KR20070117537A - An expression vector encoding coronavirus-like particle - Google Patents

Abstract

The present invention provides an expression vector encoding coronavirus-like particles, which comprises two transcriptional units, wherein the first unit comprises genes encoding membrane M protein and envelope E protein from coronavirus, while the second unit comprises SpikeCT coding gene from coronavirus. Said expression vector is useful for cloning viral genes from Type I membrane fusion mechanism, and also is useful as DNA vaccine candidate.

Description

Expression vectors encoding coronavirus-like particles {AN EXPRESSION VECTOR ENCODING CORONAVIRUS-LIKE PARTICLE}

The present invention relates to the field of recombinant DNA technology, and more specifically to the field of DNA vaccines. In particular, the present invention relates to a novel expression vector encoding a virus like particle for cloning the Class I viral fusion protein gene, as a DAN vaccine candidate.

Vaccination has played an important role in controlling viral diseases for the last 30 years. Vaccination is based on the simple principle of immunology. Once exposed to an infectious agent, the animal begins an immune response to protect against infection by the same infectious agent. The purpose of vaccination is to induce animals to increase their defense mechanisms before infection. This vaccination has conventionally been carried out using an attenuated live or dead form of the virus as an immunogen. The success of this past approach is due in part to the presence of innate antigens and the ability of the attenuated virus to elicit the full range of immune responses obtained from natural infection. However, traditional vaccine methods always have some potential limitations. Attenuated strains can be latent and more virulent or non-immunogenic; Improperly inactivated vaccines may cause the disease intended to be prevented.

Recombinant DNA technology has led to the development of vaccines based on the use of defined antigens instead of complete infectious agents as immunogens, offering the possibility of eliminating some of the limitations of conventional vaccines. It is a chemically synthesized, peptide vaccine composed of immunoreactive epitopes; Subunit vaccine vaccines formed by expression of viral proteins in recombinant heterologous cells and the use of live viral vectors for presenting one or multiple regulatory antigens. Both peptides and subunit vaccines have potential limitations. The main problem is that it is difficult to be convinced that modifications of genetic engineering proteins mimic that of antigens in their natural environment. In the case of suitable adjuvants and peptides, carrier proteins should be used to increase the immune response. In addition, these vaccines mainly induce humoral resoponse, and thus fail to elicit effective immunity.

Many other methods have been developed to introduce new genetic information into target cells. Currently, the most effective way to introduce DNA into target cells is by a modified virus called a recombinant viral vector. The most frequently used viral vector systems are retroviruses, adenoviruses, herpes viruses or adeno-associated viruses (AAV). All systems have their own advantages and disadvantages. Some vector systems have the ability to integrate their DNA into the host cell genome, while others do not. While still impossible in other systems, viral genes can be completely removed from some vector systems. Some vector systems have very good propagation characteristics in Vivo. Some vector types can produce very large capacities very easily, while others are difficult to produce.

Coronaviruses deliver three or four proteins in their envelopes. M protein is the most abundant component. Small E proteins are small but essential viral components. The importance of S proteins in pathogenesis harmonizes with their biologic functions, both in the introduction and viral propagation of viruses ( Collins , AR, et. al ., 1982, Virology 119 : 358-371 ; Williams , RK, et al ., 1991, Proc . Natl . Acad . Sci . USA 88 : 5533-5536 ). When expressed in a virion envelope, the S protein binds to cellular receptors and induces fusion of the virus and cell membranes when the virus is introduced. Following infection, S protein expressed on the plasma membrane of infected cells induces cell fusion. S proteins are targets that neutralize antibodies ( Collins , AR, et. . al, 1982, Virology 119: 358-371) and cell-mediated (cell-mediated) immune inducer (Bergmann, CC, et al., 1996, J. Gen. Virol . 77: 315-325; Castro , RF, and S. Perlman , 1995, J. Virol . 69 : 8127-8131 ) and plays an important role in the immune response to viral infections. M and E proteins are the smallest protein units for viral aggregation ( Baudoux, P., et al ., 1998, J. Virol . 72 : 8636-8643 ; Bos , EC, 1996, Virology 218 : 52-60 ; de Haan , CA M., et al ., 1998, J. Virol . 72 : 6838-6850 ; Godeke , G.-J., et al ., 2000, J. Virol . 74 : 1566-1571 ; Vennema, H., et al ., 1996, EMBO J. 15 : 2020-2028 ). Both are integrated membrane proteins. Expression of both M and E proteins is sufficient to induce virus-like particles (VLP). When the S protein is expressed with the M and E proteins, the S protein binds to the VLP, perhaps with authentic conformation. This mouse hepatitis virus (MHV, mouse hepatitis virus) ( Bos, EC, 1996, Virology 218: 52-60; de Haan , CA M., et al., 1998, J. Virol . 72 : 6838-6850 ; Vennema , H., et al., 1996, EMBO J. 15 : 2020-2028) , transmissible gastroenteritis virus gastroenteritis virus ( Baudoux, P., et al ., 1998, J. Virol . 72 : 8636-8643) and feline infectious peritonitis virus ( feline infectious peritonitis virus ( Godeke , G.-J., et al ., 2000, J. Virol . 74: 1566-1571) . The membrane of the coronavirus consists essentially of a dense matrix that laterally interacts with the M protein, which in many cases requires an E protein for budding and, if available, the transmembrane of the carboxyl end and the spike. Specific interactions with M through the transmembrane region integrate S and HE glycoproteins into it ( de Haan , CA M., 1999, J. Virol . 73 : 7441-7452 ; Nguyen, V.-P., and BG Hogue . 1997, J. Virol . 71 : 9278-9284; Opstelten , D.-JE, et al ., 1995, J. Cell Biol . 131 : 339-349 ; Vennema , H., et al ., 1996, EMBO J. 15 : 2020-2028 ). VLPs containing these spikes can infect cells with the same infectivity as a true virus ( Bos , EC, 1996, Virology 218 : 52-60 ). However, no effective DNA vector was developed as a DNA vaccine.

Thus, there is a need to develop effective DNA vectors encoding virus-like particles capable of expressing functional viral fusion protein genes on the surface of VLPs, which will be the most excellent candidates for potential vaccines against viral diseases. will be.

Composition of the Invention

The present invention relates to an expression vector for cloning the Class I viral fusion protein gene as a DNA vaccine candidate, wherein the expression vector is

Iii) the membrane protein gene (M protein gene) of the coronavirus, the envelope protein gene (E protein gene) of the coronavirus and the internal ribosome entry sites (IRES) sequence. A first transcription unit, said IRES comprising: a first transcription unit inserted into a junction of said membrane protein gene and said envelope protein gene;

Ii) a first eukaryotic promoter coupled to act on said membrane protein gene, said first eukaryotic promoter located upstream of said M protein gene and inducing expression of said first transcriptional unit;

Iii) multiple cloning sites (MCSs) for the cloning of spikena genes of coronaviruses and for in-frame insertion or cloning of class I viral fusion protein genes. A second transcription unit comprising, the MCS is located at the beginning of the Spike CT gene, the second transcription unit having restriction enzymes cutting sites (restriction enzymes cutting sites); And

Iii) a second eukaryotic promoter coupled to operate on a SpikeCT gene, the second eukaryotic promoter located on top of the SpikeCT gene and inducing expression of a second transcriptional unit,

The first eukaryotic promoter has a stronger transcription activity than the second eukaryotic promoter.

