US 5,654,136 AGrant
Attenuated Measles Virus Vaccine, Containing Specific Nucleotide Sequence and a Method for Its Absolute Identification
Issue Date:1997-08-05
•3 Claims
•3 Drawing Sheets
Abstract
A measles vaccine virus AIK-C strain comprises genomic RNA that produces by reverse transcription cDNA having a specific nucleotide sequence indicative of the viral genomic RNA in the seed virus for measles vaccine or measles virus vaccine. The complete sequence of 15,894 nucleotides has been determined.
Metadata
Assignee
- The Kitasato Institute
Inventors
- Keiko Sasaki
- Takayuki Mori
- Satoshi Makino
Application Information
Application Number:US 3488918
Filing Date:1994-11-25
Priority Date:1991-10-14
Art Unit:185
Classifications
IPC:
C12Q 170C12N 1545
Field of Search:
4355365;6;237;320.123.72
Patent Drawings (3 sheets)
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a strain of measles virus vaccine comprising a specific nucleotide sequence, and a method for its absolute identification.
2. Description of the Prior Art
Measles virus is the causative virus of measles, and belongs to the Paramyxoviridae family of RNA virus.
The first isolation of measles virus from patients using a primary culture of human kidney cells was made by J. F. Enders et al. in 1954 (Enders, J. F. et al., Proc. Soc. Exp. Biol. Med., Vol. 86, pp. 227-286, 1954). Attenuated measles virus vaccine was developed using the isolated Edmonston strain by Enders et al. (Enders, J. F. et al., New England J. Med., Vol. 263, pp. 153-259, 1960). However, the vaccine developed by Enders et al. frequently induced adverse effects including pyrogenicity and exanthema.
Many strains of attenuated measles virus having been established by further attenuation of the Edmonston strain. Among these strains the Schwarz strain established by A. J. F. Schwarz has been commonly used for live measles vaccines.
The present inventors have isolated four strains of a cold variant derived from attenuated measles virus of the Edmonston strain, supplied by Dr. Enders (Makino, S. et al., Jap. J. Microbiol., Vol 14, pp. 501-504, 1970).
Reduction of immunogenicity, i.e. effectiveness, according to a development of attenuation of measles virus has generally been known. Among the strains of the isolated cold variant, a viral strain which grows adaptively at 33.degree. C. was found to be a further attenuated measles virus with high immunogenicity having properties different from those generally observed in the conventional measles virus (Makino, S. et al., Jap. J. Microbiol., Vol. 17, pp. 75-79, 1973).
In order to develop the seeds for live measles vaccines from the cold variant, one of the present inventors has isolated clone virus which is a strain adapted with chick embryo cells obtained from specific pathogen-free eggs, having the same temperature marker, and designated as the AIK-C strain (Sasaki K., Kitasato Arch. Exp. Med., Vol. 47, pp. 1-12, 1974).
The pyrogenicity ratio (.ltoreq.37.5.degree. C.) of an AIK-C strain live vaccine produced from the seeds of AIK-C strain in measles-sensitive infants approximately 1/3-1/4 as compared with that of Schwarz strain vaccine. The AIK-C strain has been found to be a further attenuated measles virus than the Schwarz strain, with the unique characteristic of having a high immunogenicity response without lowering immunogenicity (Makino, S. et al., Kitasato Arch. Exp. Med., Vol. 47, pp. 13-21, 1974).
Encephalitis that seems to be caused by administered live measles vaccine has been observed in 1-3 persons per million treated infants. However, this neurological complication has never been reported in the case of the AIK-C strain in spite of the administration of AIK-C strain live measles vaccine to ten million people in Japan (Hirayama, M. et al., Inf. Dis., Vol. 5, pp. 495-503, 1983; Makino, S., Vol. 5, pp. 504-505, 1983).
