis apis clastic enzyme of above-mentioned any one record.

Contain thing and Lac regis apis clastic enzyme by Lac regis apis clastic enzyme of the present invention, can not damage the physiologically active that functional peptide contained in Lac regis apis etc. has and Lac regis apis is decomposed.

In addition, the Lac regis apis analyte that contains the decomposition such as thing by Lac regis apis clastic enzyme of the present invention is owing to containing functional peptide that abundant physiologically active is high etc., in the research of the physiologically active of Lac regis apis, research, can provide very effective sample.Further, the Lac regis apis analyte of the application of the invention, can provide with the in the past edible food that contains Lac regis apis etc. and compare, the food of functional and the property digested and assimilated excellence etc.

In addition, by Lac regis apis decomposition method of the present invention, can effectively decompose Lac regis apis.

The accompanying drawing explanation

Fig. 1 is for after the enzyme reaction solution that interpolation is mixed with to Lac regis apis protein and the solution that contains Lac regis apis clastic enzyme reacted under 37 ℃, carries out the figure that dyeing picture that the result of SDS-PAGE obtains means according to pH in order to separate the Lac regis apis protein in enzyme reaction solution.In Figure 1A pH be 4, in Figure 1B pH be 5, in Fig. 1 C pH be 6, in Fig. 1 D pH be 6.5, in Fig. 1 E pH be 7, in Fig. 1 F pH be 7.5, in Fig. 1 G pH be 8 and Fig. 1 H in pH be 9.Numeral on the swimming lane of each figure response time separately.In addition, M means molecular weight marker (marker).Arrow a means the band of about 51kDa, and arrow b means the band of about 46kDa.

The figure that Fig. 2 means according to pH for the SDS-PAGE result to embodiment 1.The dyeing picture that the dyeing picture that Fig. 2 A is pH7, Fig. 2 B are pH9.Numeral on the swimming lane of each figure response time, M separately means molecular weight marker.Arrow a means the band that the band of about 51kDa, band that arrow b means about 46kDa, band that arrow c means about 33kDa and arrow d mean about 25kDa.

Fig. 3 is measured for the intensity to the band of the dyeing picture of Fig. 2 in embodiment 1, and the band strength of take under the reaction time started 0 is 1, means the figure that the passing band strength along with the response time changes.Fig. 3 A means the strength ratio of the band of about 51kDa, and Fig. 3 B means the strength ratio of the band of about 46kDa, and Fig. 3 C means the strength ratio of the band of about 33kDa and about 25kDa.In Fig. 3 A and Fig. 3 B ● mean the band of pH7, zero means the band of pH9.In addition, the △ in Fig. 3 C means the band of about 33kDa under pH7, the band of about 25kDa under ▲ expression pH7.

Fig. 4 is illustrated in the figure that decomposes the result of Lac regis apis protein under the condition of pH9 in embodiment 2.Fig. 4 A means the dyeing picture of the gel that the result by SDS-PAGE obtains, the strength ratio that Fig. 4 B means the band of about 46kDa.Numeral on swimming lane in Fig. 4 A response time, M separately means molecular weight marker.In addition, arrow a means that about 51kDa, arrow b mean about 46kDa.

Fig. 5 is for meaning the figure of the result of the absorbance of the 280nm of mensuration in embodiment 3.Fig. 5 A starts front result for reacting, Fig. 5 B is that the reaction beginning result of latter 8 hours, Fig. 5 C are for reacting the beginning result of latter 16 hours and Fig. 5 D for reacting the result started latter 24 hours.In addition, the arrow a in figure means the fraction of the fraction of the protein of about 350kDa, fraction that arrow b means the protein of about 60kDa, fraction that arrow c means the protein of about 5kDa, protein that arrow d means about 3kDa and the fraction that scope e means the protein of hundreds of Da.

The use HiTrap that Fig. 6 is embodiment 8 ^tMin the column chromatography of Desalting chromatographic column, the figure that the result of mensuration absorbance (280nm) and conductivity obtains.Solid line means that absorbance, the dotted line of Lac regis apis analyte (pH9) mean that absorbance, the double dot dash line of Lac regis apis analyte (pH7) mean that conductivity, the chain-dotted line of Lac regis apis analyte (pH9) mean the conductivity of Lac regis apis analyte (pH7).In figure, " D1 " is the fraction taken out as fraction D1, and " D2 " is the fraction taken out as fraction D2.

The figure that Fig. 7 is the cell proliferation amount while in expression embodiment 8, using each culture fluid to cultivate.Fig. 7 A contains fraction D1(pH9 for adding) culture fluid the time result, Fig. 7 B contains fraction D1(pH7 for adding) culture fluid the time result.In figure, " NC " means that contrast culture liquid, " PC " mean the culture fluid that contains 10%FCS.In addition, each concentration means the protein concentration from fraction D1 in each culture fluid.

The use Cosmosil(registered trade mark that Fig. 8 is embodiment 9) 5C ₁₈in the column chromatography of-AR chromatographic column, the figure that the result of mensuration absorbance (215nm) and conductivity obtains.Solid line means fraction D2(pH9) absorbance, dotted line mean fraction D2(pH7) absorbance, double dot dash line mean fraction D2(pH9) conductivity, chain-dotted line mean fraction D2(pH7) conductivity.In figure, " C1(pH7) " is as fraction C1(pH7) fraction that takes out, " C1(pH9) " be for as fraction C1(pH9) fraction that takes out, " C2(pH7) " be for as fraction C2(pH7) fraction that takes out, " C2(pH9) " be for as fraction C2(pH9) fraction that takes out.

The figure that Fig. 9 is the cell proliferation amount while in expression embodiment 9, using each culture fluid to cultivate.Fig. 9 A contains fraction C1(pH9 for adding) culture fluid the time result, Fig. 9 B contains fraction C1(pH7 for adding) culture fluid the time result.In figure, " NC " means that contrast culture liquid, " PC " mean the culture fluid that contains 10%FCS.

The figure that Figure 10 is the cell proliferation amount while in expression embodiment 9, using each culture fluid to cultivate.Figure 10 A contains fraction C2(pH9 for adding) culture fluid the time result, Figure 10 B contains fraction C2(pH7 for adding) culture fluid the time result.In figure, " NC " means contrast culture liquid.In addition, each concentration means the protein concentration from fraction C2 in each culture fluid.

Figure 11 is for meaning the figure of the BSA concentration results of each PBS sample solution in embodiment 10.Result when result, Figure 11 C when result, Figure 11 B when Figure 11 A is interpolation Lac regis apis analyte is the interpolation ascorbic acid is the interpolation carnosine.

Figure 12 is for meaning the figure of the cell-proliferation activity of Lac regis apis analyte in embodiment 7.The result of Figure 12 A when under pH7, Lac regis apis clastic enzyme being contained thing and processed, the result of Figure 12 B when under pH9, Lac regis apis clastic enzyme being contained thing and processed.

Figure 13 is for meaning the figure of SDS-PAGE result in embodiment 11.Arrow a means the band of about 55kDa, the band that arrow b means about 46kDa.

The specific embodiment

If the queen bee nit of 2～3 ages in days of the queen bee nit Apis mellifera in the present invention without particular limitation of.Can be the fresh larva that lives or the larva after freezing preservation.Owing to thinking that (hereinafter referred is RJ to Lac regis apis.) activity of catabolic enzyme etc. is high, preferably uses the fresh larva lived.The freezing preservation of larva can be undertaken by conventional method, but in order to prevent that the RJ catabolic enzyme from losing activity, and after preferably using liquid nitrogen etc. cooling rapidly, under-80 ℃, preserves.

Soma's float in the present invention refers to the soma of larva, mainly is the float of internal organs and body fluid.To above-mentioned internal organs etc. without particular limitation of, but be preferably the float of whole somas of larva individuality.

If the method that the method for preparing soma's float by larva prepares suspensions of tissues by animal etc. usually without particular limitation of.For example, can use sieve to wait whole somas or internal organs or the body fluid that filters larva, also can use the preparations such as mortar, blender, homogenizer.Owing to can under the condition relaxed, preparing soma's float, preferably use sieve to wait and filtered.The preferred mesh of above-mentioned sieve is of a size of 80 orders～120 orders.While using blender etc., in order to prevent excessive heat, preferably add normal saline etc. and carry out.And, in order to prevent from sneaking into impurity, larva preferably before preparation soma float, when freezing preservations before preservation, in advance with washings such as normal saline.