Detailed description of the invention

Genes that encode viral polypeptides can be self-assembled into defective, non-self propagating viral particles, so that the gene that encodes the viral polypeptide is the genomic DNA or RNA of the DNA virus. Genomic cDNA of the virus, or valid subgenomic clones containing genes can be obtained. Such genes include encoding viral envelope glycoproteins for viral capsid proteins (ie, proteins comprising viral protein shells) and for enveloped viruses such as retroviruses. Virus-like particles can be used for vaccination against pathogenic viruses, for therapeutic purposes such as enhancing an immune response in an infected individual, or for targeting therapeutic drugs such as cytotoxic drugs. To be delivered to the human body, it can be isolated and used as an immunogen to a particular cell type.

The present invention provides an expression vector for cloning a class I viral fusion protein gene as a DNA vaccine candidate. The expression vector is,

A first transcription unit comprising a membrane protein gene (M protein gene) of the coronavirus, an envelope protein gene (E protein gene) of the coronavirus and an internal ribosomal introduction site (IRES) sequence, wherein the IRES is the membrane protein gene and the A first transcription unit inserted into a junction of the envelope protein gene;

A first eukaryotic promoter coupled to said membrane protein gene, said first eukaryotic promoter positioned above said M protein gene and inducing expression of said first transcriptional unit;

A second transcription unit comprising a SpikeCT gene of Coronavirus and a plurality of cloning sites (MCS) for in-frame insertion or cloning of a Class I viral fusion protein gene, wherein the MCS is the beginning of the SpikeCT gene. A second transcription unit located at and having a restriction enzyme cleavage site; And

A second eukaryotic promoter coupled to act on said SpikeCT gene, said second eukaryotic promoter located on top of said SpikeCT gene and inducing expression of a second transcription unit,

The first eukaryotic promoter has a stronger transcriptional activity than the second eukaryotic promoter.

According to the invention, the expression vector comprises a first transcription unit comprising a membrane protein gene (M protein gene) of the coronavirus, an envelope protein gene (E protein gene) of the coronavirus and an internal ribosomal introduction site (IRES) sequence. The IRES is inserted into the junction of the membrane protein gene and the envelope protein gene.

According to the present invention, the envelope protein gene of the present invention encodes the envelope protein (E protein) of coronavirus. E proteins are small, envelope-dependent proteins. E proteins are small but essential viral components. It accumulates in the cell and induces coalescence of the membrane of the intermediate compartment (IC), forming a typical structure. Segments of proteins may appear extracellularly in unknown membrane structures. Preferably, the coronaviruses of the invention are swine, human or avian coronaviruses. More preferably the corona virus is swine TGEV coronavirus, human 229E corona virus or human SARS virus. More preferably, the E protein of the present invention has a sequence as shown in SEQ ID NO: 1.

SARS SARS ) The coding sequence of the E gene (SEQ ID NO: 1)

ATGGCATACT CTTTTGTGTC TGAGGAAACT GGCACTCTGA TCGTGAACTC TGTACTGCTG

TTTCTCGCTT TTGTGGTATT CCTGCTGGTC ACTCTCGCTA TCCTCACTGC TCTTCGTCTG

TGTGCCTACT GTTGTAATAT CGTGAACGTG TCTCTGGTTA AGCCTACTGT GTATGTGTAT

TCTCGTGTGA AAAATCTCAA TTCTTCTGAA GGAGTTCCCG ATCTGCTGGT CTAG

According to the present invention, the membrane protein gene of the present invention encodes the membrane protein (M protein) of coronavirus. M protein is the most abundant component. It is one short amino-terminal ectodomain, three consecutive transmembrane domains, and one long carboxyl-terminal domain inside the virion (or inside the cytoplasm). It is composed of type III glycoproteins. Preferably, the coronaviruses of the invention are swine, human or avian coronaviruses. More preferably the coronavirus is swine TGEV coronavirus, human 229E corona virus or human SARS virus. More preferably, the M protein of the present invention has a sequence as shown in SEQ ID NO: 2.

 Coding Sequence of the SARS M Gene (SEQ ID NO: 2)

ATGGCAGATA ACGGCACTAT TACTGTGGAG GAACTGAAAC AACTGCTGGA ACAATGGAAC

CTCGTAATCG GCTTTCTCTT TCTGGCTTGG ATTATGTTGT TACAGTTTGC GTATTCTAAT

CGTAACCGTT TCCTCTACAT TATTAAGCTC GTTTTCCTGT GGTTGTTGTG GCCTGTAACT

CTTGCTTGCT TTGTGCTTGC TGCTGTCTAT CGTATCAACT GGGTTACTGG TGGTATTGCT

ATCGCTATGG CTTGTATTGT AGGCTTGATG TGGCTGTCTT ATTTCGTTGC TTCTTTCCGT

CTGTTTGCTC GTACTCGCTC TATGTGGTCC TTTAATCCTG AGACTAATAT CCTGCTGAAT

GTTCCGCTCC GTGGTACTAT CGTTACTAGA CCGCTGATGG AATCTGAACT GGTTATTGGT

GCCGTCATTA TCCGTGGTCA TTTGCGTATG GCTGGTCACT CTCTGGGTCG TTGCGATATT

AAGGATCTGC CAAAGGAAAT CACTGTAGCC ACTTCTCGTA CTCTGTCTTA CTATAAACTC

GGTGCATCGC AACGTGTGGG AACTGATTCG GGCTTCGCTG CGTATAATCG TTATCGTATT

GGCAACTATA AACTGAACAC CGACCACGCA GGCTCTAATG ACAACATCGC TCTCCTCGTT

CAGTGA

According to the invention, only M and E proteins are required for the assembly of the coronavirus envelope ( Cornelis AM de Haan et al ., Journal of Virology, June 2000, p. 4967-4978) . Intracellular expression of genes encoding these proteins results in the formation and release of viruslike particles (VLPs) that are similar in size and shape to true virions.

According to the invention, the first transcription unit comprises an internal ribosome entry site (IRES) sequence inserted into the junction of the M gene and the E gene. IRES allows for more than one genetic translation from di- or polycittronic mRNA. The IRES unit is then fused to the 5 ′ end of one or more additional coding sequences that will be inserted into the vector at the end of the original coding sequence, so that the coding sequence is separated from the other by the IRES. According to the invention any IRES derivative may also be used in the plasmind construct of the invention. IRES sequences enable ribosomal machinery to initiate genetic decryption from the second site within a single transcript.

According to the present invention, the expression vector of the present invention comprises a first eukaryotic promoter that is linked to operate on a membrane protein gene and is located on top of the M protein gene and induces expression of a first transcription unit. Preferably, the first eukaryotic promoter is CMV, SV40, RSV, HIV-1 LTR, hybrid beta-actin / CMV promoter enhancer, muscle-specific desmin, creatine It is a viral derived promoter, such as creatine kinase, beta-Actin promoter, and other ubiquitous expression promoters such as EF1alpha.