SUMMARY AND OBJECTS OF THE INVENTION
The biological properties of the AIK-C strain are unique, and differ markedly from those of the other strains of measles virus. However, the virus is easily mutated and even in an attenuated measles virus strain such as the AIK-C strain, a small degree of formation of variant strains can be observed during the growth phase. Accordingly, during the production of live measles vaccine, the quality control for comparing the respective identities of the seed virus and the vaccine virus produced therefrom is the most important procedure.
As noted above, a temperature marker test for the AIK-C strain has been applied as a quality control test (Sasaki, K., Kitasato Arch. Exp. Med., Vol. 47, pp. 1-12, 1974). However that biological assay method does not always provide absolute identification of the AIK-C strain.
The fundamental biological properties of the virus depend on its genome, which consist of nucleic acid of viral particles. The nucleic acid of measles virus consists of single strain (-)RNA, and each viral strain has its own nucleic acid made up of a specific nucleotide sequence.
Complete differential identification between viral trains is, therefore, necessary to determine the nucleotide sequence of viral nucleic acid in the strain. The present inventors have focused on this point, and have analyzed the nucleotide sequence of nucleic acid relating to measles virus infection. In the course of that analysis, the inventors have provided an administrative control method for producing a stable AIK-C strain of measles virus without variants or mutation.
The known identification methods for various measles strain apply a specific biological response test for the virus strain in question; however, these are not methods capable of absolute identification. For quality control during production of the AIK-C strain vaccine, the aforementioned temperature marker test has been applied. The present inventors, on the other hand, have determined the entire nucleotide sequence of the AIK-C strain virus genome, thereby to establish an absolute identification method for the said identification test. Since the entire specific nucleotide sequence, consisting of 15,894 bases in AIK-C strain, has been clearly determined, the virus can be identified at the genetic level, and the identification technique can therefore provide an absolute determination, so that quality control on a stable AIK-C strain vaccine can easily and exactly be performed.
Therefore an object of the present invention is to provide an attenuated live measles vaccine comprising a specific nucleotide sequence.
Another object of the present invention is to provide an absolute identification method for a measles virus strain.
A further object according to the present invention is to provide a measles vaccine virus genomic DNA consisting of the nucleotide sequence described below, with partial insertion mutational sequence or defective sequence.
A still further object of the invention is to provide an absolute identification method for a measles vaccine virus strain, which comprises detecting a specific nucleotide sequence consisting of the 15,894 nucleotides listed below coded by the genomic RNA of measles vaccine virus strain.
A yet still further object of the invention is to provide an absolute identification method comprising detecting a part of the nucleotide sequence of viral genomic DNA by the Northern blot technique and polymerase chain reaction method.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects and advantages according to the invention will be more readily apparent from a reading of the following detailed discussion taken with reference to the accompanying drawings, in which:
FIG. 1 is an outline of cDNA construction.
FIG. 2 is a mapping of cDNA clones of the AIK-C.
FIG. 3 is a genomic DNA sequence of the synthetic oligonucleotides (SEQ ID NOS:8-14) as complementary genome DNA.
DETAILED DISCUSSION
The inventors have determined that the complete cDNA sequence coded by the RNA genome of the AIK-C strain is as follows as shown in SEQ ID NO:1. The coding regions of SEQ ID NO:1 encode the amino acid sequences of SEQ ID NOS:2-7
The physical characteristics of the sequence are:
Sequence type: nucleic acid
Strandedness: single
Topology: linear
Molecule type: antigenomic DNA
Original source
Organism: attenuated measles vaccine
Strain: AIK-C
According to the present invention, a seed virus of the measles virus AIK-C strain is first used. This vaccine virus is propagated in chick embryo cells derived from specific pathogen-free hen's eggs to prepare so-called bulk vaccine, from which a virus suspension can be prepared by conventional purification techniques. The bulk vaccine can be a part of the virus pool prepared for commercial measles AIK-C strain vaccine.
