CN107228894B - Method for detecting protein content and purity in oil emulsion protein subunit vaccine and application thereof - Google Patents
Method for detecting protein content and purity in oil emulsion protein subunit vaccine and application thereof Download PDFInfo
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Abstract
The invention provides a method for detecting protein content and purity in an oil emulsion protein subunit vaccine and application thereof, wherein the method comprises the following steps: 1) precipitating proteins in the vaccine using cold acetone; 2) SDS-PAGE electrophoresis; 3) carrying out gray scanning on each strip by using a gray scanner; 4) and calculating the content and purity of the protein in the vaccine by combining the results of the gray scanning. The method solves the problems of low dissociation efficiency and easy degradation of the antigen protein in the oil emulsion vaccine, also solves the problem of experiment by using a target animal in the quality inspection of the finished product of the oil emulsion protein subunit vaccine for livestock, and has the accuracy rate of more than 90 percent in the quantitative detection and the purity detection of the antigen protein content; the antigen protein is not denatured and degraded and is easy to repeat; meanwhile, the content and purity of the antigen protein in the oil emulsion protein subunit vaccine can be obtained by performing SDS-PAGE detection on the obtained antigen protein once, so that the cost, the labor force and the time are saved.
Description
Technical field
The present invention relates to the detection methods and application of protein content and purity in a kind of oil emu protein subunit vaccine, belong to
In field of biotechnology.
Background technique
Vaccine is one of current infection prevention disease most efficient method.In addition to safety, the another of a vaccine is evaluated
One important indicator is exactly its immunogenicity.Traditional attenuated live vaccine and inactivated vaccine is still presently the most common epidemic disease
Seedling, because two kinds of vaccines all maintain good virion character and virus surface proteins, and attenuated live vaccine can also be
Duplication in vivo, effectively can induce neutralizing antibody to generate.Although these two types of vaccines are all widely used at present,
Its virulence is returned the strong and incomplete safety issue of inactivation and still be can not be ignored, very high especially for those biological safety levels
Virus；In addition, these two types of production of vaccine techniques are all complex, higher cost.Therefore, it is researched and developed using recombinant technique sub- single
Position vaccine is a new direction and the trend of modern vaccination, and genome of this kind of vaccine without virus is a kind of opposite peace
Full vaccine.And the virus that we can't cultivate at present and effectively expand for those, or the bio-safety grade of culture
Not exigent virus, subunit vaccine are primary selections.
Genetic engineering subunit vaccine (Subunitvaccine) is also known as recombinant subunit vaccine (Recombinant
Subunit Vaccines) or biosynthesis subunit vaccine, it is that will encode pathogenic microorganism protectiveness using DNA recombinant technique
The channel genes recipient bacterium (such as Escherichia coli) of antigen or cell (such as 293T cell), make its high efficient expression in receptor, secretion
Protective antigens, isolates and purifies protective antigens and adds adjuvant genetic engineering subunit vaccine is made.Utilize genetic engineering
Method, can not only clone to obtain the gene of coding viral protective antigens, and it can be transformed in vitro or
Modification, and be transferred back in heterologous organisms host or cultivate in cell, so that virus protein is obtained great expression.Genetic engineering
Subunit vaccine exactly expresses protective antigen gene in protokaryon or eukaryotic, then is produced with the gene of this biosynthesis
Subunit vaccine made of object.Therefore, genetic engineering subunit vaccine is easy to be mass produced, and low in cost.Made with albumen
For the diagnostic method that antigen is established, immune animal and natural infected animal can be distinguished and, not interfere epidemiological survey；
The disease effects anxious for morbidity, the course of disease is short are preferable；Therefore it will become the most biological high-technology of kind that puts goods on the market in the recent period
One of product.
