CN102380094B - Application of specific polypeptide to preparation of rabies vaccine - Google Patents
Application of specific polypeptide to preparation of rabies vaccine Download PDFInfo
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- CN102380094B CN102380094B CN201110306464.1A CN201110306464A CN102380094B CN 102380094 B CN102380094 B CN 102380094B CN 201110306464 A CN201110306464 A CN 201110306464A CN 102380094 B CN102380094 B CN 102380094B
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Abstract
The invention discloses application of specific polypeptide to preparation of a rabies vaccine. The invention provides application of a polypeptide containing a polypeptide fragment shown in as a sequence 1 in the sequence table to preparation of a rabies vaccine. The vaccine (synthesized peptide vaccine) provided by the invention comprises B cell epitope and T cell epitope. The vaccine can induce body humoral immunity to generate protective antibody and cause cellular immunity reaction, thereby removing virus effectively and playing a role of immunization protection. As no rabies active virus is related, the vaccine is safe and reliable; and active component short peptide of the vaccine can be artificially synthesized, so as to reduce production costs substantially.
Description
Technical field
The present invention relates to the application of a kind of specific polypeptides in the preparation rabies vaccine.
Background technology
Rabies are that the people beast of the central nervous system infection that caused by rabies virus suffers from infectious disease altogether, and its death rate of the onset is 100%.China's rabies death toll is in the second place of the world, is only second to India.
Causing rabic pathogen is the rabies virus (Rabies Virus) of Rhabdoviridae lyssavirus.Complete rabies virus is bullet shaped, and it is about 70 nanometers that length is approximately 200 nanometer left and right sides diameters.Whole virus is made of the RNA molecule of outermost lipid bilayer adventitia, structural protein shell and load hereditary information.
Rabies virus is at first infected the myocyte after entering human body, tide over incubation period in the myocyte, the back enters neurocyte by the acetylcholinergic receptor between myocyte and the neurocyte, enters spinal cord along identical path then, and then go into brain, do not prolong the blood diffusion.Viral brain inner infection hippocampus, cerebellum, brain stem and even whole central nervous system, and at the grey matter massive duplication, sprawl through positions such as descending arrival salivary gland, cornea, nasal mucosa, lung, skins.Rabies virus from negri body, is its discarded protein coat is assembled formation in cell acidophilia's granule to the main infringement of host.
The rabies vaccine of China's use at present all is cell culture vaccines, mainly comprises two kinds of inactivated vaccine and attenuated live vaccines.Because based on live virus, therefore there is certain potential safety hazard in the production of vaccine in producing and using.It is higher to adopt the cell culture mode to produce the vaccine cost in addition, causes domestic rabies vaccine price higher.
Summary of the invention
The purpose of this invention is to provide the application of a kind of specific polypeptides in the preparation rabies vaccine.
The invention provides the application of polypeptide in the preparation rabies vaccine of polypeptide fragment shown in the sequence 1 that contains ordered list.
Whether the polypeptide that the present invention also provides polypeptide fragment shown in the sequence 1 that contains ordered list infects application in the reagent of rabies virus preparation assistant identification animal.
The present invention also protects a kind of rabies vaccine, and its active component is the polypeptide that contains polypeptide fragment shown in the sequence 1 of ordered list.
Described vaccine also can comprise Freund's complete adjuvant or aluminum hydroxide adjuvant.
Described vaccine can be the vaccine for preparing by the following method: with described polypeptide and the complete emulsifying of Freund's complete adjuvant, obtain vaccine.Described vaccine specifically can be the vaccine for preparing by the following method: (1) with the PBS solution dissolving of described polypeptide with pH7.4, making the concentration of polypeptide is 1mg/ml, is antigenic solution; (2) isopyknic Freund's complete adjuvant and described antigenic solution are sucked respectively in two syringes, link to each other with a thin sebific duct between two syringes, alternately promote needle tubing then, till the Emulsion that forms thickness, be vaccine.
Described vaccine specifically can be the vaccine for preparing by the following method: (1) with the PBS solution dissolving of described polypeptide with pH7.4, making the concentration of polypeptide is 1mg/ml, is antigenic solution; (2) with isopyknic described antigenic solution and the abundant mixing of aluminum hydroxide adjuvant, be vaccine.Described aluminum hydroxide adjuvant specifically can prepare by the following method: get 5% (g/100ml) aluminum sulfate aqueous solution 250ml, under strong agitation, add 5% (g/100ml) sodium hydrate aqueous solution 100ml, with normal saline centrifuge washing precipitation 2 times, precipitation is hung into make in the normal saline again and reaches 250ml.