Contain thing in order to prepare RJ catabolic enzyme of the present invention, at first, as operation (a), by the soma's float to queen bee nit below 6 ℃, carrying out centrifugal treating, be separated into the middle level of upper strata, the yellow-white turbid solution of white solid, containing adularescent to three layers of the lower floors of the precipitation of brown slightly.Before carrying out centrifugal treating, buffer that preferably uses neutrality and low concentration etc. is diluted, thereby suitably viscosity or the concentration of soma's float is adjusted.This is due to the viscosity of soma's float or concentration when high, likely reduce the RJ catabolic enzyme extraction efficiency, operability is worsened.As above-mentioned buffer, such as phosphate buffer (50mM, pH7.0) or normal saline etc. are arranged.And inferring above-mentioned upper strata is the insoluble composition that lipid components, above-mentioned lower floor are soma.

If for the condition of above-mentioned centrifugal treating can be by soma's float centrifugalize three layers without particular limitation of, but preferably at 8000～12000 * g, carry out the centrifugal treating more than 5 minutes below 6 ℃.Particularly preferably in the centrifugal treating of carrying out 10 minutes under 10000 * g, 5 ℃.In the short situation of the situation that centrifugal speed is low or centrifugation time, likely can not successfully be separated into three layers.

Then, as operation (b), the middle level in three layers is contained thing as Lac regis apis clastic enzyme and is reclaimed, thereby can prepare RJ catabolic enzyme of the present invention, contains thing.Above-mentioned recovery method can be undertaken by conventional method.In order to improve extraction accuracy, preferably under the condition identical with operation (a), above-mentioned middle level is carried out to centrifugal treating again.

In operation (b) afterwards, in order to remove the insoluble matters such as insoluble tissue that are blended in above-mentioned middle level, preferably above-mentioned middle level is filtered.Owing to can preventing the pollution caused because of Institute of Micro-biology, preferably in above-mentioned filtration, use the filter of 0.2 μ m etc.

And, owing to preparing larva body fluid extract or preparing Lac regis apis clastic enzyme from larva body fluid extract, containing thing, in larva body fluid extract, contained enzyme likely loses activity, and centrifugal treating is preferably carried out under 3～6 ℃.In addition, other operation is preferably carried out under ice-cooled.

RJ analyte of the present invention can be by the decomposition method of RJ of the present invention, with RJ catabolic enzyme of the present invention, contain thing prepares.Above-mentioned RJ can be fresh RJ or the RJ that dried powder RJ reduction is obtained.In addition, above-mentioned RJ can be the RJ in any place of production, can be the RJ that the honeybee kind produces arbitrarily.Further, above-mentioned RJ also can be for being separated the protein obtained by conventional method by RJ by conventional method.Be particularly preferably the water soluble protein of RJ.This is owing to can expecting to contain a large amount of materials with physiologically active.

If the separation of the water soluble protein of RJ can separating water-soluble protein and the method for hydrophobic protein can use any means.As said method, the suspension that is mixed with water-soluble solvent and RJ is carried out to allocation process to organic solvents such as useful ethyl acetate or butanols and the method for abstraction purification, by carrying out centrifugal treating after above-mentioned suspension is standing, obtain method of supernatant etc.

For example, can contain the method that thing cultivated and prepare the RJ analyte by adding RJ catabolic enzyme of the present invention in the water soluble protein to RJ or RJ.RJ etc. also can be used phosphate buffer etc. to be diluted in advance.In addition, also can use with the RJ catabolic enzyme of the adjusting protein concentrations such as phosphate buffer or viscosity and contain thing.

The temperature of above-mentioned cultivation or time, if the condition that does not suppress the RJ degrading activity that RJ catabolic enzyme of the present invention contains thing without particular limitation of, but the reaction temperature condition optimization is 30～40 ℃, is particularly preferably 34～38 ℃.In addition, pH is preferably 6.5～10, pH and is particularly preferably 7～9.5.While carrying out under the reaction condition that is 7～7.5 at pH, in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein meaned with the form of being with on the position of about 51kDa mainly is decomposed, while carrying out under the reaction condition that is 8.5～9 at pH, in SDS-PAGE, the protein meaned with the form of being with on the position of about 46kDa mainly is decomposed.

Below the decomposition method of RJ of the present invention is carried out to more specific description.

At first, use the Lowry method to take after protein concentration that RJ catabolic enzyme prepared the method by record in aftermentioned embodiment 1 as standard substance by BSA contains thing measured, use the 50mM phosphate buffer of pH7 to be diluted, prepare thus the solution that contains the RJ catabolic enzyme of 6mg/mL.

Then, the solution (6mg/mL) of the RJ protein solution (30mg/mL) prepared to the method for passing through record in aftermentioned reference example 2 of adding 1mL in the buffer of 8mL and the above-mentioned RJ of the containing catabolic enzyme of 1mL, be mixed with enzyme reaction solution.Above-mentioned enzyme reaction solution is reacted under 37 ℃, after ice-cooled, stopped reaction, confirm the variation of RJ protein by SDS-PAGE.Use CBB R-250 dyeing for the RJ protein separated.Buffer is used respectively the acetate buffer of the 50mM that pH is 4,5 and 5.5, the phosphate buffer of the 50mM that pH is 6,6.5,7 and 7.5, the Tri-HCl buffer of the 50mM of pH8 and pH9.

Fig. 1 is for meaning the figure of the result of SDS-PAGE according to pH.In Figure 1A pH be 4, in Figure 1B pH be 5, in Fig. 1 C pH be 6, in Fig. 1 D pH be 6.5, in Fig. 1 E pH be 7, in Fig. 1 F pH be 7.5, in Fig. 1 G pH be 8 and Fig. 1 H in pH be 9.Numeral on the swimming lane of each figure response time separately.In addition, M means molecular weight marker.If the RJ protein solution that will prepare by the method for record in aftermentioned reference example 2 carries out SDS-PAGE, detect stronger band on the position of about 51kDa and about 46kDa.Arrow a means the band of about 51kDa, and arrow b means the band of about 46kDa.Result is known, and the processing that time RJ protein is undertaken by the solution by containing the RJ catabolic enzyme in pH6～9 reduces, and along with the increase decomposition rate of pH is accelerated.Particularly, confirmed that, under the reaction condition of pH7～7.5, the protein of about 51kDa mainly is decomposed, under the reaction condition of pH8.5～9, the protein of about 46kDa mainly is decomposed.That is, from the result of Fig. 1, the RJ catabolic enzyme prepared by the method for putting down in writing in aftermentioned embodiment 1 contains in thing, contains the enzyme that decomposes RJ protein.In addition, neutrallty condition is with to contain by above-mentioned RJ catabolic enzyme the proteolytic pattern of RJ that thing carries out under alkali condition different, and therefore above-mentioned RJ catabolic enzyme contains in thing and at least contains two kinds of catabolic enzymes.That is, above-mentioned RJ catabolic enzyme contains in thing the best pH that contains enzymatic activity is 6.5～7.5, preferably pH is 7～7.5, more preferably pH is 7 RJ catabolic enzyme and the RJ catabolic enzyme that best pH is 8～9, preferably pH is 9 of enzymatic activity.

The RJ catabolic enzyme of the application of the invention contains thing, under pH9, to RJ, preferably carries out more than 2 hours, more preferably more than 4 hours, further preferably the enzyme of 4 hours～30 hours, particularly preferably 4 hours～24 hours is processed and the RJ analyte that obtains has various physiologically actives.Above-mentioned physiologically active, compare with the physiologically active that the RJ water soluble fraction that does not carry out the enzyme processing has, and has several features.

For example, above-mentioned RJ analyte, inhibitory action for the nerve cell death of being induced by amyloid, with the RJ water soluble fraction that does not carry out the enzyme processing, equate, but, for nonspecific cytopathy effect of neurocyte, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, compare reduction.More particularly, Neuronal Survival rate when the above-mentioned RJ analyte that protein concentration is 30～125 μ g/ml and the amyloid peptide of 2mg/ml coexist, Neuronal Survival rate while coexisting with the amyloid peptide of the RJ water soluble fraction that does not carry out the enzyme processing of same protein concentration and 2mg/ml is roughly the same, be that ratio between two (RJ analyte/do not carry out the RJ water soluble fraction of enzyme processing) is 1 left and right, be preferably 0.9～1.1.On the other hand, when the above-mentioned RJ analyte that protein concentration is 60～1000 μ g/ml and the amyloid peptide of 2mg/ml coexist, compare while coexisting with the amyloid peptide of the RJ water soluble fraction that does not carry out the enzyme processing of same protein concentration and 2mg/ml, reduce the neurocyte number that presents cytomorphosis.