The clarified virus suspension is purified by ultra-centrifugation through continuous sucrose gradients. Virus RNA is extracted by the SDS-phenol method. Synthetic primer can be synthesized with approx. 25 mer synthetic primer by the amidide method.
The deoxyoligonucleotide, a synthetic primer, can be synthesized, for example, on a CYCLONE DNA SYNTHESIZER (Biosearch, Inc.)
The AIK-C virus genome RNA is used as a template for the synthesis of cDNA using the above synthetic oligonucleotide primer. The AIK-C virus genome RNA is reversely transcribed by reverse transcriptase to prepare single-strand cDNA, which is thereafter treated by the RNase H method to prepare double-strand cDNA. The double-stranded cDNAs are treated with appropriate restriction enzymes and are inserted into pUC plasmids (pUC 18 or pUC 19).
The above process is illustrated schematically in FIG. 1.
E. coli K 12 strain HB101 is transformed with the resulting recombinant plasmid. The transformants are selected according to their ampicillin resistance. Colonies containing recombinant plasmids are screened by measuring the size of plasmic DNA by agarose gel electrophoresis. To determine the AIK-C genome sequence, the cDNA are subcloned into the bacteriophage M 13 series, and the single-strand M 13 phage DNAs are isolated from candidate subclones.
Determination of the obtained cDNA clone can be made using several restriction enzyme cleavage types. In the cDNA obtained using a synthetic primer such as MP-1, the restriction enzyme sites of BamHI, E coRV and Xbal are located. In the plasmid extracted from an ampicillin resistant strain, in case this type of cDNA is observed, approximately 1504 base fragments are identified by splitting with BamHI and Xbal in an agarose gel electrophoresis. Further treatment of these fragments with EcoRV provides two fragments of 650 and 854 base fragments.
Accordingly, an identification of the obtained cDNA clone is made by cleaving the inserted fragment in the plasmid and determining its size, or by determining the specific restriction site of measles DNA in the fragments.
To determine the base sequence, the cDNA, which are split by restriction enzymes to prepare cohesive ends or blunt ends, is inserted into M 13 phage vectors whose cloning parts are cleaved by the appropriate restriction enzymes. The phage containing the thus-prepared recombinant DNA is infected into E. coli. Single-strand recombinant DNA is extracted from the infected phage plaques, and the nucleotide sequence thereof is determined by the dideoxy chain termination method (dideoxy sequence method).
A primer such as pMN 2, the cDNA obtained from MP-1, is split by BamHI and Xbal, and is cloned with pUC plasmid. After determining the cDNA, the DNA fragment originated from AIK-C strain by splitting with the same restriction enzyme, further the fragment is split using EcoRV to prepare two fragments consisting of 650 bp and 854 bp.
These fragments are ligated into the M 13 replicative form double-strand DNA, which was previously split by BamHI and Xbal transfected into E. coli. After the recombinant phage is selected, the phage is infected in E. coli and allowed to multiply. Single-strand recombinant DNA is extracted from the supernatant phase of the phage solution and the nucleotide sequence thereof is determined by the dideoxy sequence method.
The viral genome of the AIK-C strain has been found to be constructed of 15,894 nucleotides and its entire sequence is shown in the sequence given above. A measles virus containing this RNA genome has the superior properties of AIK-C virus strain. The specific nucleotide sequence of AIK-C virus strain genome is determined by the PCR method and can be compared with the virus strain whether the strain is to be used for AIK-C strain virus vaccine or whether the prepared AIK-C strain virus vaccine is simply the maintaining properties of AIK-C strain. Furthermore, if an infant to whom the measles vaccine has been administered exhibits exanthema and antibodies against measles virus are observed in the lymphocyte of the infant, the causes thereof can be determined by comparing with the nucleotide sequence, as to whether the exanthema is caused by the administered vaccine or not.
The following examples are provided to illustrate the present invention but are not to be construed as limiting.
