CN1050561A - Recombinant dna order, express the recombinant adenovirus of this DNA sequence and contain the rhabdovirus vaccine of this recombinant adenovirus - Google Patents
Recombinant dna order, express the recombinant adenovirus of this DNA sequence and contain the rhabdovirus vaccine of this recombinant adenovirus Download PDFInfo
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Abstract
The invention provides the recombinant dna order that contains coding rabies virus antigenic insertion DNA sequence and in conjunction with the recombinant adenovirus of this DNA sequence.
The present invention also provides the vaccine of the recombinant adenovirus that contains significant quantity and pharmaceutically acceptable vehicle and has made Mammals produce the method for immunological competence to the disease of being brought out by rabies virus, comprises virus or vaccine administration in Mammals.
Description
The present invention relates to recombinant dna order, express the recombinant adenovirus of this DNA sequence and contain the rhabdovirus vaccine of this recombinant adenovirus.
Although saved countless life owing to inoculated the anti-rabies virus vaccine since the bus moral epoch, this disease still continues to become serious worldwide problem.Rabies are to be caused by the virus in the rhabdoviridae of virus (Rhabdoviridae family) Lyssavirus (Lyssavirus genera).Still have every year people more than 20,000 attacked by rabies virus according to estimates, mainly occur in south east asia.Even in the people's of this area death condition and not serious, but hundreds of thousands of them's vaccination then makes us feeling tired brain, and a large amount of domestic animal dies of illness and caused serious economy loss.The Wildlife that was subjected to infect has formed the main source of rabies virus, and the people and the domestic animal that can utilize had at present all proved safely and effectively already with vaccine, yet only limited to these vaccines are used in the rabic plan of mass control from economy with in fact.In addition, the known recombinant virus vaccine that has the coding antigenic gene of rabies and be expressed in infection carrier virus has very big potentiality.The recombinant chou vaccine virus carrier of expressing glycoprotein of rabies virus had proved already that induce immune response was avoided the attack of lethality rabies with the various animals of protection effectively.Adenovirus hominis is studied as a kind of recombinant rhabdovirus glycoprotein carrier with potential possible non-rabies virus, and can bring out the neutralizing antibody to the stomatitis herpesvirus in mouse, dog, pig and the ox (VSV) glycoprotein effectively.Adenovirus has such as being defined host range, stability and can making it be specially adapted to the development of rabies virus vaccine by many character such as oral infections.
One of task of the present invention is to provide the recombinant dna that contains the antigenic insertion DNA sequence of coding rabies virus order.The adenovirus DNA order can be derived by adenovirus hominis, and described adenovirus hominis is good with 5 type adenovirus hominiss especially.
Inserting the expression of DNA sequence can be controlled by adenovirus promoter, and adenovirus promoter can be the E3 promotor or the main late promotor of adenovirus hominis.
Insert DNA sequence and can replace adenovirus DNA order and early stage adenovirus DNA order, insertion sequence preferably replaces part E3 adenoviral gene at least.
Inserting DNA can be included in the another kind of viral DNA box.Described another kind of virus can be simian virus, is good with SV40 especially, inserts DNA and can be inserted between SV40 promotor and the early stage poly A attachment site of distinguishing.
Insert the DNA sequence rabies virus antigen of can encoding, this rabies virus antigen can be glycoprotein.
Two of task of the present invention is to provide the recombinant virus that contains above-mentioned recombinant dna order, and this recombinant virus can be that the Ad5RGILP(preserving number that is deposited in ATCC is ATCC No.VR2204).
Three of task of the present invention is to provide the vaccine of acceptable vehicle on the above-mentioned recombinant virus that contains significant quantity and the medicine.The significant quantity of virus is at least every 0.1ml vaccine and contains 10
4Plaque forming unit (PFU).
Four of task of the present invention is to provide the immunization method of the disease of Mammals to being brought out by rabies virus, and this method comprises to above-mentioned recombinant virus of administration or vaccine.
Above-mentioned vaccine can be used by mouth and nose.
Now describe expressible dna recombinant adenovirus in proper order and the rhabdovirus vaccine that contains this recombinant adenovirus in detail by embodiment and with reference to accompanying drawing.
Fig. 1 illustrates the structure of recombinant adenovirus;
Fig. 2 illustrates the test-results of vaccine drug effect of the present invention.
1. the structure that contains 5 type adenovirus hominiss of glycoprotein gene of rabies virus recombinant chou
The construction step that is used to produce the 5 type recombinant chou adenovirus hominiss that contain rabies virus glucoprotein is shown in Fig. 1.