The present invention also protects a kind of assistant identification animal whether to infect the reagent of rabies virus, and its active component is the polypeptide that contains polypeptide fragment shown in the sequence 1 of ordered list.
Described polypeptide can also comprise polypeptide fragment (the position relation of each polypeptide fragment is variable) shown in the sequence 4 of polypeptide fragment shown in the sequence 3 of sequence table and/or sequence table.
Described polypeptide also can comprise polypeptide fragment shown in the sequence 2 of sequence table (the position relation of each polypeptide fragment is variable).
Concrete available two glycine residues (Gly Gly) connect between the described polypeptide fragment of in the described polypeptide each.
Described polypeptide specifically can be any one in following (1) to (9):
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
Described polypeptide can be the polypeptide after the activation.The method of described activation is specific as follows: described polypeptide is dissolved in containing Tris-HCl (pH8.0) buffer of 15% (volume ratio) DMSO and 0.9mM oxidisability glutathion, and 4 ℃ left standstill 48 hours.
The present invention also protects the polypeptide of polypeptide fragment shown in the sequence 1 that contains ordered list, is in following (1) to (9) any one:
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
Vaccine provided by the invention (synthetic peptide vaccine) is made up of B cell epitope and t cell epitope.This vaccine can bring out humoral immunity of organism and produce protection antibody, and causes cell immune response, thereby effectively removes virus, plays immanoprotection action.Owing to do not relate to the rabies live virus, this vaccine safety is reliable.And the active component of this vaccine is small peptide, can synthetic, reduce production costs greatly.
Description of drawings
Fig. 1 is that the mass spectrum of polypeptide III is identified collection of illustrative plates.
Fig. 2 is that the mass spectrum of polypeptide VI is identified collection of illustrative plates.
Fig. 3 is that the mass spectrum of polypeptide VIII is identified collection of illustrative plates.
Fig. 4 is that the mass spectrum of polypeptide IX is identified collection of illustrative plates.
Fig. 5 is that the mass spectrum of polypeptide I is identified collection of illustrative plates.
Fig. 6 is the reversed-phase HPLC spectrogram of polypeptide II.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Standard C VS rabies virus: Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Embodiment 1, synthetic rabies antigenic peptides
One, polypeptide is synthetic
By the Merrifield solid phase synthesis technique, automatically mate on the peptide synthesizer in the applying biological system, synthesize following polypeptide (nine kinds): polypeptide I (shown in the sequence 1 of sequence table) respectively with the Fmoc chemical compound, polypeptide II (shown in the sequence 5 of sequence table), polypeptide III (shown in the sequence 6 of sequence table), polypeptide IV (shown in the sequence 7 of sequence table), polypeptide V (shown in the sequence 8 of sequence table), polypeptide VI (shown in the sequence 9 of sequence table), polypeptide VII (shown in the sequence 10 of sequence table), polypeptide VIII (shown in the sequence 11 of sequence table) and polypeptide IX (shown in the sequence 12 of sequence table).
Two, the activation of polypeptide
Each polypeptide that step 1 is synthetic carries out following activation respectively:
1, uses trifluoroacetic acid according to the standardization program process resin, excise polypeptide from resin, and make the function group on the amino acid side chain take off sealing.
2, get the polypeptide of step 1, dissolve in containing Tris-HCl (pH8.0) buffer of 15% (volume ratio) DMSO and 0.9mM oxidisability glutathion, 4 ℃ left standstill 48 hours, promoted the formation of disulfide bond between the cysteine.
3, the polypeptide with step 2 carries out the HPLC purification
The used pillar length of HPLC purification is 250 millimeters, and internal diameter is 20 millimeters, and implant is C18 silica gel, and the implant model is Hedera ODS-2 10um.
Co-elute 40 minutes, flow velocity are 15ml/min.Eluent A is 0.1% (volume ratio) trifluoroacetic acid aqueous solution, and eluent B is 0.1% (volume ratio) trifluoroacetic acid acetonitrile solution.The eluent cumulative volume is 100%, the eluent in the initial moment is eluent A, stopping eluent constantly is eluent B, the initial moment and the eluent that stops between the moment are the mixed liquor of eluent A and eluent B, in the elution process, eluent A drops to 0% by 100% linearity, and eluent B rises to 100% by 0% linearity, collect eluent B account for the eluent cumulative volume 20%-60% cross solution behind the post.
