CN1570115A - Optimized SARS coronavirus spike protein gene - Google Patents
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Abstract
The optimized SARS coronavirus spike protein gene encodes SARS coronavirus S protein or immunogenic fragment, and its identity to natural SARS coronavirus S protein coding sequence is 77 +/-3% (as SEQ ID NO: 1). Vectors, host cells, and vaccine compositions containing the optimized S protein coding sequences are also provided. The optimized S protein coding sequence has higher expression efficiency in mammalian cells, so that the S protein is more efficiently produced, and the mammals can be more effectively stimulated to generate immune response against SARS.
Description
Technical field
The present invention relates to virusology, genetic engineering and vaccinology, the sars cov spike protein gene that relates more specifically to optimize.This gene can apply to efficiently express the generation spike protein in mammal especially human cell's environment.The invention still further relates to the carrier and the vaccine that contain this gene.
Background technology
Atypical pneumonia, be called for short SARS, claim SARS (Severe Acute Respiratory Syndrome) (SARS) to spread to more than 30 countries and regions, the whole world again, particularly serious China, Hong-Kong, Singapore, Canada, Vietnam, TaiWan, China and the U.S. of comprising has caused serious life and property loss in a short time.Nearest epidemiological study estimates that mortality ratio can be up to nearly half patient more than 60 years old.
It is the pathogenic inducement of SARS that a kind of new sars coronavirus (SARS Coronavirus) has been confirmed to be, and this viral genome is checked order and announces.Sars coronavirus is ssRNA (+) virus, duplicates the intermediate without DNA, uses standard cipher.Under Electronic Speculum, virion is irregular shape, and diameter is about 60-220nm, and there is quincuncial peplomer on its surface, shape such as imperial crown.The particle center is indefinite form under the negative staining Electronic Speculum, nucleocapsid is rarefaction.Two kinds of glycoprotein are arranged: S albumen and M albumen on the coating.S albumen is spike glycoprotein, is main antigen, with receptors bind, makes cytogamy.M albumen is transmembrane protein, participates in coating and forms.In addition, N albumen also has immunogenicity.
The development vaccine is the effective means of prophylaxis of viral infections, and is proved and successfully applies to multiple fatal virus infection, as smallpox, poliomyelitis, measles, parotitis, hepatitis A, hepatitis B.In order to control and prevent SARS effectively, people are developing various SARS vaccines, wherein mainly contain five types: inactivated vaccine, attenuated live vaccine, recombinant subunit vaccine, dna vaccination and attenuated virus carrier bacterin.
Yet still inreal up to now effectively SARS vaccine comes out.Therefore, this area presses for the effective SARS vaccine of exploitation, especially can introduce or produce the vaccine of immunogenicity SARS polypeptide in host in a large number.
Summary of the invention
Purpose of the present invention just provides and a kind ofly can produce the sars cov spike protein gene of the optimization of immunogenicity SARS polypeptide in a large number in host, contains the carrier and the vaccine of this gene.
In a first aspect of the present invention, a kind of isolated nucleic acid molecule is provided, S albumen or the immunogenic fragments of its encoding SARS coronavirus, and the homogeny of the S albumen coded sequence of itself and natural sars coronavirus is 77 ± 3%.
In another preference, the sars coronavirus S albumen shown in the described nucleic acid molecule encoding SEQ ID NO:2.
In another preference, described nucleic acid molecule contains the nucleotide sequence shown in the SEQ ID NO:1.
In another preference, described nucleic acid molecule is selected from down group:
(a) nucleotide sequence shown in the SEQ ID NO:1;
(b) with SEQ ID NO:1 shown in the nucleotide sequence homogeny greater than 95%, and the coding same acid sequence nucleotide sequence.
In another preference, compare with the S albumen coded sequence of natural SARS virus, in mammalian cell, the proteic expression amount of the S of described nucleic acid molecule encoding improves at least 100%.
In a second aspect of the present invention, a kind of carrier is provided, it contains said nucleic acid molecule of the present invention.