In addition, above-mentioned RJ analyte, for the cel l proliferation of neurogliocyte, is compared increase with the Lac regis apis water soluble fraction that does not carry out the enzyme processing.More particularly, when the above-mentioned RJ analyte that protein concentration is 60～1000 μ g/ml and the amyloid peptide of 2mg/ml coexist, while coexisting with the amyloid peptide of the RJ water soluble fraction that does not carry out the enzyme processing of same protein concentration and 2mg/ml, compare, the neurogliocyte number increases.

In addition, the infection inhibitory action that above-mentioned RJ analyte infects for Human reoviruslike agent, compare increase with the Lac regis apis water soluble fraction that does not carry out the enzyme processing.More particularly, while under the existence of Human reoviruslike agent and above-mentioned RJ analyte, carrying out cell culture, and compare when carrying out cell culture under the RJ water soluble fraction that does not carry out the enzyme processing exists in Human reoviruslike agent, the rotavirus infection cell number reduces.

The RJ catabolic enzyme of the application of the invention contains thing, under pH9, to RJ, carries out preferably more than 2 hours, more preferably more than 4 hours, further preferably the enzyme of 4 hours～30 hours, particularly preferably 4 hours～24 hours is processed and the RJ analyte that obtains also has antioxidant activity.Therefore, above-mentioned RJ analyte can be used as antioxidant.For example, the protein that above-mentioned RJ analyte can suppress to cause due to free radicals such as Dichlorine monoxide (ClO) by its antioxidant activity decomposes.RJ is owing to being the food of ingesting since ancient times; so above-mentioned RJ analyte is also very safe; with ascorbic acid etc. similarly, can expect as adding the antioxidant in the diet product to or take the effective ingredient etc. of the not oxidated enriching substance that stress ingest as purpose of protection organism.

The RJ catabolic enzyme of the application of the invention contains thing, under pH9 to RJ carry out preferably more than 2 hours, more preferably more than 4 hours, further preferably the enzyme of 4 hours～30 hours, particularly preferably 4 hours～24 hours process and the RJ analyte that obtains owing to thering is above-mentioned physiologically active, so can be used as antioxidant, cell proliferating agent, (virus) infection inhibitor, induce the effective ingredient of nerve cell death inhibitor etc. and exist.The protein concentration that above-mentioned RJ catabolic enzyme in above-mentioned cell proliferating agent contains thing is preferably 0.5mg/ml～1mg/ml.These physiological agents can be used as the diet product such as enriching substance and ingest separately, also can with other composition for beverage or food or Pharmaceutical composition similarly as the additive in diet product or medicine.

If the preparation method using above-mentioned RJ analyte as these physiological agents of effective ingredient do not suppress above-mentioned RJ analyte physiologically active method without particular limitation of, the composition for beverage or food that usually can use preparation to contain RJ or RJ analyte or the method for using during Pharmaceutical composition are prepared.To the dosage form of these physiological agents without particular limitation of, but be preferably the dosage form that is suitable for oral agents.For example, can be soft capsule, hard capsule, tablet or syrup etc.In addition, if in these physiological agents the physiologically active of the contained above-mentioned RJ analyte of above-mentioned RJ analyte amount can play a role effect amount without particular limitation of, physiologically active, dosage form that can be considered as the kind of the RJ of raw material, required above-mentioned RJ analyte etc. suitably determines.

If the physiologically active using above-mentioned RJ analyte as the above-mentioned RJ analyte of the intake of these physiological agents of effective ingredient can play a role the amount of effect without particular limitation of, can suitably determine according to the human or animal's of picked-up body weight, age, sex etc.For example, preferred every day 1 time or be divided into and absorb 300mg～3g for several times.

Embodiment

Below enumerate embodiment the present invention is carried out to more specific description, but the present invention is not limited to following examples.

The preparation of [reference example 1] larva float

After gathering the queen bee nit of 3 age in days Apis mellifera of about 15g, with ice-cooled normal saline washing.Then, by the average 100 purpose cloth (" テ ト ロ Application ", DongレShe system) that use polyester, filter queen bee nit, destroy the epidermis of larva, squeeze out body fluid and internal organs.Prepare thus milky larva float.

The preparation of [reference example 2] RJ protein solution

To domestic in RJ(: the permanent bright bee product company limited system in rivers and mountains, Zhejiang Province) in 10ml, add the 50mM phosphate buffer 20mL of pH7.0 to be mixed, preparation RJ dilute solution.Above-mentioned RJ dilute solution after centrifugal 20 minutes, is removed to insoluble composition under 25000 * g, 5 ℃, reclaim supernatant.Then, with the 50mM phosphate buffer of pH7.0, dilute above-mentioned supernatant, the RJ protein solution of preparation 30mg/mL.

[embodiment 1]

1.RJ the preparation that catabolic enzyme contains thing

Will be by reference to the method larva float that prepare of record in example 1 with after 2 times of the 50mM phosphate buffer dilutions of pH7, under 10000 * g, 5 ℃ centrifugal 10 minutes.By above-mentioned centrifugal treating, be separated into the middle level of upper strata, the yellow-white turbid solution of white solid, containing adularescent to three layers of the lower floors of the precipitation of brown slightly.To infer above-mentioned upper strata and be lipid components, above-mentioned lower floor be insoluble soma etc.After reclaiming above-mentioned middle level, further under 10000 * g, 5 ℃, within centrifugal 10 minutes, be separated into three layers, reclaim middle level and contain thing as the RJ catabolic enzyme.Contain thing for above-mentioned RJ catabolic enzyme, in order to remove the insoluble tissue do not eliminated and to carry out sterilizing, use the cellulose acetate esters filter (DISMIC-25CS, ADVANTEC company system) of 0.2 μ m to be filtered.Thus, the RJ catabolic enzyme that obtains yellow transparent contains thing.

2.RJ the decomposition of protein

Use the Lowry method to take BSA and measured as the protein concentration that standard substance contains thing to above-mentioned RJ catabolic enzyme, use the 50mM phosphate buffer of pH7 to prepare the solution that contains the RJ catabolic enzyme of 3mg/mL.

The solution that contains the RJ catabolic enzyme (6mg/mL) of 1mL prepared by the RJ protein solution (30mg/mL) prepared to the method for passing through record in above-mentioned reference example 2 of adding 1mL in the buffer of 8mL and the method by record in above-described embodiment 1, be mixed with enzyme reaction solution.The Tri-HCl buffer of the phosphate buffer of the 50mM of above-mentioned buffer use pH7 or the 50mM of pH9.Above-mentioned enzyme reaction solution is reacted under 37 ℃, after ice-cooled, stopped reaction, by SDS-PAGE, separate RJ protein.After the gel of Dryly use CBB R-250 dyeing, use fluorescent scanning instrument Typhoon9400(ア マ シ ャ system バ イ オ サ イ エ Application ス society system) obtaining dyeing looks like.

Fig. 2 is for meaning the figure of the result of SDS-PAGE according to pH.The dyeing picture that the dyeing picture that Fig. 2 A is pH7, Fig. 2 B are pH9.Numeral on the swimming lane of each figure response time, M separately means molecular weight marker.Arrow a means the band that the band of about 51kDa, band that arrow b means about 46kDa, band that arrow c means about 33kDa and arrow d mean about 25kDa.Its result, confirmed that, under pH7 and pH9, the protein of about 51kDa and about 46kDa is decomposed.Particularly known, under pH9, within the reaction beginning short time of latter 4 hours, the protein of about 46kDa is broken down into can't detected degree.In addition, under pH7, along with the passing in response time, detect the protein of about 33kDa and about 25kDa.

Use image analysis software Image Quant(GE ヘ Le ス ケ ア バ イ オ サ イ エ Application ス society system), band strength of each band of the dyeing picture of Fig. 2 is carried out quantitatively.It is 1 that Fig. 3 be take the band strength of reaction under the time started 0, has meaned the variation along with the passing band strength in response time.Fig. 3 A means the strength ratio of the band of about 51kDa, and Fig. 3 B means the strength ratio of the band of about 46kDa, and Fig. 3 C means the strength ratio of the band of about 33kDa and about 25kDa.In Fig. 3 A and Fig. 3 B ● mean the band of pH7, zero means the band of pH9.In addition, the △ in Fig. 3 C means the band of about 33kDa under pH7, the band of about 25kDa under ▲ expression pH7.