The new SV2(Southern of plasmid, P.J. and Berg, P. distinguish in early days under the promotor control mammalian cell to be converted at SV40 and have antibiotic resistance with bacterial gene, J.Molecular App-lied Genetics, 1982,1, modification 327-334) can be passed through NdeI(N) site is converted into XbaI(X) site, replace BamI(B by the XbaI linker) to EcoRI(E) fragment, by with containing the Hind III, BamHI, EcoRI, SmaI(S) and XhoI(Xh) the synthetic multi-joint knot of restriction site replaces Hind III to HpaI plasmid sequence all new genes and SV40 is lacked in proper order.In the plasmid pSV2X3 that forms, SV40 early promoter order (representing with full box among Fig. 1) is separated with SV40 poly A additional signal (representing with discontented box among Fig. 1) by the synthetic multi-joint knot order that can insert required gene.SV40 promotor, multi-joint knot and SV40 poly A additional signal are connected to form one " box " by the XbaI site.
The complete cDNA copy of the glycoprotein gene of the rabies virus ERA strain that is obtained by Connaught medical research laboratory in pBR322 is as the glycoprotein gene of rabies virus source that is inserted into pSV2X3.Clone's rabies virus DNA is by Malek, and L.T., Soostmeyer, G., Gar-vin, R.T. and James.E. did to introduce.Glycoprotein gene of rabies virus is expressed as the sex change polypeptide and (consults Chanock in intestinal bacteria (E.coli), R. and Lerner, R. compiles: ModernApproaches to Vaccines, Cold Spring Harbor Laboratoy, 1984,203-208).By isolating gene with EcoRI and BamHI digested plasmid, and the gene that after filling up blunt end with the Ke Lienuofu polysaccharase separation is obtained is inserted in the SmaI site of multi-joint knot of pSV2X3.
One of the plasmid that forms pSV2X3RG has rabies virus DNA, can be transcribed by the SV40 promotor on correct direction.Containing the box that inserts rabies virus glucoprotein can separate by XbaI digestion removal and by the agarose separation method.Then box is inserted into (Haj-Ahmad in the XbaI site of plasmid pFGDX1, Y. and Graham, F.L.Development of a helper-independent humen adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene, J.of Virology, 1986,57,267-274).Plasmid pFGDX1 contains the genome of 5 type adenovirus hominiss, comprises the E3 promotor on the BamHI site (except the XbaID fragment of 78.5%-84.3%) of 59.5%-100%.
In one of the plasmid that forms thus pBCRG, the box that contains glycoprotein gene of rabies virus is inserted on the correct direction, makes the transcriptional orientation of SV40 keep identical direction with adenovirus E3 promotor.Select plasmid pBCRG to cultivate and purifying.293 cell (Graham, F.L., Smiley, J., Russell, W.C. and Nairn, R., Characteristics of a human Cell line transformed by DNA from human adenovirus type 5.J.of General Virology, 1977,36,59-72) adopt 5 type adenovirus DNAs of calcium phosphate precipitation technology and pBCRG and EcoRI digestion to carry out transfection.Recombinant adenovirus (A5RGILP) with required rabies virus gene insertion is by identifying with Hind III, XbaI and EcoRI restriction enzyme analysis and purifying.Recombinant adenovirus A5RGILP carries out the plaque purifying twice in 293 cells.The expansion and growing in KB cell (consult H.Eagle show in " Proc.Soc.Exp.Biol.and Med., 1955,89; 362 " " Human; oral, epidermoid carcinema, HeLamarkers(ATCC CCL 17KB) suspension culture of final plaque isolate.A5GRILP is by extracting in the cells infected, and on the CsCl density gradient by twice in conjunction with carrying out purifying.Recombinant virus carries out dialysis and titration before use.The rabies virus gene insert be with the equidirectional that replaces the adenovirus E3 gene that forms recombinant virus on.Rabies virus gene among the A5RGILP is to express on the transcriptional start point of adenovirus E3 promotor or main late promotor.
2. use the antibody response after the Ad5RGILP virus
When animal experiment begins recombinant chou Ad5RGILP virus is introduced in mouse and the dog.Be seeded on the mouse by oral or parenteral, by in skin or nose, giving dog virus.Before and after the inoculation, take serum sample, before analysis, be numbered earlier, suppress the titre that micromethod (FIMT) is measured the rabies virus neutralizing antibody by fluorescence.As shown in table 1, it is all fine that both use replying of vaccine to parenteral.Therefore in the mouse that oral vaccine is replied,, can not affirm oral or whether can cause seroconversion through enteral administration because some inoculum can cause gastric disorder causing nausea in animal body or be stored in the oral cavity during taking out pipe.Observed particularly importantly that by nasal cavity virus to be applied to dog very effective to bringing out neutralizing antibody.Equally also obtain above-mentioned observation with the adenovirus carrier of expressing stomatitis herpesvirus glycoprotein.Because recombinant chou stomatitis herpesvirus antibody in bringing out ox and pig of the latter also is effectively, therefore think that Ad5RGILP virus also can be used as very effective immunogen in many animals.