In another preference, described carrier is plasmid or virus vector, and more preferably described virus vector is an adenovirus.
In a third aspect of the present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention, is perhaps transformed by said nucleic acid molecule of the present invention.
In a fourth aspect of the present invention, a kind of composition of knowing clearly is provided, it contains the above-mentioned carrier of the present invention or nucleic acid molecule and pharmaceutically acceptable excipient or thinner.
Embodiment
The inventor is through deeply and extensive studies, and the encoding sequence of the spike protein of sars coronavirus has been carried out series of optimum.Encoding sequence after the optimization is more suitable for expressing in the cell or tissue environment of and other mammals for example human Mammals, thereby not only be more suitable for efficiently expressing the production spike protein on a large scale, and be more suitable for as dna vaccination at the mammal cell line system.
The nucleotide sequence of a kind of optimization of the present invention contains 3768bp shown in SEQ ID NO:1, the aminoacid sequence identical with original coronavirus spike protein of encoding is to keep its immunizing antigen character.Yet this optimizes synthetic gene only has 77.6% homogeny (or 22.4% different) with original coronavirus spike protein gene.795 codons (or 63% codon) in its 1256 codons are changed, but have kept the aminoacid sequence mutually identical with former spike protein with the expression of optimization in cells of mamma animals, and the purposes that is used for vaccine.
Nucleotide sequence shown in the SEQ ID NO:1 is called as " COSO ", mean optimization coronavirus spike protein gene (
CoRonavirus
SpIke protein
OpTimized gene).
Should understand, because SARS virus also can morph as other virus, therefore, the S albumen that is different from the variation of SEQ ID NO:2 for aminoacid sequence, can be on the basis of SEQ ID NO:1, replace the codon of corresponding SARS virus in the corresponding position with the preferred codon of corresponding mammalian cell, thus to optimize, the proteic nucleotide sequence of encoding mutant S.
As used herein, term " the S albumen coded sequence of optimization " is meant the nucleotide sequence shown in the SEQ ID NO:1, and with SEQ ID NO:1 homogeny greater than 95%, preferably greater than 96%, more preferably greater than 98% nucleotide sequence.Compare with the S pyrenoids nucleotide sequence of former SARS virus, in mammalian cell (as Chinese hamster ovary celI etc.), the expression amount of the S albumen coded sequence of optimization improves at least 100%, more preferably improves at least 200% or higher.
In addition, the present invention also comprises its fragment except the S albumen coded sequence of the optimization that comprises total length, for example based on the nucleotide sequence of sequence shown in the SEQ ID NO:1 and coding immunogenicity S protein fragments.
The S albumen coded sequence that the present invention optimizes, can import vertebra class animal by the DNA recombinant technology, especially in the cell or tissue environment of for example human and other mammals of Mammals, thereby express spike protein, immunogenic fragments or its derived products efficiently.
The present invention also provides the plasmid or the virus vector of the S albumen coded sequence that contains all or part of optimization, thereby and because of being changed over to by described plasmid or virus vector is transfected into the mammalian host cell that contains the preferred S albumen coded sequence of the present invention.
A kind of preferred virus vector is an adenovirus carrier, for example has been used at present the replication defective adenoviral carrier of clinical vaccine test, comprises E1 zone absence type, E3 zone or disappearance or 5 types or 2 types or other hypotype adenovirus that keep.In some cases, adenovirus carrier also can comprise the E1 zone, but is lacked in other zones.Other adenovirus carriers can comprise the carrier of multizone disappearance, for example assistant's dependent form or full absence type adenovirus carrier.
In E1 zone or E3 zone or other appropriate area of adenoviral gene group, can insert the S protein expression box of a SARS virus.This expression cassette from 5 ' → 3 ' contain successively: 1) express promotor (promoter); 2) the coronavirus S albumen coded sequence (comprising its varient or derived fragment) of the optimization that links to each other with described promotor operability; 3) the polyadenylation signal sequence that links to each other with the encoding sequence of this optimization.