As shown in Fig. 3 A and Fig. 3 B, can confirm quantitatively that the protein of about 51kDa and about 46kDa is decomposed under pH7 and pH9.In addition, by the results verification of Fig. 3 C under pH7, until reaction starts latter 16 hours, the protein of about 33kDa and about 25kDa increases.This result has implied that the protein of about 33kDa and about 25kDa is likely that the protein of about 51kDa or about 46kDa decomposes the analyte obtained by RJ catabolic enzyme contained in the solution that contains the RJ catabolic enzyme.

[embodiment 2]

Be 60 minutes except making the response time, all operate with reacting similarly under the pH9 condition of embodiment 1, decompose RJ protein, by SDS-PAGE, separate RJ protein, the intensity of each band is carried out quantitatively.Fig. 4 A means the dyeing picture of gel, the strength ratio that Fig. 4 B means the band of about 46kDa.Numeral on swimming lane in figure response time, M separately means molecular weight marker.In addition, arrow a means that about 51kDa, arrow b mean about 46kDa.

Results verification the protein of about 46kDa by RJ catabolic enzyme contained in the solution that contains the RJ catabolic enzyme, decompose about 80% in reaction starts short time of latter 1 hour.And, from Fig. 4 B, the moment started latter 5 minutes in reaction has obtained almost not producing the result of decomposing, but infers that this is because before starting to carry out enzyme reaction by the RJ catabolic enzyme, do not cultivate in advance the RJ protein solution under 37 ℃, therefore until start enzyme reaction, occurred time lag.

[embodiment 3]

Be 24 hours except making the response time, all operate with reacting similarly under the pH7 condition of embodiment 1, decompose RJ protein.Then, enzyme reaction solution ice-cooled, stopped reaction is used to Super dex200HR10/30(Off ァ Le マ シ ア society system) implement gel permeation chromatography, separate contained protein in above-mentioned enzyme reaction solution.Gel permeation chromatography, used the phosphate buffer (pH7) of the 50mM that contains 0.15M sodium chloride as eluent, at flow velocity 0.5mL/min, under the condition of 5 ℃, carries out.In order to detect the protein of separation, use the UV detector to measure the absorbance of 280nm.

Fig. 5 is for meaning the figure of the absorbance result of the 280nm of mensuration in embodiment 3.Fig. 5 A starts front result for reacting, Fig. 5 B is that the reaction beginning result of latter 8 hours, Fig. 5 C are for reacting the beginning result of latter 16 hours and Fig. 5 D for reacting the result started latter 24 hours.In addition, the arrow a in figure means the fraction of the fraction of the protein of about 350kDa, fraction that arrow b means the protein of about 60kDa, fraction that arrow c means the protein of about 5kDa, protein that arrow d means about 3kDa and the fraction that scope e means the protein of hundreds of Da.

From the result of Fig. 5, along with the passing in response time, when the protein of about 350kDa and about 60kDa reduces, the protein of about 5kDa, about 3kDa and hundreds of Da increases.The analyte of the protein that the protein of the approximately 5kDa infer increased thus etc. is about 350kDa etc.In other words, by the result of separating by gel permeation chromatography, can be confirmed, contain thing by RJ catabolic enzyme of the present invention, the hmw protein of RJ is decomposed.

[embodiment 4]

Similarly prepare RJ protein solution (30mg/mL) with reference example 2.In addition, the concentration after making dilution is all to operate and prepared the solution (3mg/mL) that contains the RJ catabolic enzyme similarly to Example 1 3mg/mL.

Add the above-mentioned RJ protein solution (30mg/mL) of 1mL and the above-mentioned solution that contains the RJ catabolic enzyme (3mg/mL) of 1mL in the buffer of 8mL, be mixed with enzyme reaction solution.The Tri-HCl buffer of the phosphate buffer of the 50mM of above-mentioned buffer use pH7 or the 50mM of pH9.Make the reaction under 4,20,37 and 50 ℃ respectively of above-mentioned reactant liquor, ice-cooled, stopped reaction.To in the temperature chamber of each temperature, mix the moment of above-mentioned enzyme reaction solution as reacting the zero hour, making the response time is 4,16,24 hours.Separate similarly to Example 1 RJ protein by the above-mentioned enzyme reaction solution to after ice-cooled, the impact of observing response temperature.

Its result similarly to Example 2, under pH7 and pH9, is observed the decomposition of the protein of about 51kDa and about 46kDa under 37 ℃.Particularly, under pH9, the protein that starts the about 46kDa of the moment of 4 hours in reaction is decomposed to detectability, and the moment that the protein of about 51kDa starts 16 hours in reaction also is decomposed to detectability.

On the other hand, under 4 ℃, under pH7 and pH9, almost do not observe the decomposition of the protein of about 51kDa and about 46kDa.Under 20 ℃, with the situation of 37 ℃, compare, although decomposition reaction is carried out slowly, observing the decomposition of the protein of about 51kDa and about 46kDa.On the other hand, under 50 ℃, with 37 ℃, compare decomposition reaction and further accelerate, under pH7 and pH9, confirmed that the most protein of the moment started 4 hours in reaction is decomposed.

Known in a word, in the temperature range of 20～50 ℃, RJ catabolic enzyme of the present invention contains thing can decompose RJ, and in the said temperature scope, and the higher decomposition reaction that by RJ catabolic enzyme of the present invention, contains the RJ that thing carries out of temperature is faster.But, usually under 50 ℃, likely cause that RJ's sends out brown or the degeneration of RJ protein etc.Therefore, while by RJ catabolic enzyme of the present invention, containing thing decomposition RJ, preferably near temperature 37 ℃, carry out.

[embodiment 5]

Studied and used RJ catabolic enzyme of the present invention to contain the physiologically active for cranial nerve cell that RJ analyte that thing obtains has.Specifically, studied the impact for the RJ analyte of the mice embryonic hippocampal neurons, the nerve cell death that induced by amyloid of initial culture.And the mice embryonic hippocampal neurons is at 37 ℃, 5%CO ₂under atmosphere, the hippocampal neurons that will take from mice embryonic by conventional method is with 4.0 * 10 ⁴cells/well is seeded on the 96 hole porous plates of having cultivated neurogliocyte, thereby is cultivated.As cell culture fluid, use the ニ ュ ー ロ バ ー サ Le メ デ ィ ウ system (ギ Block コ society system) that contains the B27 enriching substance.Use the cell after a week after cultivating in experiment.

At first, to domestic in RJ(: the permanent bright bee product company limited system in rivers and mountains, Zhejiang Province) in 5mL, add the 50mM phosphate buffer 15mL of pH7.0 to be mixed, preparation RJ dilute solution.Above-mentioned RJ dilute solution after centrifugal 20 minutes, is removed to insoluble composition under 25000 * g, 5 ℃, reclaim supernatant.Then, by by above-mentioned supernatant lyophilization, obtain the thick fraction of RJ water solublity.Use the 50mM phosphate buffer of pH7.0 to dilute the thick fraction of above-mentioned RJ water solublity, the thick fraction solution of RJ water solublity of preparation 30mg/mL.

Then except substituting the RJ protein solution for preparing of method by record in above-mentioned reference example 2 with the thick fraction solution of above-mentioned RJ water solublity and making the response time, be 4 hours, all operations similarly to Example 1, decompose contained RJ protein in the thick fraction solution of above-mentioned RJ water solublity, obtain the RJ analyte.

After adding amyloid peptide (ペ プ チ De institute system) and the thick fraction solution of RJ water solublity or RJ analyte in the 96 hole porous plates of cultivating the mice embryonic hippocampal neurons, cultivate 48 hours.After the amyloid peptide is incubated 48 hours with the concentration of 2mg/mL under 37 ℃, adding into ultimate density is 20 μ g/mL.In addition, decompose under the RJ analyte obtain or pH9 the ultimate density of decomposing in the cell culture fluid that the RJ analyte obtained adds into each protein under the thick fraction solution of RJ water solublity, pH7 and be respectively 0,31.25,62.5,125,250,500 or 1000 μ g/mL.Use in contrast any one that do not add amyloid peptide and RJ analyte etc., add the cell culture fluid of equivalent and obtain sample.

