KR101457977B1 - Recombinant cell expressing capsid protein of swine hepatitis E virus - Google Patents

Abstract

The present invention relates to a recombinant cell expressing a capsid protein of a porcine E hepatitis virus, which produces a capsid protein very similar to the infection state of a porcine hepatitis E virus, which is difficult to artificially cultivate in a laboratory, The capsid protein can be used in a final confirmation antibody test method in view of the characteristics that can be usefully used as a vaccine and difficult to artificially cultivate the virus, and thus it is very useful for accurate disease diagnosis.

Description

Recombinant cell expressing capsid protein of swine hepatitis E virus.

The present invention relates to a recombinant cell expressing a capsid protein of a porcine hepatitis E virus.

Swine hepatitis E virus (swHEV) is a non-enveloped virus with a positive strand of about 7.5 kb and a single-stranded RNA genome. It belongs to the genus Hepeviridae and Hepevirus.

The gene of hepatitis virus (HEV) is composed of three ORFs (Open reading frame), of which ORF1 encodes an unstructured protein and ORF2 encodes a capsid protein. ORF3 encodes a protein whose function has not yet been clearly identified.

In the past, diseases caused by hepatitis virus were recognized to occur only in India, China and other developing countries. However, in recent years, the number of patients infected with hepatitis virus has increased in advanced countries such as the United States, Japan, and Europe. In addition, a survey of antibodies against hepatitis virus in the serum of Korean people showed that about 18% of the subjects had antibodies to hepatitis virus. As a result, Of the infected person.

The first case of hepatitis E virus isolated from pigs in the United States in 1997 and the genetic linkage between hepatitis E virus isolated from humans has shown that both viruses have very similar nucleic acid and amino acid sequences. Based on the data, it was confirmed that infection of hepatitis E virus isolated from humans with pigs and infection of primate with hepatitis E virus isolated from pigs resulted in interspecific cross infection. In addition, it has been confirmed that antibodies against the hepatitis E virus are highly present in sera of wild animals such as pigs and cats as well as pets such as dogs and rats, and most of the animals are already infected with hepatitis virus It has been recognized that the hepatitis E virus is highly likely to be transmitted to humans through animals as it is discovered.

It is estimated that the probability of infection through contact with pigs, especially animals, is very high because the viruses of the pigs and humans are close to the phylogenetic class, and veterinarians who frequently come into contact with pigs, Of the patients with hepatitis B virus antibodies were significantly higher than those of the control group.

Hepatitis virus is not a clinical symptom when it is infected with pigs, but when it is infected with human, it shows acute hepatitis symptoms, especially when it is infected with pregnant woman, it also leads to death.

Meanwhile, pig hepatitis E virus has been reported as a common infectious disease and it is an important cause of public health. In order to prevent the spread of the virus from animals to humans, it is urgent to develop a preventive vaccine to be applied to animals such as pigs. . Although a vaccine for the prevention of phosphorylation of the hepatitis E virus, which is a common infectious disease, is being developed in an artificial expression form in insect cells or Escherichia coli, there has not been developed a vaccine against animals for the prevention of animal E hepatitis virus. In addition, enzyme immunoassay (ELISA) has been used for antibody testing in infected animals, but there is no method available for the final confirmation antibody test because of the inability to carry out viral artificial cultivation.

The inventors of the present invention completed the present invention by preparing a cell line expressing ORF2 (open reading frame 2) protein of porcine hepatitis E virus and confirming that the cell line expresses the ORF2 protein.

It is an object of the present invention to provide a lentiviral vector comprising a gene coding for an ORF2 (open reading frame 2) protein which is a capsid protein of porcine E virus.

Another object of the present invention is to provide a recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector.

Another object of the present invention is to provide a vaccine composition comprising an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein.

It is still another object of the present invention to provide a porcine hepatitis E virus detection kit comprising an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein.

It is another object of the present invention to provide a method for detecting porcine E hepatitis virus in an infected or infected cell through an antigen-antibody reaction using an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein have.

In order to achieve the above object, there is provided a lentivirus vector comprising a gene coding for an ORF2 (open reading frame 2) protein which is a capsid protein of a porcine E type virus of the present invention.

The present invention also provides a recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector.

The present invention also provides a vaccine composition comprising an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein.

The present invention also provides a porcine hepatitis E virus detection kit comprising an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein.

The present invention also provides a method for detecting porcine hepatitis E virus in a cell infected or infected through an antigen-antibody reaction using an ORF2 protein expressed by a recombinant animal cell comprising a gene encoding an ORF2 protein.

The present invention can be used as a vaccine against porcine E hepatitis virus, producing a capsid protein very similar to the infection state of porcine E hepatitis virus, which is difficult to artificially cultivate in a laboratory, and considering the difficult characteristics of artificial cultivation of virus , The capsid protein can be used in a final confirmation antibody test method and is very useful for accurate disease diagnosis.