Except adenovirus carrier, other other virus vector are including, but not limited to adeno-associated virus (AAV) (AAV, adem-assocated virus), poxvirus (vaccinia virus) etc.
Another kind of preferred carrier is a plasmid vector.The same S protein expression box that inserts SARS virus in this plasmid vector.
By cultivating the mammalian cell of the S albumen coded sequence that imports the present invention's optimization, can produce S albumen on a large scale expeditiously, obtain the S albumen of purifying then with conventional separation and purification process.The S albumen of purifying can be used for producing antibody, SARS diagnostic kit and other purposes.
Vaccine composition
The present invention also provides a kind of vaccine composition, and it comprises the plasmid or the virus vector of the S albumen coded sequence that carries all or part of optimization, and pharmaceutically acceptable carrier.When vaccine composition of the present invention is applied to Mammals (as the people); these plasmids or virus vector can efficiently express spike protein, immunogenic fragments or its derived products in vivo, thereby induce body generation humoral immunization and cellular immunization to prevent and treat the infection of sars coronavirus.
Medicinal compositions can be prepared into various formulations, as granula, tablet, capsule, suspension, spraying, suppository, transdermal drug (as paster etc.), ointment, lotion etc.
In addition, the preparation of vaccine composition of the present invention also can contain other composition, comprises as adjuvant, stablizer, pH regulator agent, sanitas etc.These compositions are that the vaccine those skilled in the art are known.
Route of administration and dosage
When as vaccine, available known method is applied to individuality with recombinant viral vector of the present invention.Usually adopt route of administration identical and/or simulation pathogenic infection path to use these vaccines with conventional vaccine.The routine of administration and pharmaceutically acceptable approach comprise: in the nose, interior, the intravenously of interior, subcutaneous, the intracutaneous of intramuscular, tracheae, lung, intranasal, oral administration or other administered parenterally approach.If desired can the combination medicine-feeding approach, or regulate by antigen peptide or disease situation.
Vaccine composition can single dose or multiple doses give, and can comprise and give booster dose to cause and/or to keep immunizing power.
Should give vaccine of the present invention with " immune significant quantity ", promptly the amount of recombinant viral vector is enough to cause immunne response in selected administration path, can impel the protection host to resist SARS virus effectively and infect or the SARS symptom.
The amount of selected virus vector in each vaccine dose part is not have the amount of significant side effects by causing protective immune response and decide.Usually, behind host cells infected, the vaccine of each agent part is enough to produce about 1-1000 μ g, preferably is 1-200 μ g, more preferably 10-100 μ g protein.With virus vector nucleic acid is the vaccine effective dose of basic calculation, generally includes about 1ug-100mg nucleic acid.In addition, the general range of the effective dose of vector-viral vaccine is about 10
2-10
7, 10
3-10
6Or 10
4-10
5Plaque forming unit (PFU).For the non-Causative virus vaccine carrier that with the adenovirus carrier is example, introduce 10 usually
6-10
12Individual adenovirus particles/everyone, preferably 10
7-10
11Individual adenovirus particles/everyone.The available research on standard method that comprises the antigen titration degree in the object of observation and other reaction is determined the optimum amount of concrete vaccine.Can determine whether to need to strengthen dosage by the immunity level that the monitoring vaccine provides.After having assessed the antigen titration degree in the serum, may need to select for use enhancing dosage immunization.Use adjuvant and/or immunostimulant and can improve immunne response SARS virus.
By introducing 10
6To 10
12The non-Causative virus vaccine carrier adenovirus particles of gene copy with in the body that carries these polynucleotide and enter for example human with Mammals especially and other mammals of vertebra class animal to induce the immune response of SARS coronary virus resistant.
Major advantage of the present invention is:
(1) expression efficiency of S albumen coded sequence in Mammals of You Huaing is higher, therefore produces the proteic efficient height of S.