According to the invention, the second transcription unit comprises a multiple cloning site (MCS) having restriction enzymes cutting sites, wherein the MCS is located at the beginning of the spike CT gene. do. The SpikeCT gene interacts with the M protein interaction domain, which is located on top of an aromatic residue-rich region parallel to the transmembrane segment of the spike protein of coronavirus. It encodes a C-terminal heptad repeat located. Many cloning sites have several restriction enzyme cleavage sites. Restriction enzyme cleavage sites include, but are not limited to, SmaI, BsaB I, EcoR V and BspE I. MCS is located at the beginning of the SpikeCT gene and can be used for in-frame insertion or cloning of class I viral fusion protein genes. Class I viral fusion protein genes include, but are not limited to, gp160 of HIV, HA of influenza viruses, and spikes of SARS virus. Class I viral protein genes are derived from viruses employing class I viral fusion mechanisms including coronavirus, HIV and influenza viruses. Fusion protein genes are inserted into the cloning / expression vectors of the invention to be cloned and used as candidates for DNA vaccines. Preferably, the SpikeCT gene has the sequence shown in SEQ ID NO: 3.

Coding Sequence of the SpikeCT Gene (SEQ ID NO: 3)

GATATCTCCGGA ATCAATGCGA GCGTAGTGAA CATCCAGAAA

GAGATTGACC GTTTGAATGA AGTTGCTAAA AATCTGAATG AACCTCTGAT TGACCTCCAA

GAACTCGGCA AATATGAGCA ATACATTAAA TGGCCTTGGT ATGTCTGGTT GGGTTTCATC

GCAGGTCTCA TCGCTATCGT TATGGTGACT ATTCTGCTGT GTTGTATGAC TTCTTGTTGC

TCTTGTCTGA AAGGTGCGTG TTCTTGTGGT TCTTGTTGTA AGTTTGATGA GGATGATTCT

GAGCCAGTTC TGAAGGGTGT GAAGCTGCAT TATACCTAGT TCGAA

According to the present invention, the expression vectors of the present invention can produce virus like particles and express functional fusion genes of class I viruses on the surface of virus like particles. Thus, there are several restriction enzyme cleavage sites (multiple cloning sites, MCS), including but not limited to SmaI, BsaB I, EcoR V and BspE I, which are located at the beginning of SpikeCT, and these sites are of the invention. The cloning / expression vector can be used to insert or clone a class I viral fusion protein gene and then be used as a DNA vaccine candidate. Viruses employing Class I viral fusion mechanisms include, but are not limited to, coronaviruses, HIV, influenza viruses, and the like.

According to the invention the second promoter is located on top of the SpikeCT gene and induces the expression of the second transcription unit. Preferably, the second eukaryotic promoter is CMV, SV40, RSV, HIV-1 LTR, hybrid beta-actin / CMV promoter enhancer, muscle specific desmin, creatine kinase), beta-actin promoter, other ubiquitous such as EF1alpha. Promoters derived from viruses such as expression promoters. Most preferably, the second eukaryotic promoters are the pCMV, SV40, beta-actin and EF1 alpha promoters.

According to the invention the transfer unit of the invention comprises a polyA signal. One skilled in the art can recognize that any transcription has a polyA signal. Preferably, the transcription unit of the invention comprises a BGH polyA signal.

According to the invention, the expression vector further comprises a replication origin for propagation plasmids in the host cell. The origin of replication is determined by the type of host cell.

According to the present invention, the expression vector of the present invention further comprises an antibiotic-resistant gene as a selection marker.

According to the present invention the expression vector of the present invention can spontaneously form virus-like particles. Virus-like particles can be used as vaccine candidates against viral infections. Virus-like particles lack the genetic material of the virus. Thus, these particles do not have infectious ability. The expression vector of the invention can be administered to an animal and can be transfected with a host cell to produce virus-like material.

Most viruses consist of a few proteins, with only structural limitations between them. In fact using these few proteins, the virus is forced to produce high repetitive surfaces, quasi-crystalline. This high repeat antigen effectively crosslinks the B cell receptor, effectively crosslinks the B cell receptor, and produces a strong activity signal in the B cell. In contrast, self-antigens are usually not highly organized and are not particularly accessible to B cells. Thus, B cells use antigenic tissue as a marker for infectious nonmagnetic. Since virus like particles (VLPs) represent a repetitive and organized surface like the surface of a virus, vaccines that rely on virus like particles take advantage of this fundamental phenomenon. As a result, VLPs, like viruses, can trigger strong B cell responses even in the absence of adjuvants. Based on this principle, virus-like particles produced through the expression of the expression vectors of the present invention in cells can be used as vaccine candidates for viral diseases.

1 shows a plasmid map of a preferred embodiment of the expression vector of the invention.

PCR E gene synthesized by

Polymerase chain reaction (PCR) was used to synthesize E gene of SARS virus. The PCR template of the SARS E gene (0.1 pmole) is a mixture of primers described below and the PCR reaction is carried out using an Innis et al . Using KOD tag polymerase (Taq polymerase (Novagene.com)). It was performed using the basic PCR method described by (PCR protocols. A guide to methods and applications, 1990, Academic Press).

PCR primers are as described below:

E1L

5'-ACC ATG GCA TAC TCT TTT GTG TCT GAG GAA ACT G-3 '* (SEQ ID NO: 4)

E2L

5'-ACA GCA GTA CAG AGT TCA CGA TCA GAG TGC CAG TTT CCT CAG ACA CAA AAG-3 '(SEQ ID NO: 5)

E3L

5'-TGA CCA GCA GGA ATA CCA CAA AAG CGA GAA ACA GCA GTA CAG AGT TCA CGA-3 '(SEQ ID NO: 6)

E4L

5'-GCA CAC AGA CGA AGA GCA GTG AGG ATA GCG AGA GTG ACC AGC AGG AAT ACC ACA-3 '(SEQ ID NO: 7)

E5L

5'-ACC AGA GAC ACG TTC ACG ATA TTA CAA CAG TAG GCA CAC AGA CGA AGA GCA G-3 '(SEQ ID NO: 8)

E6L

5'-GAT TTT TCA CAC GAG AAT ACA CAT ACA CAG TAG GCT TAA CCA GAG ACA CGT TCA CGA T-3 '* (SEQ ID NO: 9)

E7L

5'-TCT AGA CCA GCA GAT CGG GAA CTC CTT CAG AAG AAT TGA GAT TTT TCA CAC GAG AAT ACA CA-3 '(SEQ ID NO: 10)

E8L

5'-TCT AGA CCA GCA GAT CGG GA-3 '(SEQ ID NO: 11)

DNA template was denatured for 3 minutes at 95 ℃. PCR conditions are as described below:

First cycle in 10 steps in 3 steps PCR  reaction

1. Annealing: 20 seconds at 58 ° C

2. Extension: 72 seconds at 72 ℃

3. Denaturation: 1 minute at 95 ° C

2nd 20 cycles of 3 steps PCR  reaction

Denaturation: 1 minute at 95 ° C

2. Annealing: 20 seconds at 62 ° C

3. Extension: 40 seconds at 72 ℃

The resulting double-stranded full-length product of the E gene product was analyzed on a 1.2% agarose gel and purified on a QIAQuick PCR Purification Kit (Qiagen Inc.). After ligating with a pGEM (T) vector (Promega Co.) to obtain a pGEM (T) / E ^{A +} clone (clones). The sequence of the E gene is shown in SEQ ID NO: 1.