EXAMPLE 1
Determination of the Nucleotide Sequence of a Virus Obtained from AIK-C Virus Strain Bulk Vaccine
Step a:
A seed virus of the AIK-C measles strain (Seed Lot 0-2) was inoculated up to an infectivity titer of 0.05 in chick embryo cells derived from specific pathogen-free hen's eggs cultured in a surface area cell culture (230 cm.sup.2) in a large Roux bottle (approx. 1 liter volume). A culture medium (150 ml) of Eagle's minimum essential medium (MEM) containing 1% calf serum was added thereto and the virus was cultured at 33.degree. C.
Step b:
On the third day after inoculation, the cultured medium was removed, and then 150 ml of new medium was added and the cultivation was continued.
Step c:
On the sixth day after inoculation of virus, the culture was removed. The layer of infected chick embryo cells was washed completely three times with 100 ml aliquots of Hank's solution. Cultivation was then continued at 33.degree. C. after adding 200 ml of Eagle's MEM for cell culture, containing 0.1% sodium glutamate.
Step d:
On the tenth day after virus seed inoculation, the specific cytopathogenic effect for measles virus was observed over the entire infected cell layer, and the culture medium was collected to produce the bulk vaccine solution (volume: approx. 200 ml, infectivity titer: 10.sup.7.2 TCID.sub.50 */ml; *medium tissue culture infective dose).
Step e:
The bulk vaccine solution was centrifuged at 3,000 rpm for 30 mins. and the supernatant solution removed therefrom was further centrifuged at 25,000 rpm for 90 mins. (Beckman. L 8-55 M, rotor: SW 28)
Step f:
After the further supernatant solution was removed, 1 ml of TEN buffer solution (10 mM-Tris HCl, 1 mM-EDTA, 100 mM-NaCl, pH 7.4) was added to the precipitate in each centrifuge tube, and the precipitate was well suspended therein to prepare concentrated virus. Each of the thus-obtained concentrated virus suspensions was adjusted to a final volume of 10 ml by adding TEN buffer solution, to prepare clarified virus suspension.
Step g:
The clarified virus suspension (5 ml) was layered on 30-60% continuous sucrose gradients in two centrifuge tubes (Beckman centrifuge SW 41 rotor), each having 60% (w/v) sucrose solution in TEN buffer solution (6.8 ml) and 30% (w/v) sucrose solution in TEN buffer solution (6.8 ml) per tube, and centrifuged for 90 mins. at 207,000 g and 4.degree. C. The suspension was fractionated and the fractions showing the highest infectivity titer (10.sup.8.3 TCID.sub.50 /ml) were collected and again centrifuged by the same technique to prepare purified virus particles.
Step h:
The purified virus particles were diluted five-fold with TEN buffer solution, and 200 .mu.l of each was added into a respective 1.5 ml microtesttube, along with 5 .mu.l of 20% SDS, 100 .mu.l phenol and 100 .mu.l chloroform, followed by complete stirring using a vortex mixer. The mixture was further centrifuged at 12,000 rpm for 5 mins. to separate the organic layer and the aqueous layer. The aqueous layer was collected into another 1.5 ml microtesttube and treated twice by phenol extraction (SDS-phenol extraction). The recovered aqueous layer was mixed with 5M-NaCl (1/25 volume), and ethanol (2.5 volume), allowed to stand at -20.degree. C. for 2 hours, then centrifuged at 12,000 rpm for 10 mins. to collect precipitated RNA which was washed with 70% ethanol and dried. The dried material was dissolved in sterilized redistilled water (50 .mu.l ) and autoclaved to prepare an RNA suspension.
Step i:
Cyclone DNA synthesizer
Synthetic primer deoxyoligonucleotide, 12 primers comprising approx. 25 mer of MP-1-MP-11 and BEP (dT).sub.7 as shown in FIG. 3 were synthesized using a Cyclone DNA Synthesizer (Biosearch Inc., U.S.A.)