In the research and development of Recombinant protein vaccine, the stability after proteantigen is adsorbed with adjuvant is also that necessary detection refers to
One of mark, this just needs the antigenic content in Accurate Determining vaccine finished product.And vaccine finished product is mostly that antigen protein and adjuvant adsorb
How colloidal compound afterwards uses method appropriate that proteantigen and adjuvant is allowed to will be completely dissociated and not destroy the property of antigen
Matter is a problem for needing emphasis to solve.In current report, have using the methods of sodium citrate, guanidine hydrochloride by albumen with
The method of adjuvant dissociation, but generally existing dissociation is not exclusively, the problems such as Dissociation time is partially long.Especially in oil emulsion vaccine,
Since vaccine sheet is as oily, the yield using commonsense method demulsification dissociation antigen protein is very low, and is easy to cause antigen protein
Denaturation is even degraded.
At present in the quality inspection of finished product vaccine, especially in the quality inspection of live vaccine, generally using finished product vaccine according to rule
Target animals are immunized in fixed immune programme, track subsequent antibody titer result or carry out Immunization experiment, to verify this batch
Can whether secondary vaccine meets the requirements, put goods on the market.Therefore, the content of antigen in a kind of alternative detection finished product vaccine is found
A research emphasis of current live vaccine with purity, this can not only save the cost and time, also can be avoided using dynamic
Object is tested, to save the usage amount of experimental animal.
Summary of the invention
The problem of and mutability low in view of antigen protein dissociation efficiency in above-mentioned oil emulsion vaccine is degraded, Yi Ji
In the quality inspection of finished product live vaccine using target animals tested so as to cause higher cost, quality inspection time it is long, and need using
The problem of many experimental animals, the present invention provides antigenic content in a kind of quickly detection oil emu subunit vaccine and purity
Method.
The method of the present invention includes the following steps: 1) using the albumen in cold acetone precipitation vaccine；2) SDS-PAGE electrophoresis；
3) gel is placed in imager to shoot, saves picture；4) picture in step 3) is placed in gray scale scanning instrument, uses software
The gray scale for defining standard protein band is 1, thus obtains sample protein band relative to the gray scale of standard protein band and compares knot
Fruit, then gray scale comparison result × standard protein content by sample protein band relative to standard protein band, to obtain
The content of sample protein；4) picture in step 3) is placed in conventional gray scale scanning instrument, chooses sample protein band using software
The swimming lane at place, then gray scale scanning is carried out to all bands of the swimming lane, to obtain sample protein band relative in sample
The ratio of all protein bands, the ratio are sample protein purity.
In technical solution of the present invention, the preferably described oil adjuvant is oil-in-water adjuvant or W/O/W adjuvant or oil
Packet water adjuvant.It is highly preferred that the oil adjuvant is 201 VG, ISA 35 VG or white oil of ISA.
In technical solution of the present invention, it is preferable that the cold acetone be acetone be placed in 20 DEG C of refrigerator precooling treatment 2h of ﹣ with
Upper acquisition.
In technical solution of the present invention, it is preferable that the content of the standard protein is 2~10 μ g.
In technical solution of the present invention, it is preferable that in the step 1), use the albumen packet in cold acetone precipitation vaccine
It includes following steps: a) taking the vaccine that theoretical protein content is 2~10 μ g in 1.5ml EP pipe, added according to the volume of taken vaccine
Enter cold acetone, wherein the volume ratio of cold acetone and vaccine is 9:1, is put into 20 DEG C of ﹣ and at least handles 1h or more；B) by sample in a)
12,000rpm, 4 DEG C, it is centrifuged 20min, supernatant liquid is removed, stays precipitating, after acetone volatile dry, 20 μ l PBS weight is added
It is outstanding；C) 5 μ l 5*loading buffer, boiling 10min in boiling water are added in every pipe after being resuspended；12,000rpm, 4 DEG C of centrifugations
5min, for use.