Cross solution behind the post with what each polypeptide HPLC purification obtained, make lyophilized powder, be polypeptide behind each purification, use it for the every test among the embodiment 2 to embodiment 5.
4, polypeptide behind the purification of step 3 being carried out mass spectrum and reversed-phase HPLC identifies.
(1) mass spectrum is identified
Expection molecular weight: 4531.39;
Flow velocity: 0.2 ml/min;
Running time: 1 minute;
Buffer A: the aqueous solution that contains 0.1% (volumn concentration) formic acid;
Buffer B: the acetonitrile solution that contains 0.1% (volumn concentration) formic acid.
The mass spectrum qualification result shows: polypeptide II is shown in the sequence 5 of sequence table, polypeptide I shown in the sequence 1 of sequence table, polypeptide III shown in the sequence 6 of sequence table (see figure 1), polypeptide IV shown in the sequence 7 of sequence table, polypeptide V shown in the sequence 8 of sequence table, polypeptide VI shown in the sequence 9 of sequence table (see figure 2), polypeptide VII shown in the sequence 10 of sequence table, polypeptide VIII (see figure 3) shown in the sequence 11 of sequence table, polypeptide IX is (see figure 4) shown in the sequence 12 of sequence table; Polypeptide I is (see figure 5) shown in the sequence 1 of sequence table.
(2) reversed-phase HPLC is identified
Parameter as follows:
Elution time is 25 minutes, and flow velocity is 1ml/min, and applied sample amount is 10uL, and the detection wavelength is 220nm.
The pillar model is: Kromasil 100-5C18,4.6mmX250mm, 5 micron.
Eluent A is 0.1% (volume ratio) trifluoroacetic acid aqueous solution, and eluent B is 0.1% (volume ratio) trifluoroacetic acid acetonitrile solution.The eluent cumulative volume is 100%, the eluent in the initial moment is eluent A, stopping eluent constantly is eluent B, the initial moment and the eluent that stops between the moment are the mixed liquor of eluent A and eluent B, in the elution process, eluent A drops to 0% by 100% linearity, and eluent B rises to 100% by 0% linearity, collect eluent B account for the eluent cumulative volume 10%-50% cross solution behind the post.
The reversed-phase HPLC spectrogram of polypeptide II is seen Fig. 6, and analysis result sees Table 1, and purity is 49.28%.
The reversed-phase HPLC analysis result of table 1 polypeptide II
| In proper order | Time | Content | Area |
| 1 | 0.123 | 0.0004733 | 603 |
| 2 | 11.873 | 6.293 | 8020232 |
| 3 | 12.168 | 13.43 | 17111776 |
| 4 | 12.612 | 49.28 | 62807635 |
| 5 | 13.212 | 9.683 | 12341462 |
| 6 | 13.563 | 4.301 | 5481860 |
| 7 | 14.657 | 0.878 | 1119053 |
| 8 | 14.908 | 12.62 | 16080533 |
| 9 | 15.195 | 1.969 | 2508874 |
| 10 | 16.117 | 1.548 | 1972895 |
| |
100 | 127444923 |
The reversed-phase HPLC analysis result of polypeptide III sees Table 2, and purity is 79.35%.
The reversed-phase HPLC analysis result of table 2 polypeptide III
| In proper order | Time | Content | Area |
| 1 | 7.576 | 1.845 | 869621 |
| 2 | 8.476 | 0.1731 | 81571 |
| 3 | 8.781 | 4.527 | 2133766 |
| 4 | 9.072 | 79.35 | 37393914 |
| 5 | 9.607 | 3.039 | 1432509 |
| 6 | 9.953 | 2.001 | 943254 |
| 7 | 10.513 | 0.6868 | 323666 |
| 8 | 10.775 | 4.71 | 2219992 |
| 9 | 11.510 | 1.037 | 488636 |
| 10 | 12.057 | 1.105 | 520663 |
| 11 | 12.637 | 0.5195 | 244823 |
| 12 | 13.166 | 1.008 | 474903 |
| |
100 | 47127318 |
The reversed-phase HPLC analysis result of polypeptide IV sees Table 3, and purity is 87.12%.