Therefore (2) expression efficiency of S albumen coded sequence in Mammals of You Huaing is higher, can produce more immunogenicity S albumen in the mammalian body of inoculation, thus the immunne response of excitating organism effectively.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually people such as condition such as Sambrook routinely, molecular cloning: the condition described in the laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment 1
Synthesizing of the S albumen coded sequence of optimizing
In the present embodiment, according to the proteic aminoacid sequence SEQ of the sars coronavirus S ID NO:2 that announces, and mammiferous preferred codon, and encoding sequence is optimized according to following principle:
(a) between different plant species codon selected preference for use;
(b) difference selected for use of codon is relevant with translation speed;
(c) context of codon (context) and a certain specific cryptosystem can have a strong impact on translation speed in the existence of a certain specific position;
Especially some codon that influences translation speed is especially replaced, designed sequence shown in the SEQ ID NO:1.
Use dna synthesizer (Mclab company) then,, promptly obtain oligonucleotide fragment earlier by ordinary method, then with each fragment annealing, and the PCR extension, thereby the synthetic dna sequence dna that obtains SEQ ID NO:1.
Optimization synthetic gene shown in the SEQ ID NO:1 only has 77.6% homogeny (or 22.4% different) with original coronavirus spike protein gene.795 codons (or 63% codon) in its 1256 codons are changed, but the aminoacid sequence mutually identical with former spike protein of encoding is to optimize the expression in cells of mamma animals.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉old, insult
Old, gram is diligent
＜120〉the sars cov spike protein gene of You Huaing
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<220>
<22?1>misc_feature
<222>(1)..(3768)
＜223〉the sars coronavirus S albumen coded sequence of You Huaing
<400>1
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acctttgatg?atgtgcaggc?ccccaactac?acccagcaca?cctcctccat?gaggggcgtc 120
tactaccctg?atgagatctt?caggtctgac?accctgtacc?tgacccagga?cctgttcctg 180
ccattctact?ccaatgtgac?aggcttccac?accatcaacc?acacctttgg?caaccctgtg 240
atcccattca?aggatggcat?ctactttgct?gccacagaga?agtccaatgt?ggtgaggggc 300
tgggtctttg?gctccaccat?gaacaacaag?tcccagtctg?tgatcatcat?caacaactcc 360
accaatgtgg?tgatcagggc?ctgcaacttt?gagctgtgtg?acaacccatt?ctttgctgtc 420
tccaagccca?tgggcaccca?gacccacacc?atgatctttg?acaatgcctt?caactgcacc 480
tttgagtaca?tctctgatgc?cttctccctg?gatgtctctg?agaagtctgg?caacttcaag 540
catctgaggg?agtttgtctt?caagaacaag?gatggcttcc?tgtatgtcta?caagggctac 600
cagcccattg?atgtggtgag?ggacctgcca?tctggcttca?acaccctgaa?gcccatcttc 660
aagctgcccc?tgggcatcaa?catcaccaac?ttcagggcca?tcctgacagc?cttctcccct 720
gcccaggaca?tctggggcac?ctctgctgct?gcctactttg?tgggctacct?gaagcccacc 780
accttcatgc?tgaagtatga?tgagaatggc?accatcacag?atgctgtgga?ctgctcccag 840
aaccccctgg?ctgagctgaa?gtgctctgtg?aagtcctttg?agattgacaa?gggcatctac 900
cagacctcca?acttcagggt?ggtgccctct?ggcgatgtgg?tgaggttccc?caacatcacc 960
aacctgtgcc?catttggcga?ggtcttcaat?gccaccaagt?tcccatctgt?ctatgcctgg 1020
gagaggaaga?agatctccaa?ctgtgtggct?gactactctg?tgctgtacaa?ctccaccttc 1080
ttctccacct?tcaagtgcta?tggcgtctct?gccaccaagc?tgaatgacct?gtgcttctcc 1140
aatgtctatg?ctgactcctt?tgtggtgaag?ggcgatgatg?tgaggcagat?tgcccctggc 1200
cagacaggcg?tgattgctga?ctacaactac?aagctgcctg?atgacttcat?gggctgtgtg 1260
ctggcctgga?acaccaggaa?cattgatgcc?acctccacag?gcaactacaa?ctacaagtac 1320
aggtacctga?ggcatggcaa?gctgaggcca?tttgagaggg?acatctccaa?tgtgccattc 1380
tcccctgatg?gcaagccatg?caccccccct?gccctgaact?gctactggcc?cctgaatgac 1440