Then, by immunostaining or He Xisite (Hoechst) 33342 pigments (Sigma company system, Cat No.M4403) the dyeing identification dead cell that has used anti-Map2 antibody (Sigma company system, Cat No.B2261), respectively the survival number of the mice embryonic hippocampal neurons in each culture dish, the survival number of neurogliocyte are measured.Further, by microscopic examination, the mice embryonic hippocampal neurons number of observing cytomorphosis is measured.And cytomorphosis refers to that the distinctive form of neurocyte that neurocyte forms from cyton with dendron and aixs cylinder changes.Cohesion between the atrophy of formation projection, aixs cylinder, prominentization of multiaxis, neurocyte, the death of neurocyte etc., in above-mentioned variation, are for example arranged.

[table 1]

The measurement result of table 1 expression mice embryonic hippocampal neurons number etc.It is 100% expression that survival neurocyte number and degeneration neurocyte number be take the neurocyte number of control sample, and in addition, it is 100% expression that the neurogliocyte number be take the neurogliocyte number of control sample.If to adding the amyloid peptide in the mice embryonic hippocampal neurons of initial culture, about 60% nerve cell death, survival rate is about 40%.If add the thick fraction solution of amyloid peptide and RJ water solublity simultaneously, the thick fraction solution volume dependent of RJ water solublity ground suppresses the nerve cell death of being induced by amyloid.The maximum keep alive rate returns to 60～70%.The above-mentioned effect that the thick fraction solution of RJ water solublity brings is also approved under the low concentration of 62.5～250 μ g/mL.But, if add the above thick fraction solution of RJ water solublity of 1000 μ g/mL, observe nonspecific neurocyte metamorphosis.

While being added on the RJ analyte that under pH7, decomposition obtains, observe effect roughly the same when adding the thick fraction solution of RJ water solublity.On the other hand, while being added on the RJ analyte that under pH9, decomposition obtains, also with the thick fraction solution of RJ water solublity, similarly observe the inhibition of the nerve cell death of being induced by amyloid.Further, with the thick fraction solution of RJ water solublity etc., compare, reduce nonspecific neurocyte metamorphosis, improve the cel l proliferation to neurogliocyte.Infer that this is because the result of the propagation of promotion neurogliocyte etc. is brought into play the neuroprotective effect better, therefore reduces the non-specific neurocyte metamorphosis that the thick fraction solution of RJ water solublity has.

As shown in Table 1, use RJ catabolic enzyme of the present invention to contain thing, carry out under pH9 that enzyme more than 4 hours is processed and the RJ analyte that obtains has following characteristics: be for the inhibitory action of the nerve cell death of being induced by amyloid, roughly the same with the Lac regis apis water soluble fraction that does not carry out the enzyme processing; For nonspecific cytopathy effect of neurocyte, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, compare remarkable reduction; And, for the cel l proliferation of neurogliocyte, with the Lac regis apis water soluble fraction that does not carry out the enzyme processing, compare raising.

[embodiment 6]

In order to study, use RJ catabolic enzyme of the present invention to contain that the RJ analyte that thing obtains is that have, infection that infect for Human reoviruslike agent suppresses active, in carrying out and activity test.Specifically, by the method for following record, in the cell culture fluid of the MA104 cell from Rhesus Macacus embryo kidney, add Human reoviruslike agent and RJ or RJ analyte simultaneously, observe thus the impact that the RJ analyte infects for Human reoviruslike agent.

As above-mentioned RJ, use the thick fraction solution of RJ water solublity of operation preparation similarly to Example 4.

As above-mentioned RJ analyte, using except making reaction temperature is that 37 ℃, response time are 24 hours, and all operations are similarly to Example 4 decomposed the RJ analyte obtained or decompose the RJ analyte obtained under pH9 under pH7.

Above-mentioned MA104 cell is at 37 ℃, 5%CO ₂under atmosphere, cultivate.After subculture during 3 days, SIGMA company system) and the 10%TPB(tryptose phosphate broth as the cell proliferation culture fluid, use to containing the 10%FCS(hyclone:: the culture fluid that in Eagle ' s MEM culture medium DIFCO company system) (day water pharmacy society system), the antibiotic of the some amounts of interpolation etc. obtains.After subculture the 3rd day, except 10%FCS is replaced by 2%FCS, use with above-mentioned cell proliferation and maintain and use culture fluid as cell with the culture fluid of culture fluid same composition.

As antibiotic of above-mentioned some amounts etc., use 1 bottle of the streptomycin sulfate (Meiji Seika Kaisha's system) of 1% penicillin streptomycin (use 1 bottle of the scotcil of 1,000,000 units/bottle (Banyu Pharmaceutical Co., Ltd.'s system) and 1g(are tired)/1 bottle to use sterilizing PBS(-) quantitatively to 100ml, the penicillin streptomycin obtained with the filter sterilised filtration) and 1% amphotericin B (Sankyo Co., Ltd's system).

In and activity test

At first, at 24cm ²in the TC culture bottle with 5.0 * 10 ⁵the cells/flask inoculation, and cultivate the MA104 cell that obtains monolayer in 5 days under 37 ℃, with 0.125% trypsin solution that is added with 0.025%EDTA, above-mentioned MA104 cell is peeled off, after carrying out centrifugalize, remove supernatant, with 2 * 10 ⁵cells/ml adds cell proliferation to prepare cell suspending liquid with culture fluid.To adding Human reoviruslike agent solution (the Human reoviruslike agent MO strain (serotype 3) that Miyagi Prefecture Cancer center, sea tall and erect three Lang Shi of old name assign: remain under-80 ℃ until while using) and the thick fraction solution of RJ water solublity or RJ analyte in this cell suspending liquid.Then, respectively inject respectively 20 μ l on 24 hole microscope slides, cultivate 22 hours under 37 ℃.

For above-mentioned Human reoviruslike agent solution, make its activation by carry out in advance 30 minutes precultures under 37 ℃ with trypsin solution, being added into ultimate density is 6.5 * 10 ⁴fCFU/mL.

In addition, for the thick fraction solution of above-mentioned RJ water solublity and above-mentioned RJ analyte (decomposing the RJ analyte obtained under the RJ analyte that under pH7, decomposition obtains or pH9), after carrying out preculture respectively under 37 ℃, be added into respectively the concentration that the ultimate density in the cell culture fluid of each protein is table 2 record.

Substitute in contrast the sample of above-mentioned RJ analyte etc. with the cell culture fluid that adds equivalent.

Then remove culture fluid with aspirator, to the sterilized deionized-distilled water that respectively adds 20 μ l in each hole, to remaining in salt on wave carrier piece, washed, air-dry after, flood and be fixed in 20 minutes in cold acetone, again carry out air-dryly, remain under-30 ℃ until measure.

Then, as an antibody, the antibody that the abundant veterinary of the applied biosystem division department of the Chinese Academy of Sciences of the university subject of the P3-1(discrimination veterinary community health lecture of identifying specifically the VP6 of Columba livia rotavirus strain PO-13 is assigned) being diluted to 5000 times of samples that obtain is placed on fixing cell, in dark moistening case, under 37 ℃, make its sensitization after 45 minutes, use PBS(-) washing 3,5 and 7 minutes of vibrating.

Then, as secondary antibodies, place FITC labelling goat anti-mouse IgG (American Qualex company system), make its sensitization 45 minutes under 37 ℃.Then, and similarly use PBS(-after an antibody sensitized) after washed cell, drip several glycerol buffer on wave carrier piece, use the coverslip sealing, by fluorescence microscope, the MA104 cell number of infection Columba livia rotavirus is measured.

[table 2]

Table 2 mean Human reoviruslike agent in and the result of activity test.The infection cell number is usingd the infection cell number of control sample as 100% expression.While adding the thick fraction solution of RJ water solublity, observe and approximately under the concentration more than 300 μ g/mL, promoting viral infection, approximately under the concentration below 300 μ g/mL, suppressing viral infection.In other words, implied and likely there is the effect dual character that promotes to suppress under infection, low concentration infection under high concentration.