The reversed-phase HPLC analysis result of table 3 polypeptide IV
| In proper order | Time | Content | Area |
| 1 | 8.958 | 1.385 | 52972 |
| 2 | 10.465 | 5.106 | 195264 |
| 3 | 11.220 | 1.025 | 39186 |
| 4 | 11.535 | 0.6526 | 24956 |
| 5 | 12.024 | 87.12 | 3331427 |
| 6 | 12.338 | 0.1558 | 5957 |
| 7 | 12.588 | 0.9158 | 35021 |
| 8 | 13.193 | 1.728 | 66064 |
| 9 | 13.983 | 0.2302 | 8801 |
| 10 | 14.288 | 0.2815 | 10763 |
| 11 | 14.788 | 1.401 | 53570 |
| |
100 | 3823981 |
The reversed-phase HPLC analysis result of polypeptide V sees Table 4, and purity is 98.56%.
The reversed-phase HPLC analysis result of table 4 polypeptide V
| In proper order | Time | Content | Area |
| 1 | 9.140 | 0.1131 | 16992 |
| 2 | 10.771 | 0.09063 | 13618 |
| 3 | 10.942 | 98.56 | 14809792 |
| 4 | 11.489 | 0.1043 | 15677 |
| 5 | 11.612 | 0.1287 | 19336 |
| 6 | 11.833 | 0.04504 | 6768 |
| 7 | 12.670 | 0.5052 | 75905 |
| 8 | 12.924 | 0.4506 | 67702 |
| |
100 | 15025790 |
The reversed-phase HPLC analysis result of polypeptide VI sees Table 5, and purity is 50.39%.
The reversed-phase HPLC analysis result of table 5 polypeptide VI
| In proper order | Time | Content | Area |
| 1 | 8.463 | 0.8312 | 276628 |
| 2 | 8.773 | 2.604 | 866587 |
| 3 | 9.231 | 4.246 | 1413209 |
| 4 | 11.354 | 3.61 | 1201528 |
| 5 | 11.611 | 7.647 | 2544952 |
| 6 | 11.901 | 50.39 | 16770288 |
| 7 | 12.491 | 0.1231 | 40982 |
| 8 | 12.702 | 2.679 | 891531 |
| 9 | 13.012 | 4.443 | 1478488 |
| 10 | 13.438 | 3.018 | 1004490 |
| 11 | 13.696 | 13.04 | 4338082 |
| 12 | 14.318 | 5.003 | 1664915 |
| 13 | 15.329 | 2.366 | 787300 |
| |
100 | 33278980 |
The reversed-phase HPLC analysis result of polypeptide VII sees Table 6, and purity is 88.19%.
The reversed-phase HPLC analysis result of table 6 polypeptide VII
| In proper order | Time | Content | Area |
| 1 | 6.434 | 0.2845 | 30537 |
| 2 | 7.570 | 0.2009 | 21565 |
| 3 | 7.705 | 0.2228 | 23912 |
| 4 | 7.839 | 0.05869 | 6299 |
| 5 | 8.033 | 88.19 | 9464793 |
| 6 | 8.299 | 10.72 | 1150370 |
| 7 | 8.575 | 0.3238 | 34756 |
| |
100 | 10732232 |
The reversed-phase HPLC analysis result of polypeptide VIII sees Table 7, and purity is 92.15%.
The reversed-phase HPLC analysis result of table 7 polypeptide VIII
| In proper order | Time | Content | Area |
| 1 | 8.988 | 2.201 | 229214 |
| 2 | 9.460 | 0.2103 | 21907 |
| 3 | 9.622 | 0.2121 | 22089 |
| 4 | 9.788 | 0.1639 | 17072 |
| 5 | 10.408 | 92.15 | 9598230 |
| 6 | 10.633 | 0.009305 | 969 |
| 7 | 11.291 | 2.824 | 294196 |
| 8 | 12.341 | 0.753 | 78433 |
| 9 | 12.590 | 1.479 | 154011 |
| |
100 | 10416121 |
The reversed-phase HPLC analysis result of polypeptide IX sees Table 8, and purity is 90.31%.