tatggcttct?acaccaccac?aggcattggc?taccagccat?acagggtggt?ggtgctgtcc 1500
tttgagctgc?tgaatgcccc?tgccacagtc?tgtggcccca?agctgtccac?agacctgatc 1560
aagaaccagt?gtgtgaactt?caacttcaat?ggcctgacag?gcacaggcgt?gctgacccca 1620
tcctccaaga?ggttccagcc?attccagcag?tttggcaggg?atgtctctga?cttcacagac 1680
tctgtgaggg?accccaagac?ctctgagatc?ctggacatct?ccccatgtgc?ctttggcggc 1740
gtctctgtga?tcacccctgg?caccaatgcc?tcctctgagg?tggctgtgct?gtaccaggat 1800
gtgaactgca?cagatgtctc?cacagccatc?catgctgacc?agctgacccc?tgcctggagg 1860
atctactcca?caggcaacaa?tgtcttccag?acacaggctg?gctgcctgat?tggcgctgag 1920
catgtggaca?cctcctatga?gtgtgacatc?cccattggcg?ctggcatctg?tgcctcctac 1980
cacacagtct?ccctgctgag?gtccacctcc?cagaagtcca?ttgtggccta?caccatgtcc 2040
ctgggcgctg?actcctccat?tgcctactcc?aacaacacca?ttgccatc1c?caccaacttc 2100
tccatctcca?tcaccacaga?ggtgatgcct?gtctccatgg?ccaagacctc?tgtggactgc 2160
aacatgtaca?tctgtggcga?ctccacagag?tgtgccaacc?tgctgctgca?gtatggctcc 2220
ttctgcaccc?agctgaacag?ggccctgtct?ggcattgctg?ctgagcagga?taggaacacc 2280
agggaggtct?ttgcccaggt?gaagcagatg?tacaagaccc?ccaccctgaa?gtactttggc 2340
ggcttcaact?tctcccagat?cctgcctgac?cccctgaagc?ccaccaagag?gtccttcatt 2400
gaggacctgc?tgttcaacaa?ggtgaccctg?gctgatgctg?gcttcatgaa?gcagtatggc 2460
gagtgcctgg?gcgacatcaa?tgccagggac?ctgatctgtg?cccagaagtt?caatggcctg 2520
acagtgctgc?cccccctgct?gacagatgac?atgattgctg?cctacacagc?tgccctggtc 2580
tctggcacag?ccacagctgg?ctggaccttt?ggcgctggcg?ctgccctgca?gatcccattt 2640
gccatgcaga?tggcctacag?gttcaatggc?attggcgtga?cccagaatgt?gctgtatgag 2700
aaccagaagc?agattgccaa?ccagttcaac?aaggccatct?cccagatcca?ggagtccctg 2760
accaccacct?ccacagccct?gggcaagctg?caggatgtgg?tgaaccagaa?tgcccaggcc 2820
ctgaacaccc?tggtgaagca?gctgtcctcc?aactttggcg?ccatctcctc?tgtgctgaat 2880
gacatcctgt?ccaggctgga?caaggtggag?gctgaggtgc?agattgacag?gctgatcaca 2940
ggcaggctgc?agtccctgca?gacctatgtg?acccagcagc?tgatcagggc?tgctgagatc 3000
agggcctctg?ccaacctggc?tgccaccaag?atgtctgagt?gtgtgctggg?ccagtccaag 3060
agggtggact?tctgtggcaa?gggctaccat?ctgatgtcct?tcccccaggc?tgccccccat 3120
ggcgtggtct?tcctgcatgt?gacctatgtg?ccatcccagg?agaggaactt?caccacagcc 3180
cctgccatct?gccatgaggg?caaggcctac?ttccccaggg?agggcgtctt?tgtcttcaat 3240
ggcacctcct?ggttcatcac?ccagaggaac?ttcttctccc?cccagatcat?caccacagac 3300
aacacctttg?tctctggcaa?ctgtgatgtg?gtgattggca?tcatcaacaa?cacagtctat 3360
gaccccctgc?agcctgagct?ggactccttc?aaggaggagc?tggacaagta?cttcaagaac 3420
cacacctccc?ctgatgtgga?cctgggcgac?atctctggca?tcaatgcctc?tgtggtgaac 3480
atccagaagg?agattgacag?gctgaatgag?gtggccaaga?acctgaatga?gtccctgatt 3540
gacctgcagg?agctgggcaa?gtatgagcag?tacatcaagt?ggccatggta?tgtctggctg 3600
ggcttcattg?ctggcctgat?tgccattgtg?atggtgacca?tcctgctgtg?ctgcatgacc 3660
tcctgctgct?cctgcctgaa?gggcgcctgc?tcctgtggct?cctgctgcaa?gtttgatgag 3720
gatgactctg?agcctgtgct?gaagggcgtg?aagctgcact?acacctaa 3768
<210>2
<211>1255
<212>PRT
＜213〉sars coronavirus (SARS Coronavirus)
<400>2
Met?Phe?Ile?Phe?Leu?Leu?Phe?Leu?Thr?Leu?Thr?Ser?Gly?Ser?Asp?Leu
1 5 10 15
Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln
20 25 30
His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg
35 40 45
Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser
50 55 60
Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Gly?Asn?Pro?Val
65 70 75 80
Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn
85 90 95
Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln
100 105 110
Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys
115 120 125
Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met
130 135 140
Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr
145 150 155 160
Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser
165 170 175
Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly
180 185 190
Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp
195 200 205
Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu
210 215 220
Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro
225 230 235 240
Ala?Gln?Asp?Ile?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
245 250 255
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile
260 265 270
Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys
275 280 285
Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn
290 295 300
Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr
305 310 315 320
Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser
325 330 335
Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr
340 345 350
Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly
355 360 365
Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala
370 375 380
Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly
385 390 395 400
Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe
405 410 415
Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
420 425 430
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu
435 440 445
Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly
450 455 460
Lys?Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp
465 470 475 480
Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val
485 490 495
Val?Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly
500 505 510
Pro?Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn
515 520 525
Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg
530 535 540
Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp
545 550 555 560
Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys
565 570 575
Ala?Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser
580 585 590
Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys?Thr?Asp?Val?Ser?Thr
595 600 605
Ala?Ile?His?Ala?Asp?Gln?Leu?Thr?Pro?Ala?Trp?Arg?Ile?Tyr?Ser?Thr
610 615 620
Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln?Ala?Gly?Cys?Leu?Ile?Gly?Ala?Glu
625 630 635 640
His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile
645 650 655
Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys
660 665 670
Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala
675 680 685
Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile
690 695 700
Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys?Thr?Ser?Val?Asp?Cys
705 710 715 720
Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu
725 730 735
Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile
740 745 750
Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys
755 760 765
Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe
770 775 780
Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile
785 790 795 800
Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met
805 810 815
Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile
820 825 830
Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr
835 840 845
Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala
850 855 860
Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe
865 870 875 880
Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn
885 890 895
Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala
900 905 910
Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly
915 920 925
Lys?Leu?Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu
930 935 940
Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn
945 950 955 960
Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp
965 970 975
Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln
980 985 990
Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser?Ala?Asn?Leu?Ala?Ala
995 1000 1005
Thr?Lys?Met?Ser?Glu?Cys?Val?Leu?Gly?Gln?Ser?Lys?Arg?Val?Asp
1010 1015 1020
Phe?Cys?Gly?Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala
1025 1030 1035