On the other hand, observe decomposition obtains under pH7 RJ analyte and have under the concentration more than 37 μ g/mL and promote viral infection, suppress the trend of viral infection under the concentration below 18.5 μ g/mL.In other words, the RJ analyte that decomposition obtains under pH7 and the thick fraction solution of RJ water solublity similarly, promote under high concentration under infection, low concentration to suppress to infect, still from suppressing infection to promoting to infect the protein concentration deflection low concentration side of changing.On the other hand, while adding the RJ analyte that decomposition obtains under pH9 of the following concentration of 40 μ g/mL, during with the thick fraction solution of interpolation RJ water solublity, compare and show stronger infection inhibition.Particularly under pH9, decompose the concentration of RJ analyte below 16.5 μ g/mL obtained and show the dependent infection inhibition of protein concentration.In addition, the response time is more than 4 hours the time, and the concentration dependent of the infection inhibition of RJ analyte under low concentration that the response time is longer is more remarkable.

As shown in Table 2, use RJ catabolic enzyme of the present invention to contain thing, carry out under pH9 that enzyme more than 4 hours is processed and the RJ analyte that obtains has following characteristics: the infection inhibitory action infected for Human reoviruslike agent, compare increase with the Lac regis apis water soluble fraction that does not carry out the enzyme processing.

[embodiment 7]

In order studying, to use RJ catabolic enzyme of the present invention to contain the cell-proliferation activity that RJ analyte that thing obtains has, to use from the IEC-6 cell of rat small intestine and implement the WST-1 method.The WST-1 method is that the metabolic activity by measuring cellular enzymes is measured viable count purpose method.When living cells carries out respiratory movement, effect lactic acid, succinic acid by dehydrogenase become acetone acid, fumaric acid, now NAD ⁺become NADH.In the WST-1 method, to the 1-Methoxy PMS that adds 0.2mM in cell and WST-1 reagent as substrate.1-Methoxy PMS converts 1-Methoxy PMS reduced form to, (NADH becomes NAD ⁺) 1-Methoxy PMS reduced form is while forming 1-Methoxy PMS, by WST-1 reagent, produces yellow first hairpin (formazane).The first hairpin produced is measured to its absorbance with micro-plate device of reading.

The WST-1 method

I) preparation of reagent

WST-1 reagent

To WST-1(Wako Pure Chemical Industries, Ltd., Cell Counting Kit.No.345-06463) add 1-Methoxy PMS(0.2mM in 16.3mg and HEPES23.8mg) 5ml, with filter (MILLEX-GV, sterilizing, the Millipore company system) filtration sterilization of aperture 0.22 μ m.

The ii) preparation of test medium

The RJ analyte is dissolved in to DMEM(and contains 1%FCS) in, with filter (MILLEX-GV, sterilizing, the Millipore company system) filtration sterilization of aperture 0.22 μ m.

Iii) experimental implementation

The IEC-6 cell is to carry out trypsin treatment to forming confluent culture (confluent) in the 10cm culture dish, and is suspended in DMEM(and contains 1%FCS) in 10ml.After within centrifugal 5 minutes, attracting to remove supernatant with 1000rpm, with 2.0 * 10 ⁵cells/ml is suspended in DMEM(and contains 10%FCS) in culture medium, every 100 μ l are seeded on 96 orifice plates.

At CO ₂incubator (5%CO ₂, 37 ℃) in cultivated before 24 hours, after confirming cell adhesion, DMEM(is contained to 10%FCS) after culture medium attracts to remove, transfer in the test medium of 100 μ l.At CO ₂further cultivate after 24 hours in incubator, attract to remove test medium, in each hole, add DMEM(to contain 10%FCS respectively) culture medium 100 μ l, WST-1 reagent 10 μ l, under 37 ℃ at 5%CO ₂in incubator, cultivate 2 hours.Then, to the HCl that respectively adds 100 μ l in each hole, by micro-read the plate device at 450nm(with reference to wavelength: 600nm) the lower absorbance of measuring.

As above-mentioned RJ analyte, except making the response time, be all operations similarly to Example 4 24 hours, use respectively the RJ analyte that under RJ analyte that under pH7, decompositions obtains or pH9, decomposition obtains.

The cultivation of IEC-6 cell is undertaken by conventional method.

Resulting result is as shown in Figure 12 A and Figure 12 B.

Its result, confirmed the RJ analyte obtain for decomposing under pH7, the concentration of RJ analyte be 1mg/mL following, specifically be that 0.1～1mg/ml promotes cell proliferation, inhibition cell proliferation while surpassing 1mg/mL when following.In addition, confirmed for decomposing the RJ analyte obtain under pH9, had a mind to free burial ground for the destitute when particularly RJ analyte concentration is 0.5～1mg/ml and promote cell proliferation.

Results presumption by embodiment 6 and 7 goes out, and promote and suppress one of two faced reason of viral infection as the concentration dependent ground of observing RJ protein in embodiment 6, may be the cell proliferation facilitation effect due to RJ protein.In other words, having implied as one of reason of controlling cell proliferation, may be that RJ protein and fragments of peptides thereof show the effect that Human reoviruslike agent infects of controlling.

[embodiment 8]

Analyzed for the cell-proliferation activity that the RJ analyte more specifically obtained using RJ catabolic enzyme of the present invention to contain thing has, studied the RJ analyte is carried out to the cell-proliferation activity after desalting processing.

1. desalting processing

At first, except making reaction temperature, be that 37 ℃, response time are all operations similarly to Example 4 24 hours, use RJ catabolic enzyme of the present invention to contain thing, prepared respectively the RJ analyte that carries out the enzyme processing under pH7 and obtain (below be sometimes referred to as RJ analyte (pH7).) and pH9 under carry out the enzyme processing and the RJ analyte that obtains (below be sometimes referred to as RJ analyte (pH9).）。

Use HiTrap ^tMdesalting chromatographic column (GE ヘ Le ス ケ ア バ イ オ サ イ エ Application ス society system), by following method, respectively RJ analyte (pH7) and the RJ analyte (pH9) obtained carried out to apart.

I) preparation of sample

In the water (hereinafter referred be " ミ リ Q water ") that uses Millipore company system " MILLI-Q(registered trade mark) " to purify after dissolving each sample to 100～150mg/mL, under 1500rpm, centrifugalize is 5 minutes, by filter (MILLEX, aperture 0.45 μ m, Millipore company system) filtering supernatant.

The ii) preparation of buffer

Use with the degassed ミ リ Q water of ultrasonic washer.

Iii) operational approach

Chromatographic column is connected to

explorer System(GE ヘ Le ス ケ ア バ イ オ サ イ エ Application ス society system), with flow rate 20% ethanol of 10ml/min, washed.Then, with the flow rate of 10ml/min, degassed ミ リ Q water carries out balance.After being filled on chromatographic column by the 1ml sample, with the flow velocity of 10ml/min, carry out eluting, respectively reclaim 0.5ml in the developmental tube on being installed on fraction collector.According to the eluting pattern of measuring absorbance (mAU) and conductivity (ms/cm) under 280nm and obtaining, the two kinds of fractions of fraction (hereinafter referred to as fraction D2) that take out respectively the fraction (hereinafter referred to as fraction D1) of desalination and contain salt.

Fig. 6 is being used HiTrap ^tMin the column chromatography of Desalting chromatographic column, the figure that the result of mensuration absorbance (280nm) and conductivity obtains.Solid line means that absorbance, the dotted line of RJ analyte (pH9) mean that absorbance, the double dot dash line of RJ analyte (pH7) mean that conductivity, the chain-dotted line of RJ analyte (pH9) mean the conductivity of RJ analyte (pH7).In figure, " D1 " is the fraction taken out as fraction D1, and " D2 " is the fraction taken out as fraction D2.

2. the mensuration of cell-proliferation activity

Except the culture fluid of cell is used the fraction D1(fraction D1(pH9 that contains RJ analyte (pH9) in the culture fluid (contrast culture liquid) that does not contain the multiplicaiton factor such as FCS)) culture fluid or contain the fraction D1(fraction D1(pH7 of RJ analyte (pH7) in contrast culture liquid)) culture fluid, operation similarly to Example 7, the cell proliferation amount when using each culture fluid to cultivate is measured.Use the culture fluid contain 10%FCS, as negative control, use contrast culture liquid as positive control.The first that cell proliferation amount while using each culture fluid to cultivate produces when adding each culture fluid

the first that the product amount produces while relatively adding contrast culture liquid

the ratio of product amount means.