The reversed-phase HPLC analysis result of table 8 polypeptide VIII
| In proper order | Time | Content | Area |
| 1 | 8.936 | 0.4289 | 17520 |
| 2 | 9.091 | 90.31 | 3688886 |
| 3 | 9.461 | 0.415 | 16951 |
| 4 | 9.553 | 0.6556 | 26781 |
| 5 | 11.265 | 3.901 | 159357 |
| 6 | 11.510 | 0.4796 | 19592 |
| 7 | 12.319 | 1.727 | 70530 |
| 8 | 12.563 | 2.083 | 85106 |
| |
100 | 4084723 |
The reversed-phase HPLC analysis result of polypeptide I sees Table 9, and purity is 99.26%%.
| In proper order | Time | Content | Area |
| 1 | 10.322 | 0.3043 | 56450 |
| 2 | 10.438 | 0.3036 | 56324 |
| 3 | 10.722 | 0.1387 | 25729 |
| 4 | 11.489 | 99.26 | 18414699 |
| Sum | 11.042 | 100 | 18553202 |
The preparation of embodiment 2, vaccine
Freund's complete adjuvant is available from Sigma company (article No. F5881).
Respectively with each lyophilized powder of embodiment 1 preparation (contain respectively corresponding purification after polypeptide) respectively with the complete emulsifying of Freund's complete adjuvant, obtain various vaccines, concrete steps are as follows:
1, with PBS solution (prescription with reference to " molecular cloning " second edition appendix table B.7) dissolving of lyophilized powder with pH7.4, making the concentration of polypeptide is 1mg/ml, is antigenic solution.
2, isopyknic Freund's complete adjuvant and antigenic solution are sucked respectively in two syringes, link to each other with a thin sebific duct between two syringes, note the emptying air, alternately promote needle tubing then, till the Emulsion that forms thickness, be vaccine.
3, vaccine need carry out quality inspection, is about to emulsifying agent and splashes in the cold water, does not disperse if be kept perfectly, and becomes to drip shape and bubbles through the water column, i.e. emulsifying is complete, is qualified Water-In-Oil vaccinating agent.
Obtain vaccine I, vaccine II, vaccine III, vaccine IV, vaccine V, vaccine VI, vaccine VII, vaccine VIII and vaccine IX respectively, the vaccine sequence number is corresponding with the polypeptide sequence number.
The PBS solution of pH7.4 is replaced antigenic solution, carry out step 2, obtain the vaccine reference substance.
The immunity of embodiment 3, mice and neutralizing antibody evaluation
One, the immunity of mice
Get 560 of the female BALB/c mouse in 7 ages in week (available from Chinese Academy of Sciences heredity and grow institute) of body condition health, be divided into three groups, 540 of immune group, 10 of negative control group, 10 of blank groups.Immune component is 9 groups, and 60 every group, the various vaccines with embodiment 2 preparations carry out subcutaneous immunity (every mice single immunization contains the vaccine of 50 μ g polypeptide, 0.1 milliliter) respectively.Negative control group is carried out immunity (0.1 milliliter of vaccine reference substance of every mice single immunization) with the vaccine reference substance of embodiment 2 preparations.The blank group is not carried out any immunity.
Immune group and negative control group were carried out initial immunity on the 1st day in experiment respectively, experiment the 7th day, carried out three times booster immunization in the 14th day and the 28th day.7 days (testing the 35th day) collects serum behind booster immunization for the third time.
Two, neutralizing antibody detects
Serum detects rabies poison neutralizing antibody with fluorescent antibody virus neutralization tests (FAVN) after inactivation treatment, rabies antibody 〉=0.5IU/ml is positive.FAVN is with reference to " terrestrial animal diagnostic test and vaccine handbook are carried out.Record serum changes positive number, the results are shown in Table 10.
The result that table 10 neutralizing antibody detects
Three, immunoprotection experiment
Behind booster immunization for the third time 7 days (testing the 35th day), every mice uses the standard C VS rabies virus of 10 * LD50 to carry out counteracting toxic substances by peritoneum, and observes the appearance of syndrome, and behind the counteracting toxic substances the 13rd day, statistics was respectively organized survival number and the survival rate of mice.The results are shown in Table 11.
Table 11 immunoprotection experimental result
The preparation of embodiment 4, vaccine
Aluminum hydroxide adjuvant: get 5% (g/100ml) aluminum sulfate aqueous solution 250ml, add 5% (g/100ml) sodium hydrate aqueous solution 100ml under strong agitation, usefulness normal saline centrifuge washing precipitates 2 times, precipitation is hung into make in the normal saline again to reach 250ml.
Respectively each lyophilized powder of embodiment 1 preparation (contain respectively corresponding purification after polypeptide) is carried out following steps:
1, with PBS solution (prescription with reference to " molecular cloning " second edition appendix table B.7) dissolving of lyophilized powder with pH7.4, making the concentration of polypeptide is 1mg/ml, is antigenic solution.