In technical solution of the present invention, it is preferable that in the step 2), the SDS-PAGE electrophoresis the following steps are included:
(1) the polyacrylamide gel electrophoresis prepared is clamping fixed into electrophoresis tank；(2) by 10 × Tris-Glycine
Buffer is diluted to 1 ×, it pours into electrophoresis tank, electrophoretic buffer liquid level need to not cross short slab, be lower than long version；(3) loading: Marker
Point sample amount is 5 μ l, and reference protein point sample amount is 25 μ l, and the sample of preparation is all added in sample cell；(4) it after point sample, presses
Red/black color Warning Mark correctly closes the lid, and 80-100V constant pressure runs concentration glue, and 120-150V constant pressure runs separation gel；(5) to
It when bromophenol blue indicator is run to electrophoresis plate lower end 5mm, powers off, stops running glue；(6) coomassie brilliant blue staining: by gel from
It carefully strips out, is immersed in coomassie brilliant blue staining liquid in glass plate, liquid level can cover gel surface, be placed in shaking table dyeing
1h；(7) it decolourizes: used dyeing liquor being poured into dedicated returnable bottle, with tap water rinse gel 3 times；Gel is immersed
In suitable destainer, decoloration is placed on shaking table to gel background transparent, until protein band capable of being obviously observed.
Antigen protein is obtained by what this method can be realized from oil emu protein subunit vaccine quickly and efficiently rate,
And it not will lead to the denaturation degradation of antigen protein, and this method is simply easy to repeat；Simultaneously by obtained antigen protein into
Row SDS-PAGE detection can obtain the content and purity of antigen protein in oil emu protein subunit vaccine as a result,
Cost, labour and time are saved.
Therefore, the present invention not only solves that antigen protein dissociation efficiency in oil emulsion vaccine is low and mutability degradation is asked
It inscribes, is tested in the quality inspection for also solving the problems, such as finished product oil emu protein subunit vaccine for animals using target animals.And this
Invention accuracy rate in the detection of the quantitative detection and purity of the antigen protein content of oil emu subunit vaccine can reach
90% or more.
Detailed description of the invention
Fig. 1 shows vaccine 1 (pig annulus Cap protein subunit vaccine) SDS-PAGE electrophoresis results: M is Marker, and 1 is
7.5 μ g standard proteins, 2 and 3 be two parallel samples of 50 μ l vaccines 1.
Fig. 2 indicates vaccine 2 (pig epidemic diarrhea S1, protein subunit vaccine) SDS-PAGE electrophoresis result: 4 are
Marker, 3 be 5 μ g standard proteins, and 1 and 2 be two parallel samples of 25 μ l vaccines 2.
Fig. 3 indicates vaccine 3 (swine fever E2 protein subunit vaccine) SDS-PAGE electrophoresis result: 1 is Marker, and 2 be 3 μ g mark
Quasi- albumen, 3 and 4 be two parallel samples of 100 μ l vaccines 3.
Specific embodiment
Below with reference to drawings and examples, the present invention will be further described, and the embodiment of the present invention is merely to illustrate this
The technical solution of invention, and the non-limiting present invention.
Agents useful for same is commercial product.
White oil is purchased from Hangzhou Refinery.
35 VG of ISA 201 VG and ISA is purchased from match BIC Corp, France.
1 cold acetone precipitation albumen of embodiment
(1) acetone is previously placed in 20 DEG C of refrigerator precooling treatment at least 2h of ﹣.
(2) (the pig annulus Cap protein subunit epidemic disease of vaccine 1 prepared in right amount according to theoretical protein in vaccine containing measurement
Seedling), vaccine 2 (pig epidemic diarrhea S1, protein subunit vaccine), vaccine 3 (swine fever E2 protein subunit vaccine) respectively at
In 1.5ml EP pipe (note: taken vaccine theoretical protein content is generally in 2-10 μ g), it is added according to the volume of taken vaccine appropriate
The good cold acetone of precooling treatment, wherein cold acetone and vaccine 1, vaccine 2, vaccine 3 volume ratio be 9:1, be put at 20 DEG C of ﹣
Manage at least 1h.
(3) 12,000rpm, 4 DEG C of sample will handled well, 20min centrifugation, carefully remove supernatant liquid, stay precipitating；If
Liquid be not easy to take it is clean, can 12,000rpm, 4 DEG C be centrifuged 5min again, after acetone volatile dries, 20 μ l PBS are added and are resuspended.