Behind the fortnight, give the vaccine of dog for the second time with 0.5ml dosage.Measure the serum antibody titration degree when 2 weeks after the vaccination first time and 4 weeks.Give by what the Challis, James river obtained and make the inoculum of female mouse 3 weeks with 0.1ml.Under our the slight anesthesia of fluorine second, No. 23 gauge needle tying the vaccine inoculation pipe with the band lung are added to vaccine and give oral medication in the micro tube.Pipe is inserted in the place above its throat 1cm.All around,, measure each serum respectively by the blood drawing of mouse tail.Mouse antigen titration degree just the results are shown in table 1 with each group serum; Numeral in the bracket is respectively organized the positive result's of serum animal sum.The antigen titration degree suppresses the microdetermination unit representation with fluorescence, measures with the fluorescence focal forming method, and the serum of a unit representation ERA rabies challenge virus is at least 50% when suppressing to duplicate 2 times of highly diluteds.All serum all heated deactivation 30 minutes down at 56 ℃ before measuring.
3.Ad5RGILP mouse is to the protection of rabies virus after the virus vaccines amount of minimizing
After beginning achieving success, the Ad5RGILP recombinant virus vaccine has been done further research in the intravital drug effect of mouse.In these researchs, will make in 5 weeks the mouse grouping, close 5 mouse in the cage, arbitrarily feed, and to give titre be 10 to Purina aggressiveness food
4-10
7The gradient recombinant virus that the 0.1ml of plaque forming unit (PFU) is purified, the intraperitoneal single administration.Control group is only given 0.1ml viral dilution liquid (PBS/2% foetal calf serum).After three weeks, blood drawing (every mouse is taken out 0.2-0.3ml) on the mouse tail, in each serum sample remittance, heating deactivation (56 ℃ are following 30 minutes) is measured the rabies virus neutralizing antibody with the FIMT method.Gathered behind the serum three days, and attacked by administration in the brain, observed 21 days with the ERA strain (20 TCID, 50/ dosage) of the rabies virus of 0.3ml.Death mainly occurred in 10 to 14 days.Typical illness all appears in all mouse, comprises that the mouse hair is mixed and disorderly, and attitude is out of control, loses weight tic increase and/or tetraplegia and death.The result of this Attack Research illustrates Fig. 2.As shown in Figure 2, injection 10
7The Ad5GRILP virus of PFU/ mouse (only) amount can both be brought out high-titer neutralizing antibody on all mouse.Reduce with replying then of in mouse body, producing of the low dosage of 10 times of diluents, and the titre of antibody and reply the also corresponding reduction of mouse number.Although the mouse of contrast (immunity) group is after rabies virus is attacked, 100% is all dead, and all mouse the symptom of rabies virus infection do not occur in 21 day observation period after using vaccine, so that can detected antibody response also extremely low.Three mouse of using low dosage Ad5GRLIP virus do not detect neutralizing antibody; and still survival after rabies virus is attacked; but this is considered to provide protection by the very low antibody of detection limit, or because the stimulation of some other defense mechanism has produced provide protection.Have the rabies specificity by the viral this immune protection stimulation that produces of Ad5GRLIP,, after 2 weeks, still be easy to infected (data do not provide) with the rabies virus attack because 15 mouse only inoculate 5 type adenovirus.
Vaccine of the present invention also can bring out a large amount of neutralizing antibodies after also having proved and having given skunk and fox oral effectively, and can avoid death after the rabies virus attack with lethal dose.
The sample of recombinant chou Ad5GRILP virus is deposited in the internationally recognized American type culture collection (ATCC of microbial preservation unit that is used for patented procedure by budapest treaty on March 4th, 1988,12301 Park Lawn Drive, Rockville, Maryland 20852, U.S.A.), the preserving number of announcing on March 15th, 1988 is ATCC No.VR 2204.
Table 1: use the antibody response after the Ad5RGILP virus
Animal dose inoculation vaccine mode antigen titration degree
(PFU) second time first time in 2 all 4 weeks of 0 week
Dog 15 * 10
5Through skin through skin a small amount of 256 512
Dog 2 through skin through skin a small amount of 1,024 1024
Dog 3 noses are interior through skin a small amount of 256 512
Nose interior a small amount of 256 4096 in dog 4 noses
Mouse 1 * 10
7Oral a small amount of-1124(4/10)
Mouse 1 * 10
6Oral a small amount of-424(4/10)
Mouse 1 * 10
7The intramuscular intramuscular on a small quantity-2925(10/10)
Mouse 1 * 10
6The intramuscular intramuscular on a small quantity-1312(8/10)
Claims (18)
1, recombinant dna order, described DNA sequence contains the antigenic insertion DNA sequence of coding rabies virus.