2, with isopyknic antigenic solution and the abundant mixing of aluminum hydroxide adjuvant, be vaccine.
Obtain vaccine I, vaccine II, vaccine III, vaccine IV, vaccine V, vaccine VI, vaccine VII, vaccine VIII and vaccine IX respectively, the vaccine sequence number is corresponding with the polypeptide sequence number.
The PBS solution of pH7.4 is replaced antigenic solution, carry out step 2, obtain the vaccine reference substance.
The immunity of embodiment 5, beasle dog and neutralizing antibody evaluation
One, the immunity of beasle dog
Get 184 of the male beasle dogs of 6 monthly ages (available from Beijing Experimental Animal Center) of body condition health, be divided into three groups, 180 of immune group, 2 of negative control group, 2 of blank groups.Immune component is 9 groups, and 20 every group, the various vaccines with embodiment 4 preparations carry out intramuscular injection immunity (every beasle dog single immunization contains the vaccine of 200 μ g polypeptide, 0.4 milliliter) respectively.Negative control group is carried out immunity (0.4 milliliter of vaccine reference substance of every beasle dog single immunization) with the vaccine reference substance of embodiment 4 preparations.The blank group is not carried out any immunity.
Immune group and negative control group were carried out initial immunity on the 1st day in experiment respectively, carried out booster immunization on the 30th day in experiment.7 days (testing the 37th day) collects serum behind booster immunization.
Two, neutralizing antibody detects
Serum detects rabies poison neutralizing antibody with fluorescent antibody virus neutralization tests (FAVN) after inactivation treatment, rabies antibody 〉=0.5IU/ml is positive.FAVN is with reference to " terrestrial animal diagnostic test and vaccine handbook are carried out.Record serum changes positive number, the results are shown in Table 12.
The result that table 12 neutralizing antibody detects
Embodiment 6, safety experiment
Each mice among the embodiment 3 is observed it in each immunity back, monitors spleen simultaneously and changes, and immune mouse is all strong deposits weight increase, no whole body abnormal response through observing.
Each beasle dog among the embodiment 5 is observed it in each immunity back, monitors spleen simultaneously and changes, and the immunization experiment dog is all strong deposits weight increase, no whole body abnormal response through observing.
The polypeptide of embodiment 7, Application Example 1 preparation detects the effect (ELISA) of vaccine immune mouse
One, the immunity of mice
Step 1 with embodiment 3.
7 days (testing the 35th day) collects serum behind booster immunization for the third time.
Two, ELISA detects
Respectively each lyophilized powder of embodiment 1 preparation (is contained polypeptide behind the corresponding purification respectively as envelope antigen in the lyophilized powder, polypeptide plays the effect of envelope antigen), detect the immune effect of vaccine, the polypeptide that adopts is corresponding with vaccine, if namely envelope antigen is polypeptide I, namely for detection of immunity the mice serum of vaccine I, concrete steps are as follows:
(1) sodium carbonate buffer (pH 9.6) that 100 μ l is contained 5 μ g/ml polypeptide places dull and stereotyped hole, 96 holes, and 37 ℃ of bags were by 1 hour.
(2) gelatin (also can adopt 1% bovine serum albumin or 10% calf serum) with 250 μ l 3% places bag by the hole, and 37 ℃ were sealed 1 hour.
(3) PBST (prescription is with reference to the molecular cloning second edition) with 250 μ l pH7.5 washes 3 times, and is dry then.Use the PBST of pH7.5 to dilute the clear ratio in 1: 200 of take a blood sample, obtain the sample diluent.
(4) respectively sample diluting liquid is added each hole of step (2), every hole 100 μ l, 37 ℃ of reactions 1 hour.
(5) then with the PBST of 250 μ l pH7.5 flushing 6 times.
(6) sheep anti-mouse antibody with 100 μ l Radix Cochleariae officinalises or oxide enzyme labelling adds in the serum hole, and 37 ℃ were reacted 1 hour.
(7) then with the PBST of 250 μ l pH7.5 flushing 6 times.
(8) contain sodium citrate buffer solution (pH 5.0) reaction 15 minutes of the hydrogen peroxide of 0.04% m-diaminobenzene. (OPD) and 0.12% then with 100 μ l.
(9) the sulphuric acid cessation reaction of adding 100 μ l1M, the absorption value of mensuration 492nm is decided to be the positive for 2.1 times with negative line control serum absorption value.