Splicing -over( splicing - over M gene synthesized by

The M gene of SARS virus was synthesized by a method similar to the above-mentioned PCR method except that the PCR template of the SARS M gene (0.1 pmole) was a mixture of primers described below. Polymerase (Taq polymerase (Novagene.com)) was used.

PCR primers are as described below:

M1-1U

5'-TGA TCA TGG CAG ATA ACG GCA C-3 '(SEQ ID NO: 12)

M1U

5'-TGA TCA TGG CAG ATA ACG GCA CTA TTA CTG TGG AGG A-3 '(SEQ ID NO: 13)

M1L

5'-GTT CCA TTG TTC CAG CAG TTG TTT CAG TTC CTC CAC AGT AAT AGT GCC G-3 '(SEQ ID NO: 14)

M2L

5'-AAG CCA GAA AGA GAA AGC CGA TTA CGA GGT TCC ATT GTT CCA GCA GTT G-3 '(SEQ ID NO: 15)

M3L

5'-AGA ATA CGC AAA CTG TAA CAA CAT AAT CCA AGC CAG AAA GAG AAA GCC GA-3 '(SEQ ID NO: 16)

M4L

5'-CGA GCT TAA TAA TGT AGA GGA AAC GGT TAC GAT TAG AAT ACG CAA ACT GTA ACA ACA-3 '(SEQ ID NO: 17)

M5L

5'-CAA GAG TTA CAG GCC ACA ACA ACC ACA GGA AAA CGA GCT TAA TAA TGT AGA GGA AA-3 '(SEQ ID NO: 18)

M6L

5'-TTG ATA CGA TAG ACA GCA GCA AGC ACA AAG CAA GCA AGA GTT ACA GGC CAC AAC A-3 '(SEQ ID NO: 19)

M7L

5'-CAA GCC ATA GCG ATA GCA ATA CCA CCA GTA ACC CAG TTG ATA CGA TAG ACA GCA GCA A-3 '(SEQ ID NO: 20)

M8L

5'-AAC GAA ATA AGA CAG CCA CAT CAA GCC TAC AAT ACA AGC CAT AGC GAT AGC AAT AC-3 '(SEQ ID NO: 21)

M9L

5'-ACA TAG AGC GAG TAC GAG CAA ACA GAC GGA AAG AAG CAA CGA AAT AAG ACA GCC ACA TC-3 '(SEQ ID NO: 22)

M10U

5'-TCC TGC TGA ATG TTC CGC TC-3 '(SEQ ID NO: 23)

M10L

5'-GAG CGG AAC ATT CAG CAG GAT ATT AGT CTC AGG ATT AAA GGA CCA CAT AGA GCG AGT ACG AGC AA-3 '(SEQ ID NO: 24)

M11L

5'-CAT CAG CGG TCT AGT AAC GAT AGT ACC ACG GAG CGG AAC ATT CAG CAG GA-3 '(SEQ ID NO: 25)

M12L

5'-ACC ACG GAT AAT GAC GGC ACC AAT AAC CAG TTC AGA TTC CAT CAG CGG TCT AGT AAC GAT-3 '(SEQ ID NO: 26)

M13L

5'-ATA CGC AAA TGA CCA CGG ATA ATG ACG GCA C-3 '(SEQ ID NO: 27)

M14L

5'-AAC GAC CCA GAG AGT GAC CAG CCA TAC GCA AAT GAC CAC GGA TAA-3 '(SEQ ID NO: 28)

M15L

5'-TAC AGT GAT TTC CTT TGG CAG ATC CTT AAT ATC GCA ACG ACC CAG AGA GTG-3 '(SEQ ID NO: 29)

M16L

5'-CCG AGT TTA TAG TAA GAC AGA GTA CGA GAA GTG GCT ACA GTG ATT TCC TTT GGC AGA-3 '(SEQ ID NO: 30)

M17L

5'-CGA AGC CCG AAT CAG TTC CCA CAC GTT GCG ATG CAC CGA GTT TAT AGT AAG ACA GAG T-3 '(SEQ ID NO: 31)

M18L

5'-CAG TTT ATA GTT GCC AAT ACG ATA ACG ATT ATA CGC AGC GAA GCC CGA ATC AGT TCC C-3 '(SEQ ID NO: 32)

M19L

5'-GAG AGC GAT GTT GTC ATT AGA GCC TGC GTG GTC GGT GTT CAG TTT ATA GTT GCC AAT ACG AT-3 '(SEQ ID NO: 33)

M20L

5'-TCA CTG AAC GAG GAG AGC GAT GTT GTC ATT AGA G-3 '(SEQ ID NO: 34)

PCR conditions are essentially as mentioned above. Double-stranded full-length products of the M gene product were analyzed on 1.0% agarose gel, purified on QIAQuick PCR purification or gel extraction kit (Qiagen Inc.) and then pGEM (T L) was ligated with the vector (Promega Co.) to obtain pGEM (T) / M ^{A +} clones. The sequence of the M gene is shown in SEQ ID NO: 2.

The transmembrane domain of the SpikeCT gene encoding heptad region 2 and the Spike protein gene of SARS virus synthesized by PCR.

PCR methods and conditions, purification and cloning of PCR products are the same as those mentioned above. The DNA template (0.1 pmole) of the SpikeCT gene is a mixture of primers described below.

PCR primers are as described below.

Spike CTup-EcoRV

5'-GAT ATC TCC GGA ATC AAT GCG AGC GT-3 '(SEQ ID NO: 35)

DI-1U / BspE I

5'-5'-TCC GGA ATC AAT GCG AGC GTA GTG AAC ATC CAG AA-3 ”(SEQ ID NO: 36)

DI-1L

5'-GCA ACT TCA TTC AAA CGG TCA ATC TCT TTC TGG ATG TTC ACT ACG CTC-3 '(SEQ ID NO: 37)

DI-2L

5'-ATC AGA GAT TCA TTC AGA TTT TTA GCA ACT TCA TTC AAA CGG TCA-3 '(SEQ ID NO: 38)

DI-3L

5-TCA TAT TTG CCG AGT TCT TGG AGG TCA ATC AGA GAT TCA TTC AGA TTT TTA-3 '(SEQ ID NO: 39)

DI-4L5'-CAA GGC CAT TTA ATG TAT TGC TCA TAT TTG CCG AGT TCT TGG A-3 '(SEQ ID NO: 40)

DI-5L

5'-ACC TGC GAT GAA ACC CAA CCA GAC ATA CCA AGG CCA TTT AAT GTA TTG CT-3 '(SEQ ID NO: 41)

DI-6L

5'-CAG AAT AGT CAC CAT AAC GAT AGC GAT GAG ACC TGC GAT GAA ACC CAA CC-3 '(SEQ ID NO: 42)

DI-7L

5'-AGA GCA ACA AGA AGT CAT ACA ACA CAG CAG AAT AGT CAC CAT AAC GAT AG-3 '(SEQ ID NO: 43)

DI-8L

5'-ACC ACA AGA ACA CGC ACC TTT CAG ACA AGA GCA ACA AGA AGT CAT ACA AC-3 '(SEQ ID NO: 44)

DI-9L

5'-GAA TCA TCC TCA TCA AAC TTA CAA CAA GAA CCA CAA GAA CAC GCA CCT TT- 3 '(SEQ ID NO: 45)

DI-10L

5'-GCT TCA CAC CCT TCA GAA CTG GCT CAG AAT CAT CCT CAT CAA ACT TAC A-3 '(SEQ ID NO: 46)

DI-11L

5'-CCT AGG TAT AAT GCA GCT TCA CAC CCT TCA GAA CT-3 '(SEQ ID NO: 47)

Spike CTdn-BstB I

5'- TTC GAA CT AGG TAT AAT GCA GCT TCA C -3 '(SEQ ID NO: 48)

PCR conditions are as mentioned above. The resultant double-stranded SCT320 gene products were purified on Qiagen / PCR purification kit (Qiagen Inc.) and then cloned with pGEM-T vector (Promega Co.). pGEM (T) / Spike CT (EcoRV / BstBI) was obtained. pGEM (T) / Spike CT (EcoRV / BstBI) The plasmid contains a gene encoding a C-terminal heptad refit located on top of a region rich in aromatic residues parallel to the membrane portion of the spike protein of SARS coronavirus. The coding sequence of the "SCT320" gene is as shown in SEQ ID NO: 3.