(4) 5 μ l 5*loading buffer, boiling 10min in boiling water are added in every pipe after being resuspended；12,000rpm, 4 DEG C from
Heart 5min, for use.
The preparation of 2 polyacrylamide gel of embodiment
(1) glass plate selection (selection of spacer i.e. gel thicknesses): plate interval 1.5mm, 10 hole glue comb specification: most
Big applied sample amount: 40 μ l,
(2) gel strength selects: selecting suitable gel strength according to table 1
Table 1
(3) loading board: selecting suitable glass plate, by glass plate wiped clean, assembles glass plate.
(4) prepare separation gel according to the prior art: plate interval 1.5mm gel configures volume: 7-8ml/ block.Such as it prepares not
The formula of 10% separation gel of same volume such as table 2:
Table 2
(5) the separation sol solution prepared in step (4) is shaken up rapidly, is poured into rapidly on one side along the gap of glass offset plate point
From glue, isopropanol sealing liquid face is used rapidly after recording separation gel.
(6) after gelling to be separated is solid, upper liquid is outwelled, with filter paper blotting non-evacuation.
(7) the concentration glue according to the prior art with 3 formula of tabulation:
Table 3
(8) it is shaken up rapidly after the completion of the configuration of concentration glue, along one side of glass sheet separation, fills concentration glue rapidly, it is noted that please don't
Bubble is squeezed into, matched stripping fork is rapidly inserted into, stands to concentration glue and solidifies completely.
3 polyacrylamide gel electrophoresis of embodiment (SDS-PAGE)
(1) the polyacrylamide gel electrophoresis prepared is clamping fixed into electrophoresis tank.
(2) 10 × Tris-Glycine Buffer is diluted to 1 ×, it pours into electrophoresis tank, electrophoretic buffer liquid level need to not have
It crosses short slab, is lower than long version (general electrophoretic buffer liquid level need to add between short slab and long slab).
(3) loading: including Marker, the sample prepared in standard control albumen and embodiment 1 is got out, wherein Marker
Point sample amount is 5 μ l, and reference protein point sample amount is 25 μ l, and the sample of preparation is all added in sample cell (possible more than 25 μ l).
(4) it after point sample, correctly closeing the lid by red/black color Warning Mark, 80-100V constant pressure runs concentration glue,
120-150V constant pressure runs separation gel.
(5) it when bromophenol blue indicator is run to electrophoresis plate lower end 5mm, powers off, stops running glue.
(6) coomassie brilliant blue staining: gel is carefully stripped out from glass plate, is immersed in coomassie brilliant blue staining liquid, liquid
Face can cover gel surface, be placed in shaking table dyeing 1h.
(7) it decolourizes: used dyeing liquor being poured into dedicated returnable bottle, with tap water rinse gel 3 times.By gel
It immerses in suitable destainer, is placed on shaking table decoloration to gel background transparent, until protein band capable of being obviously observed.
(8) gel conventional imaging instrument is placed in be shot, save data.
(9) picture in (8) being placed in conventional gray scale scanning instrument, the gray scale using software definition standard protein band is 1,
Thus gray scale comparison result of the sample protein band relative to standard protein band is obtained, then by sample protein band relative to mark
Gray scale comparison result × standard protein content of quasi- protein band, to obtain the content of sample protein.
(10) picture in (8) is placed in conventional gray scale scanning instrument, the swimming where sample protein band is chosen using software
Road, then gray scale scanning is carried out to all bands of the swimming lane, to obtain sample protein band relative to albumen all in sample
The ratio of band, the ratio are sample protein purity.
4 three kinds of vaccine testing results of embodiment
(1) to better illustrate the present invention, this laboratory is implemented according to the method for embodiment 1, embodiment 2 and embodiment 3
The protein content and purity detecting of 3 kinds of vaccines, specific vaccine information are as shown in table 4:
Table 4
(2) specific testing result is as shown in Figure 1, Figure 2, Figure 3 shows.