2, by the recombinant dna order of claim 1, wherein adenovirus DNA is by the adenovirus hominis deutero-in proper order.
3, by the recombinant dna order of claim 2, wherein adenovirus hominis is 5 type adenovirus hominiss.
4, by the recombinant dna order of one of aforesaid right requirement, wherein insert the expression of DNA sequence and control by adenovirus promoter.
5, by the recombinant dna order of claim 4, wherein adenovirus promoter is the E3 promotor or the main late promotor of adenovirus hominis.
6,, wherein insert DNA sequence and replace the adenovirus DNA order by the recombinant dna order of one of aforesaid right requirement.
7,, wherein insert DNA sequence and replace early stage adenovirus DNA order by the recombinant dna order of claim 6.
8, by the recombinant dna order of claim 7, wherein insert DNA sequence and replace part E3 gene at least.
9,, wherein insert DNA and be included in the another kind of viral DNA box by the recombinant dna order of one of aforesaid right requirement.
10, by the recombinant dna order of claim 9, wherein another kind of virus is simian virus.
11, by the recombinant dna order of claim 10, wherein simian virus is SV40.
12,, wherein insert DNA and be inserted between the early stage promotor of distinguishing and poly A attachment site of SV40 by the recombinant dna order of claim 11.
13, a kind of recombinant virus, this virus contain the recombinant dna order of one of aforesaid right requirement.
14, a kind of vaccine, this vaccine contain the described recombinant virus of claim 13 and the pharmaceutically acceptable vehicle of significant quantity.
15, make Mammals produce the method for immunological competence to the disease of being brought out by rabies virus, this method comprises to administration claim 13 or 14 described virus or vaccines.
16, by the method for claim 15, wherein virus or vaccine are by oral or nasal administration.
17, the method for preparing recombinant dna order, this method comprise with the antigenic DNA sequence of coding rabies virus be inserted into adenovirus DNA in proper order in.
18, the method for preparing vaccine, this method comprise that the recombinant virus that will contain the recombinant dna order mixes mutually with pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB898919102A GB8919102D0 (en) | 1989-08-22 | 1989-08-22 | A recombinant adenovirus dna sequence,a recombinant adenovirus expressing the dna sequence and a rhabdovirus vaccine including the recombinant adenovirus |
| GB8919102.7 | 1989-08-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1050561A true CN1050561A (en) | 1991-04-10 |
Family
ID=10661965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN90107300.8A Pending CN1050561A (en) | 1989-08-22 | 1990-08-22 | Recombinant dna order, express the recombinant adenovirus of this DNA sequence and contain the rhabdovirus vaccine of this recombinant adenovirus |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1050561A (en) |
| GB (1) | GB8919102D0 (en) |
| WO (1) | WO1991002804A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310677C (en) * | 2004-02-05 | 2007-04-18 | 中国农业科学院兰州兽医研究所 | Method of preparing rabies live carrier vaccine using gland virus carrier expression rabies virus bigene |
| CN108025058A (en) * | 2015-06-12 | 2018-05-11 | 葛兰素史密丝克莱恩生物有限公司 | Adenovirus polynucleotides and polypeptides |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2201055T3 (en) * | 1992-08-07 | 2004-03-16 | Wyeth | RECOMBINANT ADENOVIRUS VACCINES. |
| AU7081896A (en) * | 1995-10-05 | 1997-04-28 | Microbix Biosystems Inc. | Rabies recombinant adenovirus |
| US6670188B1 (en) * | 1998-04-24 | 2003-12-30 | Crucell Holland B.V. | Packaging systems for human recombinant adenovirus to be used in gene therapy |
| CN1718242A (en) * | 2004-07-07 | 2006-01-11 | 中国人民解放军军事医学科学院军事兽医研究所 | The canine adenovirus type 2 recombiant vaccine of rabies virus sugar/structural protein such as nuclear |
-
1989
- 1989-08-22 GB GB898919102A patent/GB8919102D0/en active Pending
-
1990
- 1990-08-03 WO PCT/GB1990/001217 patent/WO1991002804A1/en unknown
- 1990-08-22 CN CN90107300.8A patent/CN1050561A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310677C (en) * | 2004-02-05 | 2007-04-18 | 中国农业科学院兰州兽医研究所 | Method of preparing rabies live carrier vaccine using gland virus carrier expression rabies virus bigene |
| CN108025058A (en) * | 2015-06-12 | 2018-05-11 | 葛兰素史密丝克莱恩生物有限公司 | Adenovirus polynucleotides and polypeptides |
| CN108025058B (en) * | 2015-06-12 | 2022-12-16 | 葛兰素史密丝克莱恩生物有限公司 | Adenovirus polynucleotides and polypeptides |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1991002804A1 (en) | 1991-03-07 |
| GB8919102D0 (en) | 1989-10-04 |
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