Step j:
10 .mu.l of the RNA suspension obtained in the above Step h (AIK-C virus genome RNA) was used as a template for the synthesis of DNA using 2 .mu.l of the above synthetic oligonucleotide primers, MP-1 or MP-11, and cDNA was prepared by reverse transcriptase treatment. The cDNA was then transferred to double-strand cDNA by using RNase H-DNA polymerase I according to the technique described by Gubler and Hoffman in GENE, Vol. 25, pp. 263-269, (1983).
Step k:
The obtained cDNA was cleaved at each of its restriction enzyme cleavage sites by BamHI-Xbal, Xbal-BamHI, BamHI-EcoRI, EcoRI-BamHI, BamHI-EcoRI, EcoRI, BgIII, BglII-SacI and SacI-NcoI-Xbal. Each fragment was inserted into a corresponding cloning site of pUC plasmid (pUC 18 and pUC 19).
Step l:
E. coli HB101 was transformed with the above recombinant plasmid to obtain ampicillin-resistant colonies. A plasmid DNA was extracted from the thus-obtained colonies, and the colonies containing recombinant plasmids were screened by measuring the size length of the plasmid DNA fragments by 0.8% agarose gel electrophoresis.
Step m:
To obtain the 3' terminal clone of the AIK-C genome, poly(A) was tailed at the 3' end of the genomic RNA, an RNA suspension obtained in the above Step h, with poly(A) polymerase and adenosine triphosphate (ATP). The thus-obtained 3'A tailed RNA suspension, i.e. the polyadenylated RNA, was reversely transcribed using BEP(dT).sub.7 primer to prepare cDNA according to the above Step j. The BEP(dT).sub.7 primer has the sequence 5'-CTGTGAATTCTGCAGGATCCTTTTTTT-3' (SEQ/ID NO:19).
Step n:
The 5' terminal clone was synthesized with the primer located close to the 5' terminus. That is, the primer contained complementary DNA in a domain of 15,592-15,615 in the measles virus genome. The synthetic primer has the sequence 5'-TGGAAGCTTATCCAGAATCTCAAGTCCGGCT-3' (SEQ/ID NO:18)
A DNA-RNA hybrid was prepared by using the said synthetic DNA as a primer with reverse transcriptase. After alkaline treatment of the hybrid, poly(dA), i.e. dATP, was tailed to the 3' end of the resulting cDNA with terminal deoxynucleotidyl transferase. It was subsequently converted to the double-stranded cDNA using the BEP(dT).sub.7 primer in the above Step m and the Klenow fragment.
Step o:
The thus-obtained cDNAs were subcloned into the bacteriophage M 13 series vector (mp 18 and mp 19), and the single-strand M 13 phage DNAs were isolated. The nucleotide sequence of those cDNAs was determined with the said single-stranded DNA by means of the dideoxy chain termination method using a 7-DEAZA-dGTP sequencing kit (Takara Shuzo).
Step p:
Computer analysis of the nucleotide and peptide sequence was performed using GENETYX software.
EXAMPLE 2
Determination of the Viral Nucleotide of AIK-C Strain Seed Virus Grown in Vero Cells (African Green Monkey Live Cells
Step a:
AIK-C strain seed virus (Seed Lot No. 0-2) was inoculated in Vero cells previously cultured in a large size Roux bottle according to the method described in Example 1, and incubated at 33.degree. C.
Step b:
On the fifth day of incubation, the infected cell layers were washed with Hank's solution, then Eagle's MEM (200 ml) without calf serum was added thereto and the culture was further incubated for two days. The incubated viral culture was collected to obtain a bulk virus suspension (approx. 200 ml, infective titer: 10.sub.6.5 TCID.sub.50 /ml).