(3) protein content gray scale comparison result is as shown in table 5 in vaccine:
Table 5
(4) purity of protein gray scale comparison result is as shown in table 6 in vaccine:
Table 6
The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institutes here
The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this field
In technical staff practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention
In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and the limit of range
System, intention, which covers, all to be included the alternative in the spirit and scope of the invention as defined by appendix claim and waits
Same scheme.
Claims (4)
1. the detection method of protein content and purity in a kind of oil emu protein subunit vaccine, which is characterized in that the oil cream
Agent protein subunit vaccine includes a kind of antigen protein and oil adjuvant, and the oil adjuvant is 201 35 VG or white of VG, ISA of ISA
Oil；It is described that detection method includes the following steps:
1) using the albumen in oil emu protein subunit vaccine described in cold acetone precipitation, wherein the cold acetone is set for acetone
It is achieved above in 20 DEG C of refrigerator precooling treatment 2h of ﹣；It is described using in oil emu protein subunit vaccine described in cold acetone precipitation
Albumen the following steps are included:
A) take the oil emu protein subunit vaccine that theoretical protein content is 2~10 μ g in 1.5ml EP pipe, according to institute
Take the volume of the oil emu protein subunit vaccine that cold acetone is added, wherein cold acetone and the oil emu protein subunit epidemic disease
The volume ratio of seedling is 9:1, is put into 20 DEG C of ﹣ processing at least 1h；
B) by sample 12,000rpm in a), 4 DEG C, be centrifuged 20min, remove supernatant liquid, stay precipitating, to acetone volatile dry it
Afterwards, 20 μ l PBS are added to be resuspended；
C) 5 μ l 5*loading buffer, boiling 10min in boiling water are added in every pipe after being resuspended；12,000rpm, 4 DEG C of centrifugations
5min, for use；
2) SDS-PAGE electrophoresis；
3) gel is placed in conventional imaging instrument to shoot, saves picture；
4) picture in step 3) being placed in conventional gray scale scanning instrument, the gray scale using software definition standard protein band is 1, by
This obtains gray scale comparison result of the sample protein band relative to standard protein band, then by sample protein band relative to standard
Gray scale comparison result × standard protein content of protein band, to obtain the content of sample protein；And
5) picture in step 3) is placed in conventional gray scale scanning instrument, the swimming lane where sample protein band is chosen using software,
Gray scale scanning is carried out to all bands of the swimming lane again, to obtain sample protein band relative to protein bands all in sample
Ratio, the ratio be sample protein purity.
2. detection method according to claim 1, which is characterized in that the content of the standard protein is 2~10 μ g.
3. detection method according to claim 1, which is characterized in that in step 2), the SDS-PAGE electrophoresis include with
Lower step:
(1) the polyacrylamide gel electrophoresis prepared is clamping fixed into electrophoresis tank；
(2) 10 × Tris-Glycine Buffer is diluted to 1 ×, it pours into electrophoresis tank, electrophoretic buffer liquid level need to not have too short
Plate is lower than long version；
(3) loading: Marker point sample amount is 5 μ l, and reference protein point sample amount is 25 μ l, and the sample of preparation is all added to sample cell
In；
(4) it after point sample, correctly closes the lid by red/black color Warning Mark, 80-100V constant pressure runs concentration glue, 120-
150V constant pressure runs separation gel；
(5) it when bromophenol blue indicator is run to electrophoresis plate lower end 5mm, powers off, stops running glue；
(6) coomassie brilliant blue staining: gel is carefully stripped out from glass plate, is immersed in coomassie brilliant blue staining liquid, liquid level energy
Gel surface is covered, shaking table dyeing 1h is placed in；
(7) it decolourizes: used dyeing liquor being poured into dedicated returnable bottle, with tap water rinse gel 3 times；Gel is immersed
In suitable destainer, decoloration is placed on shaking table to gel background transparent, until protein band capable of being obviously observed.
4. a kind of detection method using as described in any claim of claim 1-3 is preparing oil emu protein subunit epidemic disease
Application in seedling in the detection of antigenic content and purity.
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