Pro?His?Gly?Val?Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln
1040 1045 1050
Glu?Arg?Asn?Phe?Thr?Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys
1055 1060 1065
Ala?Tyr?Phe?Pro?Arg?Glu?Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser
1070 1075 1080
Trp?Phe?Ile?Thr?Gln?Arg?Asn?Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr
1085 1090 1095
Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly?Asn?Cys?Asp?Val?Val?Ile?Gly
1100 1105 1110
Ile?Ile?Asn?Asn?Thr?Val?Tyr?Asp?Pro?Leu?Gln?Pro?Glu?Leu?Asp
1115 1120 1125
Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys?Tyr?Phe?Lys?Asn?His?Thr?Ser
1130 1135 1140
Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser?Gly?Ile?Asn?Ala?Ser?Val
1145 1150 1155
Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu?Asn?Glu?Val?Ala?Lys
1160 1165 1170
Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu?Leu?Gly?Lys?Tyr
1175 1180 1185
Glu?Gln?Tyr?Ile?Lys?Trp?Pro?Trp?Tyr?Val?Trp?Leu?Gly?Phe?Ile
1190 1195 1200
Ala?Gly?Leu?Ile?Ala?Ile?Val?Met?Val?Thr?Ile?Leu?Leu?Cys?Cys
1205 1210 1215
Met?Thr?Ser?Cys?Cys?Ser?Cys?Leu?Lys?Gly?Ala?Cys?Ser?Cys?Gly
1220 1225 1230
Ser?Cys?Cys?Lys?Phe?Asp?Glu?Asp?Asp?Ser?Glu?Pro?Val?Leu?Lys
1235 1240 1245
Gly?Val?Lys?Leu?His?Tyr?Thr
1250 1255
Claims (10)
1. an isolated nucleic acid molecule is characterized in that, S albumen or the immunogenic fragments of its encoding SARS coronavirus, and the homogeny of the S albumen coded sequence of itself and natural sars coronavirus is 77 ± 3%.
2. nucleic acid molecule as claimed in claim 1 is characterized in that, the sars coronavirus S albumen shown in its coding SEQ ID NO:2.
3. nucleic acid molecule as claimed in claim 1 is characterized in that, it contains the nucleotide sequence shown in the SEQ ID NO:1.
4. nucleic acid molecule as claimed in claim 1 is characterized in that, it is selected from down group:
(a) nucleotide sequence shown in the SEQ ID NO:1;
(b) with SEQ ID NO:1 shown in the nucleotide sequence homogeny greater than 95%, and the coding same acid sequence nucleotide sequence.
5. nucleic acid molecule as claimed in claim 1 is characterized in that, compares with the S albumen coded sequence of natural SARS virus, and in mammalian cell, the proteic expression amount of the S of described nucleic acid molecule encoding improves at least 100%.
6. a carrier is characterized in that, it contains the described nucleic acid molecule of claim 1.
7. carrier as claimed in claim 6 is characterized in that it is a plasmid.
8. carrier as claimed in claim 6 is characterized in that it is a virus vector.
9. a host cell is characterized in that, it contains the described carrier of claim 6.
10. a composition is characterized in that, it contains the described carrier of claim 6 and pharmaceutically acceptable excipient or thinner.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03141619 CN1570115A (en) | 2003-07-16 | 2003-07-16 | Optimized SARS coronavirus spike protein gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03141619 CN1570115A (en) | 2003-07-16 | 2003-07-16 | Optimized SARS coronavirus spike protein gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1570115A true CN1570115A (en) | 2005-01-26 |
Family
ID=34470986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03141619 Pending CN1570115A (en) | 2003-07-16 | 2003-07-16 | Optimized SARS coronavirus spike protein gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1570115A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006136084A1 (en) * | 2005-06-20 | 2006-12-28 | Chinese Academy Of Medical Sciences, Institute Of Basic Medical Sciences | Fusion proteins of recombinant sars coronavirus structural proteins, their production and uses |