Step c:
The bulk vaccine was centrifuged at 3,000 rpm for 30 mins., whereafter the supernatant suspension removed therefrom was centrifuged at 25,000 rpm for 90 mins. (Centrifuge: Beckman L8-55M , rotor: SW 28)
Step d:
After the further supernatant solution was removed, 1 ml of TEN buffer solution (10 mMTris-HCl 1 mM EDTA, 100 mM NaCl, pH 7.4) was added to the precipitate in each centrifuge tube, and the precipitate was suspended completely to prepare concentrated virus material, which was adjusted to a volume of 10 ml by adding TEN buffer solution to prepare the starting material virus suspension.
Step e:
5 ml of the virus suspension starting material was layered on a 30-60% continuous sucrose gradient in each of two tubes consisting of 60% (w/v) sucrose solution (TEN buffer solution) (6.8 ml) and 30% (w/v) sucrose solution (TEN buffer solution) (6.8 ml), then centrifuged at 207,000 g for 90 mins. at 4.degree. C.
The centrifuged suspension in each tube was fractionated by means of a fraction collector and fractions showing high infectivity (10.sup.8.3 TCID.sub.50 /ml) were collected. The collected fractions were again centrifuged in the same manner to recover purified virus.
Step f:
200 .mu.l of the purified virus, diluted five-fold with TEN buffer solution, was added to each of a series of 1.5 ml microtesttubes. To each tube was then added 5 .mu.l of 20% SDS, 100 .mu.l of phenol and 100 .mu.l chloroform, and the contents of the tubes were completely stirred using a vortex mixer. The organic layer and aqueous layer were separated by centrifuging at 12,000 rpm for 5 mins. The aqueous layer was collected in another 1.5 ml microtesttube, and extracted twice with the same SDS-phenol extractant as above.
The recovered aqueous layer was mixed with 5M NaCl (1/25 volume) and ethanol (2.5 volume), allowed to stand at -20.degree. C. for 2 hours, and centrifuged at 12,000 rpm for 10 mins. to collect precipitated RNA. The precipitated RNA was then washed with 70% ethanol, and dried. An RNA suspension was prepared with sterilization of the dried material and dissolved in redistilled water (50 .mu.l).
Step g:
Cyclone DNA Synthesizer
Synthetic primer deoxyoligonucleotide, 12 primers, comprising approx. 25 mer of MP-1-MP-11 and BEP(dT).sub.7 as shown in FIG. 3 were synthesized using a cyclone DNA synthesizer (Biosearch Inc., U.S.A.)
Step h:
The RNA suspension (10 .mu.l ) obtained in the above Example 1 (AIK-C virus genome RNA) was used as a template for the synthesis of cDNA using synthetic oligonucleotide primers (the above synthetic DNA, 2 .mu.l of synthetic primer, MP-1 or MP-11) and cDNA was prepared by reverse transcriptase treatment. The cDNA was then transferred to double-strand cDNA using RNaseH-DNA polymerase I according to the method of Gubler and Hoffman cited above.
Step i:
The obtained cDNA was cleaved at each of its restriction enzyme cleavage sites by BamHI-Xbal, Xbal-BamHI, BamHI-EcoRI, EcoRI-BamHI, BamHI-EcoRI, EcoRI-BglII, BglII-SacI and SacI-NcoI-Xbal. Each fragment was inserted into a corresponding cloning site of pUC plasmid (pUC 18 and pUC 19).
Step j:
E. coli HB101 was transformed with the above recombinant plasmid to obtain ampicillin-resistant colonies. A plasmid DNA was extracted from the thus-obtained colonies, and the colonies containing recombinant plasmids were screened by measuring the size length of plasmid DNA fragments by 0.8% agarose gel electrophoresis.
Step k:
To obtain the 340 terminal clone of the AIK-C genome, poly(A) was tailed at the 3' end of the genomic RNA, an RNA suspension obtained in the above Step f, with poly(A) polymerase and adenosine triphosphate (ATP). The thus-obtained 3' tailed RNA suspension, i.e. the polyadenylated RNA, was reversely transcribed using BEP(dT).sub.7 primer to prepare cDNA according to the above Step h. The BEP(dT).sub.7 primer has the sequence 5'-CTGTGAATTCTGCAGGATCCTTTTTTT-3' (SEQ/ID NO:19).