Structure of Expression Vector of the Present Invention Construction )

Manipulation of DNA was introduced by "Molecular cloning third edition" by Sambrook and Russell et al, Cold Spring Harbor Laboratory Express. Restriction enzymes, T4DNA ligase, Klenow enzyme were purchased from New England Biolab and performed according to the manufacturer's recommendations.

I. pCMV  Structure of the promoter-derived SARS M gene plasmid construction )

The pGEM (T) / M clone was used as a DNA template for PCR with T7 and SP6 promoter primers, and the PCR conditions were the same as mentioned above except that the PCR conditions were fixed at 50 ° C. for 20 seconds at the annealing temperature. The M gene PCR product was purified with a QIAquick PCR purification kit (Qiagen Inc.) and digested with Bcl I and NotI to obtain a DNA insert containing SARS M gene. pEGFP-N1 plasmid (Clontech Co.) was digested with BglII, Not I. The resulting pEGEP-N1 plasmid digested with Bgl II, Not I provided a DNA vector for ligation with SARS M DNA insert digested with Bcl I, Not I to obtain a pN1 / M DNA plasmid. The pN1 / M DNA plasmid was digested with NheI and Not I to obtain a DNA insert containing the SARS M gene. The pACT / M DNA plasmid was digested with Bgl II and Asp 718 to obtain a DNA insert containing the CMV promoter and SARS M gene. The pCDNA3.1 (+) plasmid (Invitrogen Co.) was digested with BglII and Asp718. The resulting pCDNA3.1 (+) DNA vector digested with BglII, Asp718 was ligated with DNA comprising the CMW promoter and SARS M gene insert digested with BglII, Asp718 to obtain a pCDNA 3.1 + M DNA plasmid.

II. IRES- SARS  E gene plasmid manipulation ( Constructing )

pIRES2-EGFP (Clontech Co.) was digested with Xho I and Nco I to become the "IRES" DNA insert. GL3basic vector (Promega Co.) digested with Xho I and Nco I. Xho I, Nco I digested with pGL3basic DNA was ligated with Xho I, Nco I digested IRES DNA insert using T4 DNA ligase (NEB Co.). The ligation mixture was transformed with E. coli DH5α constitutive cells to obtain the pGL3B / IRES-luciferase DNA plasmid (pGL3B / IRES-luciferase DNA plasmid). T / E ^{A +} clones containing SARS E gene with a second Alanine insertion mutant were digested with NcoI and XbaI to obtain SARS E gene DNA insert. The pGL3B / IRES-luciferase DNA plasmid was digested with NcoI and XbaI. Nco I, Xba I digested with pGL3B / IRES-luciferase DNA vector was ligated with Nco I, Xba I digested with SARS E ^{A +} gene DNA insert and became a pGL3B / IRES-E ^{A +} DNA plasmid.

III. pCMV -Induced M + E gene expression plasmid ( pCMV -driven gene expression plasmids (coronavirus-like particles ( Co rona V irus - L ike P article) ( CoVLP Expression vector)

The pGL3B / IRES-E ^{A +} DNA plasmid was digested with BamHI and XbaI to obtain a DNA insert containing the IRES and SARS E ^{A +} genes. The pCDNA3.1 + M DNA plasmid was digested with BamH I, Xba I. BamH I, Xba I results digested with pCDNA3.1 + M DNA plasmid were ligated with BamH I, Xba I digested with DNA inserts containing IRES and SARS E ^{A +} fused genes, resulting in pCDNA3.1 + M / IRES-E ^{A +} plasmid was obtained.

IV. Manipulation of the DNA Vector of the Invention

The pGEM (T) / SpikeCT (EcoRV / BstBI) plasmid contains a gene encoding a C-terminal heptad refit located on top of a region rich in aromatic residues parallel to the membrane portion of the spike protein of SARS corona virus, which is Digested with Spe I and filled-in with Klenow enzyme and dNTP. After separation on 1.2% agarose gel, DNA fragments were purified with a QIAquick gel extraction kit (QIAGEN. Com.) And further digested by BstB I. The resulting “spikeCT” gene fragment was used as DNA insert, ligated with pCDNA3.1 + M / IRES-E ^{A +} DNA vector plasmid digested with BsaB I, BstB I and purified by gel. After transformation into E. coli DH5 alpha cells, a so-called CoVLP-cloning vector of the present DNA vector was obtained. There are several restriction enzyme cleavage sites (multiple cloning sites, MCS), including SmaI, BsaB I, EcoR V, and BspE I, which are located at the beginning of the "SpikeCT" gene, which can be cloned or It can be used as a class I viral fusion protein gene in-frame fused with the SpikeCT gene in the vector of the invention, and the resulting DNA plasmid can then be used as a DNA vaccine candidate. A map of the CoVLP-cloning vector is illustrated in FIG. 1. CoVLP-cloning vector is as shown in SEQ ID NO: 49.

DNA sequence of CoVLP-cloning vector (pCDNA3.1 + M + IRES-E + spike CT) (SEQ ID NO: 49)