| CN110951756A (en) * | 2020-02-23 | 2020-04-03 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence for expressing SARS-CoV-2 virus antigen peptide and its application |
| CN110974950A (en) * | 2020-03-05 | 2020-04-10 | 广州恩宝生物医药科技有限公司 | Adenovirus vector vaccine for preventing SARS-CoV-2 infection |
| CN112206318A (en) * | 2020-03-16 | 2021-01-12 | 广州恩宝生物医药科技有限公司 | Ad7 vector vaccine for preventing SARS-CoV-2 infection |
| CN112358533A (en) * | 2020-10-30 | 2021-02-12 | 上海泽润生物科技有限公司 | Recombinant spike protein and preparation method and application thereof |
| WO2021000969A3 (en) * | 2020-02-23 | 2021-02-18 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence expressing sars-cov-2 virus antigen peptide and use thereof |
| CN112390863A (en) * | 2020-09-23 | 2021-02-23 | 厚朴生物科技（苏州）有限公司 | Modified new coronavirus Spike protein extracellular domain and application thereof |
| CN112695057A (en) * | 2020-05-11 | 2021-04-23 | 广东珩达生物医药科技有限公司 | SARS-COV-2 antigen polypeptide and its recombinant adeno-associated virus and application in preparing vaccine |
| CN113248576A (en) * | 2020-02-12 | 2021-08-13 | 北京科兴中维生物技术有限公司 | Nucleic acid vaccine for coronavirus and preparation method thereof |
| CN113599512A (en) * | 2020-05-04 | 2021-11-05 | 国光生物科技股份有限公司 | Immunity composition |
-
2003
- 2003-07-16 CN CN 03141619 patent/CN1570115A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006136084A1 (en) * | 2005-06-20 | 2006-12-28 | Chinese Academy Of Medical Sciences, Institute Of Basic Medical Sciences | Fusion proteins of recombinant sars coronavirus structural proteins, their production and uses |
| CN113248576A (en) * | 2020-02-12 | 2021-08-13 | 北京科兴中维生物技术有限公司 | Nucleic acid vaccine for coronavirus and preparation method thereof |
| CN110951756A (en) * | 2020-02-23 | 2020-04-03 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence for expressing SARS-CoV-2 virus antigen peptide and its application |
| CN110951756B (en) * | 2020-02-23 | 2020-08-04 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence for expressing SARS-CoV-2 virus antigen peptide and its application |
| WO2021000969A3 (en) * | 2020-02-23 | 2021-02-18 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence expressing sars-cov-2 virus antigen peptide and use thereof |
| CN110974950A (en) * | 2020-03-05 | 2020-04-10 | 广州恩宝生物医药科技有限公司 | Adenovirus vector vaccine for preventing SARS-CoV-2 infection |
| CN110974950B (en) * | 2020-03-05 | 2020-08-07 | 广州恩宝生物医药科技有限公司 | Adenovirus vector vaccine for preventing SARS-CoV-2 infection |
| CN112206318A (en) * | 2020-03-16 | 2021-01-12 | 广州恩宝生物医药科技有限公司 | Ad7 vector vaccine for preventing SARS-CoV-2 infection |
| CN113599512A (en) * | 2020-05-04 | 2021-11-05 | 国光生物科技股份有限公司 | Immunity composition |
| CN112695057A (en) * | 2020-05-11 | 2021-04-23 | 广东珩达生物医药科技有限公司 | SARS-COV-2 antigen polypeptide and its recombinant adeno-associated virus and application in preparing vaccine |
| CN112695057B (en) * | 2020-05-11 | 2022-04-26 | 广东珩达生物医药科技有限公司 | SARS-COV-2 antigen polypeptide and its recombinant adeno-associated virus and application in preparing vaccine |
| CN112390863A (en) * | 2020-09-23 | 2021-02-23 | 厚朴生物科技（苏州）有限公司 | Modified new coronavirus Spike protein extracellular domain and application thereof |
| CN112390863B (en) * | 2020-09-23 | 2022-11-01 | 厚朴生物科技（苏州）有限公司 | Modified new coronavirus Spike protein extracellular domain and application thereof |
| CN112358533A (en) * | 2020-10-30 | 2021-02-12 | 上海泽润生物科技有限公司 | Recombinant spike protein and preparation method and application thereof |
| CN112358533B (en) * | 2020-10-30 | 2023-07-14 | 上海泽润生物科技有限公司 | Recombinant spike protein and preparation method and application thereof |
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