Step l:
The 5' terminal clone was synthesized with the primer located close to the 5' terminals. That is, the primer contained complementary DNA in a domain of 15,592-15,615 in the measles virus genome. The synthetic primer has the sequence 5'-TGGAAGCTTATCCAGAATCTCAAGTCCGGCT-3' (SEQ/ID NO:18).
A DNA-RNA hybrid was prepared by using the said synthetic DNA as a primer with reverse transcriptase. After alkaline treatment of the hybrid, poly(Da), i.e. dATP, was tailed to the 3' end of the resulting cDNA with terminal deoxynucleotidyl transferase. It was subsequently converted to the double-stranded cDNA using the BEP(dT).sub.7 primer in the above Step k and the Klenow fragment.
Step m:
The thus-obtained cDNA were subcloned into the bacteriophage M 13 series vector (mp 18 and mp 19), and the single-stranded M 13 phage DNAs were isolated. The nucleotide sequence of those cDNAs was determined with the said single-stranded DNA by means of the dideoxy chain termination method using a 7-DEAZA-dGTP sequencing kit (Takara Shuzo).
Step n:
Computer analysis of the nucleotide and peptide sequences was performed using GENETYX software.
EXAMPLE 3
Identification by the PCR Method of Measles Virus Obtained from a Patient on the First Day When Measles Symptoms Appeared
Step a:
A blood specimen (approx. 5 ml) was collected from a patient on his first day displaying measles exanthema. Lymphocytes were separated using Ficoll (trade name).
Step b:
The lymphocytes were washed twice with PBS. 200 .mu.l of a denaturation solution D (4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.5; 0.5% sarcosine, 0.1M 2-mercapto ethanol) was added thereto. After gentle stirring, 2M sodium acetate (20 .mu.l, pH 4), phenol (200 .mu.l) and chloroform (100 .mu.l), were added in that order.
The mixture was treated with a vortex mixer for 10 sec. and allowed to stand in ice water for 15 mins. Then the mixture was centrifuged at 10,000 g for 20 mins. to recover an aqueous layer. An equal volume of isopropanol was added thereto, and the mixture was allowed to stand at -20.degree. C. for one hour, then centrifuged at 10,000 g for 20 mins. to precipitate RNA. This procedure was performed according to the technique described by Chomoczynski and Sacchi in Anal. Biochem., Vol. 162, pp. 156-159 (1987).
Step c:
The thus-obtained RNA was subjected to a reverse transcriptase reaction and the PCR method.
Step d:
Nucleotide sequencing and identification of the measles virus AIK-C vaccine strain and the naturally-occurring strain performed.
Although the present invention has been described in connection with various preferred embodiments thereof, it will be appreciated that these embodiments are provided solely for purposes of illustration, and should not be construed as limiting the scope of the invention. Other embodiments and applications of the invention will be readily apparent to those skilled in the art from reading the present specification and practicing the techniques described herein, without departing whatsoever from the scope and spirit of the appended claims.
Claims
What is claimed is:
1. A purified and isolated genomic RNA sequence which is the full length complement of the sequence set forth as SEQ ID NO:1.
2. An isolated and purified complementary DNA consisting of the sequence set forth as SEQ ID NO:1.
3. An absolute identification method for a measles vaccine virus strain which comprises isolating the genomic RNA of said measles vaccine virus strain, and comparing the nucleotide sequence of said genomic RNA to the sequence set forth as SEQ ID NO:1 to assess identity therewith.
Patent Citations (3)
Non-Patent Literature (14)
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- "Studies on an Attenuated Measles-Virus Vaccine", New England Journal of Medicine, vol. 263, 1960, By J. Enders et al., pp. 153-184.
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