The result that table 13ELISA detects
The polypeptide of embodiment 8, Application Example 1 preparation detects the effect (ELISA) of vaccine immunity beasle dog
One, the immunity of beasle dog
Step 1 with embodiment 5.
Two, ELISA detects
Respectively each lyophilized powder of embodiment 1 preparation (is contained polypeptide behind the corresponding purification respectively as envelope antigen in the lyophilized powder, polypeptide plays the effect of envelope antigen), detect the immune effect of vaccine, the polypeptide that adopts is corresponding with vaccine, if namely envelope antigen is polypeptide I, namely for detection of immunity the beasle dog serum of vaccine I.
Concrete steps are with the step 2 of embodiment 7, and difference only is to adopt the goat-anti dog antibody of Radix Cochleariae officinalis or oxide enzyme labelling.
The result that table 14ELISA detects
The polypeptide of Application Example 1 preparation detects animal when whether infecting rabies virus (ELISA), only need get the serum of tested animal, detects with reference to the ELISA method of embodiment 7 or embodiment 8 to get final product.And by adopting different polypeptide as envelope antigen, the rabies virus strain that can the auxiliary judgment tested animal infects.
Claims (13)
1. the application of any one polypeptide in the preparation rabies vaccine in following (1) to (9);
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
2. application as claimed in claim 1 is characterized in that: described polypeptide is the polypeptide after activating.
3. application as claimed in claim 2 is characterized in that: the method for described activation is as follows: described polypeptide is dissolved in the Tris-HCl buffer that contains 15%DMSO and 0.9mM oxidisability glutathion, and 4 ℃ left standstill 48 hours.
4. whether any one polypeptide infects application in the reagent of rabies virus preparation assistant identification animal in following (1) to (9);
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
5. application as claimed in claim 4 is characterized in that: described polypeptide is the polypeptide after activating.
6. application as claimed in claim 5 is characterized in that: the method for described activation is as follows: described polypeptide is dissolved in the Tris-HCl buffer that contains 15%DMSO and 0.9mM oxidisability glutathion, and 4 ℃ left standstill 48 hours.
7. rabies vaccine, its active component is any one polypeptide in following (1) to (9);
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
8. vaccine as claimed in claim 7 is characterized in that: the polypeptide of described polypeptide after for activation.
9. vaccine as claimed in claim 8, it is characterized in that: the method for described activation is as follows: described polypeptide is dissolved in the Tris-HCl buffer that contains 15%DMSO and 0.9mM oxidisability glutathion, and 4 ℃ left standstill 48 hours.
10. whether an assistant identification animal infects the reagent of rabies virus, and its active component is any one polypeptide in following (1) to (9);
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
11. reagent as claimed in claim 10 is characterized in that: described polypeptide is the polypeptide after activating.
12. reagent as claimed in claim 11 is characterized in that: the method for described activation is as follows: described polypeptide is dissolved in the Tris-HCl buffer that contains 15%DMSO and 0.9mM oxidisability glutathion, and 4 ℃ left standstill 48 hours.
13. contain the polypeptide of polypeptide fragment shown in the sequence 1 of ordered list, be in following (1) to (9) any one:
(1) polypeptide shown in the sequence 8 of sequence table;
(2) polypeptide shown in the sequence 5 of sequence table;
(3) polypeptide shown in the sequence 6 of sequence table;
(4) polypeptide shown in the sequence 12 of sequence table;
(5) polypeptide shown in the sequence 11 of sequence table;
(6) polypeptide shown in the sequence 7 of sequence table;
(7) polypeptide shown in the sequence 10 of sequence table;
(8) polypeptide shown in the sequence 9 of sequence table;
(9) polypeptide shown in the sequence 1 of sequence table.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110306464.1A CN102380094B (en) | 2011-10-11 | 2011-10-11 | Application of specific polypeptide to preparation of rabies vaccine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110306464.1A CN102380094B (en) | 2011-10-11 | 2011-10-11 | Application of specific polypeptide to preparation of rabies vaccine |
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| Publication Number | Publication Date |
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| CN102380094A CN102380094A (en) | 2012-03-21 |
| CN102380094B true CN102380094B (en) | 2013-08-07 |
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| US6090388A (en) * | 1998-06-20 | 2000-07-18 | United Biomedical Inc. | Peptide composition for prevention and treatment of HIV infection and immune disorders |
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