GACGGATCGG GAGATCTTCA ATATTGGCCA TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT ACGTTGTATC TATATCATAA TATGTACATT TATATTGGCT CATGTCCAAT ATGACCGCCA TGTTGGCATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTCCGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA CGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACACCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTGCGA TCGCCCGCCC CGTTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG TCAGATCACT AGAAGCTTTA TTGCGGTAGT TTATCACAGT TAAATTGCTA ACGCAGTCAG TGCTTCTGAC ACAACAGTCT CGAACTTAAG CTGCAGTGAC TCTCTTAAGG TAGCCTTGCA GAAGTTGGTC GTGAGGCACT GGGC AGGTAA GTATCAAGGT TACAAGACAG GTTTAAGGAG ACCAATAGAA ACTGGGCTTG TCGAGACAGA GAAGACTCTT GCGTTTCTGA TAGGCACCTA TTGGTCTTAC TGACATCCAC TTTGCCTTTC TCTCCACAGG TGTCCACTCC CAGTTCAATT ACAGCTCTTA AGGCTAGAGT ACTTAATACG ACTCACTATA GGCTAGCGCT ACCGGACTCA GATCATGGCA GATAACGGCA CTATTACTGT GGAGGAACTG AAACAACTGC TGGAACAATG GAACCTCGTA ATCGGCTTTC TCTTTCTGGC TTGGATTATG TTGTTACAGT TTGCGTATTC TAATCGTAAC CGTTTCCTCT ACATTATTAA GCTCGTTTTC CTGTGGTTGT TGTGGCCTGT AACTCTTGCT TGCTTTGTGC TTGCTGCTGT CTATCGTATC AACTGGGTTA CTGGTGGTAT TGCTATCGCT ATGGCTTGTA TTGTAGGCTT GATGTGGCTG TCTTATTTCG TTGCTTCTTT CCGTCTGTTT GCTCGTACTC GCTCTATGTG GTCCTTTAAT CCTGAGACTA ATATCCTGCT GAATGTTCCG CTCCGTGGTA CTATCGTTAC TAGACCGCTG ATGGAATCTG AACTGGTTAT TGGTGCCGTC ATTATCCGTG GTCATTTGCG TATGGCTGGT CACTCTCTGG GTCGTTGCGA TATTAAGGAT CTGCCAAAGG AAATCACTGT AGCCACTTCT CGTACTCTGT CTTACTATAA ACTCGGTGCA TCGCAACGTG TGGGAACTGA TTCGGGCTTC GCTGCGTATA ATCGTTATCG TATTGGCAAC TATAAACTGA ACACCGACCA CGCAGGCTCT AATGACAACA TCGCTCTCCT CGTTCAGTGA AATCACTAGT GCGGCCGCA G GTACCGAGCT CGGATCCGCC CCTCTCCCTC CCCCCCCCCT AACGTTACTG GCCGAAGCCG CTTGGAATAA GGCCGGTGTG CGTTTGTCTA TATGTTATTT TCCACCATAT TGCCGTCTTT TGGCAATGTG AGGGCCCGGA AACCTGGCCC TGTCTTCTTG ACGAGCATTC CTAGGGGTCT TTCCCCTCTC GCCAAAGGAA TGCAAGGTCT GTTGAATGTC GTGAAGGAAG CAGTTCCTCT GGAAGCTTCT TGAAGACAAA CAACGTCTGT AGCGACCCTT TGCAGGCAGC GGAACCCCCC ACCTGGCGAC AGGTGCCTCT GCGGCCAAAA GCCACGTGTA TAAGATACAC CTGCAAAGGC GGCACAACCC CAGTGCCACG TTGTGAGTTG GATAGTTGTG GAAAGAGTCA AATGGCTCTC CTCAAGCGTA TTCAACAAGG GGCTGAAGGA TGCCCAGAAG GTACCCCATT GTATGGGATC TGATCTGGGG CCTCGGTGCA CATGCTTTAC ATGTGTTTAG TCGAGGTTAA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT CCTTTGAAAA ACACGATGAT AATATGGCCA CAACCATGGC ATACTCTTTT GTGTCTGAGG AAACTGGCAC TCTGATCGTG AACTCTGTAC TGCTGTTTCT CGCTTTTGTG GTATTCCTGC TGGTCACTCT CGCTATCCTC ACTGCTCTTC GTCTGTGTGC CTACTGTTGT AATATCGTGA ACGTGTCTCT GGTTAAGCCT ACTGTGTATG TGTATTCTCG TGTGAAAAAT CTCAATTCTT CTGAAGGAGT TCCCGATCTG CTGGTCTAGA GGGCCCGTTT AAACCCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTT GCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGCTTC TGAGGCGGAA AGAACCAGCT GGGGCTCTAG GGGGTATCCC CACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT AATTCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG C TGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCTGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTCC CGGGAGCTTG TATATCCATT TTCGGATCTG ATCAAGAGAC AGGATGACTA GTGATTGATA TCTCCGGAAT CAATGCGAGC GTAGTGAACA TCCAGAAAGA GATTGACCGT TTGAATGAAG TTGCTAAAAA TCTGAATGAA CCTCTGATTG ACCTCCAAGA ACTCGGCAAA TATGAGCAAT ACATTAAATG GCCTTGGTAT GTCTGGTTGG GTTTCATCGC AGGTCTCATC GCTATCGTTA TGGTGACTAT TCTGCTGTGT TGTATGACTT CTTGTTGCTC TTGTCTGAAA GGTGCGTGTT CTTGTGGTTC TTGTTGTAAG TTTGATGAGG ATGATTCTGA GCCAGTTCTG AAGGGTGTGA AGCTGCATTA TACCTAGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC CACCCCAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTATC ATGTCTGTAT ACCGTCGACC TCTAGCTAGA GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTA A TGAGTGAGCT AACTCACATT AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA

<110> DNASHUTTLE Biopharm Co., Ltd. <120> AN EXPRESSION VECTOR ENCODING CORONAVIRUS-LIKE PARTICLE <130> NONE <140> US 10 / 855,490 <141> 2004-05-28 <160> 49 <170> PatentIn version 3.3 <210> 1 <211> 234 <212> DNA <213> SARS E gene <400> 1 atggcatact cttttgtgtc tgaggaaact ggcactctga tcgtgaactc tgtactgctg 60 tttctcgctt ttgtggtatt cctgctggtc actctcgcta tcctcactgc tcttcgtctg 120 tgtgcctact gttgtaatat cgtgaacgtg tctctggtta agcctactgt gtatgtgtat 180 tctcgtgtga aaaatctcaa ttcttctgaa ggagttcccg atctgctggt ctag 234 <210> 2 <211> 666 <212> DNA <213> SARS M gene <400> 2 atggcagata acggcactat tactgtggag gaactgaaac aactgctgga acaatggaac 60 ctcgtaatcg gctttctctt tctggcttgg attatgttgt tacagtttgc gtattctaat 120 cgtaaccgtt tcctctacat tattaagctc gttttcctgt ggttgttgtg gcctgtaact 180 cttgcttgct ttgtgcttgc tgctgtctat cgtatcaact gggttactgg tggtattgct 240 atcgctatgg cttgtattgt aggcttgatg tggctgtctt atttcgttgc ttctttccgt 300 ctgtttgctc gtactcgctc tatgtggtcc tttaatcctg agactaatat cctgctgaat 360 gttccgctcc gtggtactat cgttactaga ccgctgatgg aatctgaact ggttattggt 420 gccgtcatta tccgtggtca tttgcgtatg gctggtcact ctctgggtcg ttgcgatatt 480 aaggatctgc caaaggaaat cactgtagcc acttctcgta ctctgtctta ctataaactc 540 ggtgcatcgc aacgtgtggg aactgattcg ggcttcgctg cgtataatcg ttatcgtatt 600 ggcaactata aactgaacac cgaccacgca ggctctaatg acaacatcgc tctcctcgtt 660 cagtga 666 <210> 3 <211> 327 <212> DNA <213> SpikeCT gene <400> 3 gatatctccg gaatcaatgc gagcgtagtg aacatccaga aagagattga ccgtttgaat 60 gaagttgcta aaaatctgaa tgaacctctg attgacctcc aagaactcgg caaatatgag 120 caatacatta aatggccttg gtatgtctgg ttgggtttca tcgcaggtct catcgctatc 180 gttatggtga ctattctgct gtgttgtatg acttcttgtt gctcttgtct gaaaggtgcg 240 tgttcttgtg gttcttgttg taagtttgat gaggatgatt ctgagccagt tctgaagggt 300 gtgaagctgc attataccta gttcgaa 327 <210> 4 <211> 34 <212> DNA <213> E1L primer <400> 4 accatggcat actcttttgt gtctgaggaa actg 34 <210> 5 <211> 51 <212> DNA <213> E2L primer <400> 5 acagcagtac agagttcacg atcagagtgc cagtttcctc agacacaaaa g 51 <210> 6 <211> 51 <212> DNA <213> E3L primer <400> 6 tgaccagcag gaataccaca aaagcgagaa acagcagtac agagttcacg a 51 <210> 7 <211> 54 <212> DNA <213> E4L primer <400> 7 gcacacagac gaagagcagt gaggatagcg agagtgacca gcaggaatac caca 54 <210> 8 <211> 52 <212> DNA <213> E5L primer <400> 8 accagagaca cgttcacgat attacaacag taggcacaca gacgaagagc ag 52 <210> 9 <211> 58 <212> DNA <213> E6L primer <400> 9 gatttttcac acgagaatac acatacacag taggcttaac cagagacacg ttcacgat 58 <210> 10 <211> 62 <212> DNA <213> E7L primer <400> 10 tctagaccag cagatcggga actccttcag aagaattgag atttttcaca cgagaataca 60 ca 62 <210> 11 <211> 20 <212> DNA <213> E8L primer <400> 11 tctagaccag cagatcggga 20 <210> 12 <211> 22 <212> DNA <213> M1-1U primer <400> 12 tgatcatggc agataacggc ac 22 <210> 13 <211> 37 <212> DNA <213> M1U primer <400> 13 tgatcatggc agataacggc actattactg tggagga 37 <210> 14 <211> 49 <212> DNA <213> M1L primer <400> 14 gttccattgt tccagcagtt gtttcagttc ctccacagta atagtgccg 49 <210> 15 <211> 49 <212> DNA <213> M2L primer <400> 15 aagccagaaa gagaaagccg attacgaggt tccattgttc cagcagttg 49 <210> 16 <211> 50 <212> DNA <213> M3L primer <400> 16 agaatacgca aactgtaaca acataatcca agccagaaag agaaagccga 50 <210> 17 <211> 57 <212> DNA <213> M4L primer <400> 17 cgagcttaat aatgtagagg aaacggttac gattagaata cgcaaactgt aacaaca 57 <210> 18 <211> 56 <212> DNA <213> M5L primer <400> 18 caagagttac aggccacaac aaccacagga aaacgagctt aataatgtag aggaaa 56 <210> 19 <211> 55 <212> DNA <213> M6L primer <400> 19 ttgatacgat agacagcagc aagcacaaag caagcaagag ttacaggcca caaca 55 <210> 20 <211> 58 <212> DNA <213> M7L primer <400> 20 caagccatag cgatagcaat accaccagta acccagttga tacgatagac agcagcaa 58 <210> 21 <211> 56 <212> DNA <213> M8L primer <400> 21 aacgaaataa gacagccaca tcaagcctac aatacaagcc atagcgatag caatac 56 <210> 22 <211> 59 <212> DNA <213> M9L primer <400> 22 acatagagcg agtacgagca aacagacgga aagaagcaac gaaataagac agccacatc 59 <210> 23 <211> 20 <212> DNA <213> M10U primer <400> 23 tcctgctgaa tgttccgctc 20 <210> 24 <211> 65 <212> DNA <213> M10L primer <400> 24 gagcggaaca ttcagcagga tattagtctc aggattaaag gaccacatag agcgagtacg 60 agcaa 65 <210> 25 <211> 50 <212> DNA <213> M11L primer <400> 25 catcagcggt ctagtaacga tagtaccacg gagcggaaca ttcagcagga 50 <210> 26 <211> 60 <212> DNA <213> M12L primer <400> 26 accacggata atgacggcac caataaccag ttcagattcc atcagcggtc tagtaacgat 60                                                                           60 <210> 27 <211> 31 <212> DNA <213> M13L primer <400> 27 atacgcaaat gaccacggat aatgacggca c 31 <210> 28 <211> 45 <212> DNA <213> M14L primer <400> 28 aacgacccag agagtgacca gccatacgca aatgaccacg gataa 45 <210> 29 <211> 51 <212> DNA <213> M15L primer <400> 29 tacagtgatt tcctttggca gatccttaat atcgcaacga cccagagagt g 51 <210> 30 <211> 57 <212> DNA <213> M16L primer <400> 30 ccgagtttat agtaagacag agtacgagaa gtggctacag tgatttcctt tggcaga 57 <210> 31 <211> 58 <212> DNA <213> M17L primer <400> 31 cgaagcccga atcagttccc acacgttgcg atgcaccgag tttatagtaa gacagagt 58 <210> 32 <211> 58 <212> DNA <213> M18L primer <400> 32 cagtttatag ttgccaatac gataacgatt atacgcagcg aagcccgaat cagttccc 58 <210> 33 <211> 62 <212> DNA <213> M19L primer <400> 33 gagagcgatg ttgtcattag agcctgcgtg gtcggtgttc agtttatagt tgccaatacg 60 at 62 <210> 34 <211> 34 <212> DNA <213> M20L primer <400> 34 tcactgaacg aggagagcga tgttgtcatt agag 34 <210> 35 <211> 26 <212> DNA <213> SpikeCTup-EcoRV primer <400> 35 gatatctccg gaatcaatgc gagcgt 26 <210> 36 <211> 35 <212> DNA <213> DI-1U / BspE I primer <400> 36 tccggaatca atgcgagcgt agtgaacatc cagaa 35 <210> 37 <211> 48 <212> DNA <213> DI-1L primer <400> 37 gcaacttcat tcaaacggtc aatctctttc tggatgttca ctacgctc 48 <210> 38 <211> 45 <212> DNA <213> DI-2L primer <400> 38 atcagagatt cattcagatt tttagcaact tcattcaaac ggtca 45 <210> 39 <211> 51 <212> DNA <213> DI-3L primer <400> 39 tcatatttgc cgagttcttg gaggtcaatc agagattcat tcagattttt a 51 <210> 40 <211> 43 <212> DNA <213> DI-4L primer <400> 40 caaggccatt taatgtattg ctcatatttg ccgagttctt gga 43 <210> 41 <211> 50 <212> DNA <213> DI-5L primer <400> 41 acctgcgatg aaacccaacc agacatacca aggccattta atgtattgct 50 <210> 42 <211> 50 <212> DNA <213> DI-6L primer <400> 42 cagaatagtc accataacga tagcgatgag acctgcgatg aaacccaacc 50 <210> 43 <211> 50 <212> DNA <213> DI-7L primer <400> 43 agagcaacaa gaagtcatac aacacagcag aatagtcacc ataacgatag 50 <210> 44 <211> 50 <212> DNA <213> DI-8L primer <400> 44 accacaagaa cacgcacctt tcagacaaga gcaacaagaa gtcatacaac 50 <210> 45 <211> 50 <212> DNA <213> DI-9L primer <400> 45 gaatcatcct catcaaactt acaacaagaa ccacaagaac acgcaccttt 50 <210> 46 <211> 49 <212> DNA <213> DI-10L primer <400> 46 gcttcacacc cttcagaact ggctcagaat catcctcatc aaacttaca 49 <210> 47 <211> 35 <212> DNA <213> DI-11L primer <400> 47 cctaggtata atgcagcttc acacccttca gaact 35 <210> 48 <211> 27 <212> DNA <213> SpikeCTdn-BstB I primer <400> 48 ttcgaactag gtataatgca gcttcac 27 <210> 49 <211> 4800 <212> DNA <213> CoVLP cloning vector <400> 49 gacggatcgg gagatcttca atattggcca ttagccatat tattcattgg ttatatagca 60 taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt 120 tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac tagttattaa 180 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 240 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 300 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 360 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc 420 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 480 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 540 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 600 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 660 aaatgtcgta acaactgcga tcgcccgccc cgttgacgca aatgggcggt aggcgtgtac 720 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcact agaagcttta 780 ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct 840 cgaacttaag ctgcagtgac tctcttaagg tagccttgca gaagttggtc gtgaggcact 900 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 960 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac 1020 tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggctagagt 1080 acttaatacg actcactata ggctagcgct accggactca gatcatggca gataacggca 1140 ctattactgt ggaggaactg aaacaactgc tggaacaatg gaacctcgta atcggctttc 1200 tctttctggc ttggattatg ttgttacagt ttgcgtattc taatcgtaac cgtttcctct 1260 acattattaa gctcgttttc ctgtggttgt tgtggcctgt aactcttgct tgctttgtgc 1320 ttgctgctgt ctatcgtatc aactgggtta ctggtggtat tgctatcgct atggcttgta 1380 ttgtaggctt gatgtggctg tcttatttcg ttgcttcttt ccgtctgttt gctcgtactc 1440 gctctatgtg gtcctttaat cctgagacta atatcctgct gaatgttccg ctccgtggta 1500 ctatcgttac tagaccgctg atggaatctg aactggttat tggtgccgtc attatccgtg 1560 gtcatttgcg tatggctggt cactctctgg gtcgttgcga tattaaggat ctgccaaagg 1620 aaatcactgt agccacttct cgtactctgt cttactataa actcggtgca tcgcaacgtg 1680 tgggaactga ttcgggcttc gctgcgtata atcgttatcg tattggcaac tataaactga 1740 acaccgacca cgcaggctct aatgacaaca tcgctctcct cgttcagtga aatcactagt 1800 gcggccgcag gtaccgagct cggatccgcc cctctccctc ccccccccct aacgttactg 1860 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat 1920 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc 1980 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag 2040 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc 2100 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac 2160 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca 2220 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt 2280 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa 2340 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat 2400 aatatggcca caaccatggc atactctttt gtgtctgagg aaactggcac tctgatcgtg 2460 aactctgtac tgctgtttct cgcttttgtg gtattcctgc tggtcactct cgctatcctc 2520 actgctcttc gtctgtgtgc ctactgttgt aatatcgtga acgtgtctct ggttaagcct 2580 actgtgtatg tgtattctcg tgtgaaaaat ctcaattctt ctgaaggagt tcccgatctg 2640 ctggtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 2700 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2760 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2820 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2880 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 2940 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 3000 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 3060 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 3120 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 3180 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 3240 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 3300 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 3360 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 3420 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 3480 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3540 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 3600 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 3660 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 3720 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgacta 3780 gtgattgata tctccggaat caatgcgagc gtagtgaaca tccagaaaga gattgaccgt 3840 ttgaatgaag ttgctaaaaa tctgaatgaa cctctgattg acctccaaga actcggcaaa 3900 tatgagcaat acattaaatg gccttggtat gtctggttgg gtttcatcgc aggtctcatc 3960 gctatcgtta tggtgactat tctgctgtgt tgtatgactt cttgttgctc ttgtctgaaa 4020 ggtgcgtgtt cttgtggttc ttgttgtaag tttgatgagg atgattctga gccagttctg 4080 aagggtgtga agctgcatta tacctagttc gaaatgaccg accaagcgac gcccaacctg 4140 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 4200 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 4260 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 4320 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 4380 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 4440 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 4500 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 4560 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 4620 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 4680 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 4740 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 4800                                                                         4800  