The figure of cell proliferation amount when Fig. 7 is used each culture fluid to cultivate for expression.Fig. 7 A contains fraction D1(pH9 for using) the result of culture fluid while cultivating, Fig. 7 B contains fraction D1(pH7 for using) the result of culture fluid while cultivating.In figure, " NC " means that contrast culture liquid, " PC " mean the culture fluid that contains 10%FCS.In addition, each concentration means the protein concentration from fraction D1 in each culture fluid.

Known as shown in the result of Fig. 7 A, the fraction D1(pH9 in culture fluid) concentration dependent ground increase cell proliferation amount.Particularly using fraction D1(pH9) the concentration culture fluid that is 50.5 μ g/mL is while cultivating, and the promotion cell proliferation degree during culture fluid that contains 10%FCS with use is roughly the same.But, at fraction D1(pH9) concentration is while being the culture fluid more than 84.2 μ g/mL, observe cell proliferation amount concentration dependent ground and reduce, so implied by fraction D1(pH9) there is optium concentration in the cell proliferation facilitation effect (cell-proliferation activity) that realizes.

On the other hand, known as shown in the result of Fig. 7 B, contain fraction D1(pH7 in culture fluid) time cell proliferation amount almost do not change, fraction D1(pH7) can appreciable impact not arranged on cell proliferation.

That is,, from the result of embodiment 8, use RJ catabolic enzyme of the present invention to contain thing, carry out the enzyme processing and the RJ analyte that obtains has cell-proliferation activity under pH9.

[embodiment 9]

Analyzed for the cell-proliferation activity that the RJ analyte more specifically obtained using RJ catabolic enzyme of the present invention to contain thing has, studied the RJ analyte is carried out to the cell-proliferation activity after thick purification.

1. thick purification

Use the Cosmosil(registered trade mark) 5C ₁₈-AR chromatographic column (Na カ ラ イ テ ス Network society system), the fraction D2(fraction D2(pH9 of RJ analyte (pH9) by following method to preparation in embodiment 8)) and the fraction D2(fraction D2(pH7 of RJ analyte (pH7))) carry out thick purification.

I) preparation of sample

Respectively by above-mentioned fraction D2(pH9) and fraction D2(pH7) with filter (MILLEX, aperture 0.45 μ m, Millipore company system), filter, add TFA to 0.05% concentration.

The ii) preparation of buffer

A) adsorption-buffering liquid: 0.05%TFA solution

Use ultrasonic washer to ミ リ Q water carry out degassed after, add TFA to 0.05% concentration.

B) elution buffer: 0.05%TFA/90% acetonitrile

Measuring respectively acetonitrile (with the pure pharmaceutical worker's industry of light) 450mL, ミ リ Q water 50mL is mixed.With filter (RC-Vliesverstarkt, aperture 0.22 μ m, Sartorius AG), filter, degassed by ultrasonic washer.Then, add TFA to 0.05% in 90% acetonitrile solution.

Iii) operational approach

Chromatographic column (capacity 0.83mL) is connected to

explorer System(GE ヘ Le ス ケ ア バ イ オ サ イ エ Application ス society system), the setting flow velocity is 1ml/min, with degassed ミ リ Q water washing.Then, carry out balance with 0.05%TFA solution.After being filled on chromatographic column by the 5ml sample, with the flow velocity eluting of 1ml/min, respectively reclaim 2ml in the developmental tube on being installed on fraction collector.Eluting pattern according to measuring absorbance (mAU) and conductivity (ms/cm) under 215nm and 280nm and obtaining, take out respectively non-adsorbed fraction (hereinafter referred to as fraction C1) and two kinds of fractions of adsorbed fraction (hereinafter referred to as fraction C2).

Fig. 8 is being used the Cosmosil(registered trade mark) 5C ₁₈in the column chromatography of-AR chromatographic column, the figure that the result of mensuration absorbance (215nm) and conductivity obtains.Solid line means fraction D2(pH9) absorbance, dotted line mean fraction D2(pH7) absorbance, double dot dash line mean fraction D2(pH9) conductivity, chain-dotted line mean fraction D2(pH7) conductivity.In figure, " C1(pH7) " is as fraction C1(pH7) fraction that takes out, " C1(pH9) " be for as fraction C1(pH9) fraction that takes out, " C2(pH7) " be for as fraction C2(pH7) fraction that takes out, " C2(pH9) " be for as fraction C2(pH9) fraction that takes out.

2. the mensuration of the cell-proliferation activity of fraction C1

Diluted fraction D2(pH9 except the culture fluid of cell is used) fraction C1(fraction C1(pH9)) culture fluid or diluted fraction D2(pH7) fraction C1(fraction C1(pH7)) culture fluid, operation similarly to Example 7, the cell proliferation amount when using each culture fluid to cultivate is measured.And, dilution fraction C1(pH9) or fraction C1(pH7) time used contrast culture liquid.

The figure of cell proliferation amount when Fig. 9 is used each culture fluid to cultivate for expression.Fig. 9 A contains fraction C1(pH9 for using) the result of culture fluid while cultivating, Fig. 9 B contains fraction C1(pH7 for using) the result of culture fluid while cultivating.In figure, " NC " means that contrast culture liquid, " PC " mean the culture fluid that contains 10%FCS.

As shown in the result of Fig. 9 A and Fig. 9 B, containing fraction C1(pH9) and fraction C1(pH7) the culture fluid of any one fraction all promoted cell proliferation.If both are compared, observe and contain fraction C1(pH7) time compares, and contains fraction C1(pH9) time have the trend that the cell proliferation facilitation effect is good.

3. the mensuration of the cell-proliferation activity of fraction C2

Contain fraction D2(pH9 except the culture fluid of cell is used in contrast culture liquid) fraction C2(fraction C2(pH9)) culture fluid or contain fraction D2(pH7 in contrast culture liquid) fraction C2(fraction C2(pH7)) culture fluid, operation similarly to Example 7, the cell proliferation amount when using each culture fluid to cultivate is measured.

The figure of cell proliferation amount when Figure 10 is used each culture fluid to cultivate for expression.Figure 10 A contains fraction C2(pH9 for using) the result of culture fluid while cultivating, Figure 10 B contains fraction C2(pH7 for using) the result of culture fluid while cultivating.In figure, " NC " means contrast culture liquid.In addition, each concentration means the protein concentration from fraction C2 in each culture fluid.

Its result, observe and contain fraction C2(pH9 in culture fluid) time, concentration in culture fluid is that 3.42 μ g/mL exist when following concentration dependent ground to increase the trend of cell proliferation amount, has the trend of concentration dependent ground reduction cell proliferation amount while surpassing 3.42 μ g/mL.On the other hand, observe and contain fraction C2(pH7 in culture fluid) time, concentration in culture fluid is that 4.67 μ g/mL exist when following concentration dependent ground to increase the trend of cell proliferation amount, has the trend of concentration dependent ground reduction cell proliferation amount while surpassing 4.67 μ g/mL.In addition, if both are compared, observe and contain fraction C2(pH7) time compares, and contains fraction C2(pH9) time have the trend that the cell proliferation facilitation effect is good.

Therefore, result from embodiment 9, use RJ catabolic enzyme of the present invention to contain that thing carries out the enzyme processing and the RJ analyte that obtains has cell-proliferation activity, and the RJ analyte obtained with under the reaction condition of pH7, carrying out the enzyme processing compares, carry out the enzyme processing and the RJ analyte that obtains has higher cell-proliferation activity under the reaction condition of pH9.

[embodiment 10]

The antioxidant activity that the RJ analyte obtained using RJ catabolic enzyme of the present invention to contain thing has is studied.

1.RJ the preparation of analyte

To domestic in RJ(: the permanent bright bee product company limited system in rivers and mountains, Zhejiang Province), add pure water to obtain float, use the dialyzer of aperture 10kD, in pure water to this float dialysis 72 hours.Reclaim liquid in dialyzer, after carrying out lyophilization, again be suspended in pure water, prepared the RJ protein solution of 30mg/mL.

The Tris-HCl buffer (pH9.0) that contains thing (3mg/mL), 8mL to the RJ catabolic enzyme that adds 1mL in the above-mentioned RJ protein solution of 1mL, cultivate 24 hours under 37 ℃.Then, use the dialyzer of aperture 500Da, in pure water, dialysis is 5 days.Reclaim liquid in dialyzer, carry out lyophilization, using the lyophilization thing as the RJ analyte.And the RJ catabolic enzyme contains thing and uses the catabolic enzyme of operation preparation similarly to Example 1 to contain thing.