Claims (18)

Iii) a first transcription unit comprising a membrane protein gene (M protein gene) of coronavirus, an envelope protein gene (E protein gene) of coronavirus and an internal ribosomal introduction site (IRES) sequence, wherein the IRES is the membrane protein gene A first transcription unit inserted into a junction of the envelope protein gene; Ii) a first eukaryotic promoter coupled to said membrane protein gene, said first eukaryotic promoter positioned above said M protein gene and inducing expression of said first transcription unit; Iii) a second transcription unit comprising a multiplicity of cloning sites (MCS) for in-frame insertion or cloning of the spike CT gene of coronavirus and the viral fusion protein gene of class I, wherein the MCS is the beginning of the spike CT gene A second transcription unit located in the portion and having a restriction enzyme cleavage site; And Iii) a second eukaryotic promoter coupled to act on said SpikeCT gene, said second eukaryotic promoter located on top of said SpikeCT gene and inducing expression of a second transcriptional unit, The electronic activity of the first eukaryotic promoter is a stronger expression vector than the second eukaryotic promoter, An expression vector for cloning a viral fusion protein gene of class I as a DNA vaccine candidate. According to claim 1, The coronavirus is a pig, human or avian coronavirus expression vector. According to claim 1, The coronavirus is a swine TGEV coronavirus, human 229E corona virus or human SARS virus. According to claim 1, The coat protein gene is an expression vector having the sequence shown in SEQ ID NO: 1. According to claim 1, The membrane protein gene is an expression vector having the sequence shown in SEQ ID NO: 2. According to claim 1, The Spike CT gene has an sequence shown in SEQ ID NO: 3. According to claim 1, An expression vector further comprising an origin of replication for the propagation plasmid in the host cell. According to claim 1, An expression vector further comprising an antibiotic-resistant gene as a selection marker. According to claim 1, An expression vector wherein the first and second transcription units have a polyA signal. The method of claim 9, Wherein said polyA signal is a BGH polyA signal. According to claim 1, Said vector having the sequence set forth in SEQ ID NO: 49; According to claim 1, Wherein said Spike CT gene encodes a C-terminal heptad refit located on top of a region rich in aromatic residues parallel to the transmembrane segment of a spike protein of coronavirus. According to claim 1, Wherein said plurality of cloning sites comprises a restriction site selected from the group consisting of SmaI, BsaB I, EcoR V and BspE I. According to claim 1, The class I viral fusion protein gene is an expression vector selected from the group consisting of gp160 of HIV, HA of influenza virus, and spike of SARS virus. According to claim 1, The first eukaryotic promoter is selected from the group consisting of CMV, SV40, RSV, HIV-1 LTR, hybrid beta-actin / CMV promoter enhancer, muscle specific desmin, creatine kinase, beta-actin promoter, EF1alpha, ubiquitin promoter vector. According to claim 1, The first eukaryotic promoter is an expression vector selected from the group consisting of pCMV, hybrid beta-actin / CMV promoter enhancer and beta-actin promoter. According to claim 1, The second eukaryotic promoter in the group consisting of CMV, SV40, RSV, HIV-1 LTR, hybrid beta-actin / CMV promoter enhancer, muscle-specific desmin, creatine kinase, beta-actin promoter, EF1alpha, ubiquitin promoter Selected Expression Vector. According to claim 1, The second eukaryotic promoter is an expression vector selected from the group consisting of pCMV, SV40, beta-actin and EF1 alpha promoter.

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