2. Antioxidative Activity Determination

Antioxidant activity for the RJ analyte, according to the method that waits (japanese food section electrotechnics meeting will, the 51st volume No. 5,238th～246 pages, 2004) in willow, by research RJ analyte, the impact of decomposing due to the caused BSA of Dichlorine monoxide (ClO) is measured.

Specifically, at first, by the PBS(phosphate buffered saline to containing BSA) in add to use the RJ analyte of PBS dilution, preparation contains BSA(ultimate density 40 μ g/mL) and the PBS sample solution of RJ analyte (ultimate density 0,0.3,0.5,1.0 or 2.0 μ g/mL).Add sodium hypochlorite to take ultimate density in above-mentioned PBS sample solution as 1.7mM, cultivate 30 minutes under 37 ℃.Then, by SDS-PAGE, separate the protein in the PBS sample solution, by CBB, dye and detected.After obtaining CBB dyeing picture by scanner, the concentration of the band that is equivalent to BSA is analyzed.For the BSA concentration of each PBS sample solution, using do not add sodium hypochlorite the PBS sample solution as blank solution, the BSA concentration of the research blank solution relative concentration that is 1 o'clock.And the analysis software Image J that the concentration analysis of band is used U.S.'s National Institutes of Health (National Institutes of Health) to provide carries out.In addition, the PBS sample solution obtained adding known antioxidants ascorbic acid (ultimate density 0,3.0,4.0,5.0 or 7.5mM) or carnosine (ultimate density 0,2.5,5.0,7.5 or 10.0mM) to substitute the RJ analyte, similarly studied BSA concentration.

Figure 11 is the figure of the result of the BSA concentration of each PBS sample solution of expression.Result when result, Figure 11 C when result, Figure 11 B when Figure 11 A is interpolation RJ analyte is the interpolation ascorbic acid is the interpolation carnosine.Its result, when not adding the RJ analyte, BSA dyes and does not detect band by CBB, and most BSA is decomposed.On the other hand, while having added the RJ analyte, with ascorbic acid or carnosine similarly, the addition dependency ground of PBS sample solution is increased to the relative concentration of BSA.Infer that this is because the decomposition of the caused BSA of Dichlorine monoxide produced by sodium hypochlorite is subject to the inhibition of RJ analyte.

In addition, result based on Figure 11, calculate and will be decomposed while suppressing to 50% concentration by the caused BSA of Dichlorine monoxide, be in 0.92 ± 0.24mg/mL, ascorbic acid to be 0.67 ± 0.01mg/mL(3.83 ± 0.08mg/mM in the RJ analyte), be 0.84 ± 0.10mg/mL(3.65 ± 0.32mg/mM in carnosine).That is, confirmed although the RJ analyte is mixture, to there is the resistance to oxidation be equal to mutually with known antioxidants ascorbic acid or carnosine for the resistance to oxidation decomposed by the caused BSA of Dichlorine monoxide.

From the result of embodiment 10, use RJ catabolic enzyme of the present invention to contain thing, carry out the enzyme processing and the RJ analyte that obtains has antioxidant activity under pH9.

[embodiment 11]

Be that 1～3 hour, reaction temperature are 37 ℃ or 55 ℃ except making the response time, all operate with the reacting similarly under the pH9 condition of embodiment 1, decompose RJ protein, by SDS-PAGE, separate the RJ analyte.Figure 13 means the dyeing picture of gel.In figure, " std " means molecular weight marker, and " 9-0 " means response time 0(pH9) sample carry out the result of electrophoresis.In addition, arrow a means that about 55kDa, arrow b mean about 46kDa.

Its result, while having confirmed to make RJ protein and RJ catabolic enzyme to contain liquid to be reacted under 55 ℃, in reaction starts short time of latter 1 hour, the protein of about 51kDa and about 46kDa all is decomposed basically.

Utilizability on industry

RJ catabolic enzyme of the present invention contains thing and contains RJ catabolic enzyme that queen bee nit has, that the RJ of take is original substrate, is suitable for decomposing RJ most, so can be for the Study on Physiological Activity of RJ, the medicine that uses RJ and/or the fields such as exploitation of food.

Claims (4)

1. the application of Lac regis apis analyte in preparing antioxidant, wherein, described Lac regis apis analyte be use by comprise following operation (a) and (b) or the Lac regis apis clastic enzyme that prepared by 2～3 age in days queen bee nits of Apis mellifera of the method that comprises following operation (a '), (b ') and (c ') contain thing, and under pH9, Lac regis apis is carried out to enzyme and process to obtain

(a) carry out centrifugal treating in the soma's float to described queen bee nit below 6 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, solution middle level, precipitation lower floor;

(b) reclaim the operation that thing is contained as Lac regis apis clastic enzyme in described middle level,

(a ') by filtration after ice-cooled normal saline washing for described queen bee nit, thus the operation of preparation soma float;

After (b ') used the 50mM phosphate buffer of pH7 to dilute described soma float, carry out the centrifugal treating of 10 minutes under 10000 * g, 5 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, aaerosol solution middle level, precipitation lower floor;

(c ') reclaims the operation that thing is contained as Lac regis apis clastic enzyme in described middle level.

2. the application of Lac regis apis analyte in preparing cell proliferating agent, wherein, described Lac regis apis analyte be use by comprise following operation (a) and (b) or the Lac regis apis clastic enzyme that prepared by 2～3 age in days queen bee nits of Apis mellifera of the method that comprises following operation (a '), (b ') and (c ') contain thing, and under pH9, Lac regis apis is carried out to enzyme and process to obtain

(a) carry out centrifugal treating in the soma's float to described queen bee nit below 6 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, solution middle level, precipitation lower floor;

(b) reclaim the operation that thing is contained as Lac regis apis clastic enzyme in described middle level,

(a ') by filtration after ice-cooled normal saline washing for described queen bee nit, thus the operation of preparation soma float;

After (b ') used the 50mM phosphate buffer of pH7 to dilute described soma float, carry out the centrifugal treating of 10 minutes under 10000 * g, 5 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, aaerosol solution middle level, precipitation lower floor;

(c ') reclaims the operation that thing is contained as Lac regis apis clastic enzyme in described middle level.

3. the application of Lac regis apis analyte in preparing infection inhibitor, wherein, described Lac regis apis analyte be use by comprise following operation (a) and (b) or the Lac regis apis clastic enzyme that prepared by 2～3 age in days queen bee nits of Apis mellifera of the method that comprises following operation (a '), (b ') and (c ') contain thing, and under pH9, Lac regis apis is carried out to enzyme and process to obtain

(a) carry out centrifugal treating in the soma's float to described queen bee nit below 6 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, solution middle level, precipitation lower floor;

(b) reclaim the operation that thing is contained as Lac regis apis clastic enzyme in described middle level,

(a ') by filtration after ice-cooled normal saline washing for described queen bee nit, thus the operation of preparation soma float;

After (b ') used the 50mM phosphate buffer of pH7 to dilute described soma float, carry out the centrifugal treating of 10 minutes under 10000 * g, 5 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, aaerosol solution middle level, precipitation lower floor;

(c ') reclaims the operation that thing is contained as Lac regis apis clastic enzyme in described middle level.

4. a Lac regis apis analyte is preparing the application of nerve cell death in inducing inhibitor, wherein, described Lac regis apis analyte be use by comprise following operation (a) and (b) or the Lac regis apis clastic enzyme that prepared by 2～3 age in days queen bee nits of Apis mellifera of the method that comprises following operation (a '), (b ') and (c ') contain thing, and under pH9, Lac regis apis is carried out to enzyme and process to obtain

(a) carry out centrifugal treating in the soma's float to described queen bee nit below 6 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, solution middle level, precipitation lower floor;

(b) reclaim the operation that thing is contained as Lac regis apis clastic enzyme in described middle level,

(a ') by filtration after ice-cooled normal saline washing for described queen bee nit, thus the operation of preparation soma float;

After (b ') used the 50mM phosphate buffer of pH7 to dilute described soma float, carry out the centrifugal treating of 10 minutes under 10000 * g, 5 ℃, thereby be separated into three layers, be i.e. the operation of solid upper strata, aaerosol solution middle level, precipitation lower floor;

(c ') reclaims the operation that thing is contained as Lac regis apis clastic enzyme in described middle level.

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