1 shows a method for producing a lentiviral expression vector and an expressing cell (swHEV-ORF2-LPK15) expressing the ORF2 gene of porcine hepatitis E virus (swHEV).
FIG. 2 shows the expression of a gene encoding the ORF2 protein of a porcine hepatitis E virus using RT-PCR. [Lane 1: swHEV-ORF2-LPK15 (parent cell line, lane 2: swHEV- ORF2-LPK15 clone 1D7; lane 3: swHEV-ORF2-LPK15 clone 1H4]
FIG. 3 shows the expression of ORF2 protein in porcine hepatitis E virus using indirect fluorescent antibody method (Fig. 3 (A): swHEV-ORF2-LPK15 clone 1D7 and Fig. 3 (B): swHEV-ORF2-LPK15 clone 1H4].
4 shows the expression of ORF2 protein in porcine hepatitis E virus using Western blotting (Fig. 4 (A); Lane 1: swHEV-ORF2-LPK15 cells before single cell cloning; Lane 2: porcine E type ORF2 protein fused with insect cell expression 6X histidine (positive control); 4 (B); Lane 1: negative control cell line PK-15; Lane 2: swHEV-ORF2-LPK15 clone 1D7; Lane 3: swHEV-ORF2-LPK15 clone 1H4]

The present invention provides a lentivirus vector comprising a gene encoding an ORF2 (open reading frame 2) protein which is a capsid protein of a porcine E type virus.

Hereinafter, the present invention will be described in more detail.

The lentiviral vector according to the present invention is characterized in that it comprises a gene encoding the ORF2 protein which is a capsid protein of the porcine E-type virus.

The gene encoding the OFR2 protein is composed of the DNA sequence of SEQ ID NO: 3, and the protein expressed by the gene consists of the amino acid sequence of SEQ ID NO: 4.

In the present invention, the gene encoding the ORF2 protein of the Chinese isolate virus is artificially synthesized, and then the gene is cloned. The gene encoding the ORF2 protein is cloned and inserted into a lentivirus vector. The recombinant lentiviral vector was transfected into a PK-15 cell line derived from the kidney of a pig, and the cell transfected with the lentiviral vector was cloned into a single cell line to obtain a single cell-derived cell line (swHEV-ORF2-LPK15 cell line clone 1D7, clone1H4) was finally selected. Among these single cell lines, the swHEV-ORF2-LPK15 cell line clone 1D7 was deposited with Korea Research Institute of Bioscience and Biotechnology on July 24, 2012 and received the accession number KCTC 12251BP.

In order to confirm that the cell line expresses the ORF2 protein, RT-PCR, indirect fluorescent antibody method and Western blotting were performed. As a result, ORF2 protein was expressed in both swHEV-ORF2-LPK15 clone 1D7 and swHEV-ORF2-LPK15 clone 1H4 . The ORF2 protein is a protein expressed in eukaryotic cells, and can be usefully used as a vaccine against animals including humans, in addition to the transcription or post-transcriptional process different from the ORF2 protein expressed in the prokaryote E. coli. It can be used for the confirmation antibody test method and is very useful for accurate disease diagnosis.

The present invention also provides a recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector.

The recombinant animal cell may be a cell derived from a porcine kidney cell.

The recombinant animal cell may be swHEV-ORF2-LPK15 (Accession No: KCTC1225BP) cells or swHEV-ORF2-LPK15 1H4 cells.

The recombinant animal cell comprises the gene of SEQ ID NO: 3 and is characterized by expressing a protein consisting of the amino acid sequence of SEQ ID NO: 4.

In addition, the present invention provides a vaccine composition for preventing porcine hepatitis comprising the ORF2 protein expressed by the recombinant cells.

The ORF2 protein according to the present invention is a protein expressed in porcine kidney cells and is a protein directly expressed in the host cell of the porcine E type virus, so that the ORF2 protein is more effective than the conventional vaccine.

The vaccine of the present invention further comprises a pharmacologically acceptable carrier or diluent. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carrier include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements, such as those based on Ringer ' s dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant. The adjuvant includes any absorption-promoting agent which refers to a compound or mixture that promotes an immune response and / or promotes the rate of absorption after inoculation. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyvalent anions, peptides, oils or hydrocarbon emulsions, , But not limited to, di-nitrophenol, and the like. The preferred adjuvant is the METASTIM adjuvant of Fort Dodge Animal Health.

The dosage of the vaccine composition for the purposes of the present invention is from 0.01 ml to 1 ml per kg of body weight, preferably from 0.2 ml to 0.4 ml per kg of body weight.

The vaccine composition of the present invention may be administered through at least one route selected from the group consisting of muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, ocular, and brain.

The present invention also provides a diagnostic kit for porcine hepatitis E virus comprising an ORF2 protein expressed by a recombinant animal cell expressing an ORF2 protein.

The diagnostic kit may be prepared using a method commonly used in the art.

These diagnostic kits include ORF2 proteins expressed in recombinant cells expressing the ORF2 protein as well as tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling agents capable of producing a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

The present invention also provides a method for detecting a porcine E-type virus, characterized in that a porcine E-type virus is detected in an infected or infected cell using an ORF2 protein expressed by a recombinant cell expressing the ORF2 protein.

The antigen-antibody reaction can be assayed by immunohistochemical staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, (Complement Fixation Assay), Fluorescence-activated cell sorter (FACS) and protein chip analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that the following examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

[ Example 1] Preparation of lentivirus vector containing gene encoding capsid protein ( ORF2 )

One. Pig E hepatitis virus ( swHEV )of The capsid protein (ORF2) gene Cloning .

The gene encoding the ORF2 protein of the Chinese isolate virus was synthesized and cloned into the pGEM-T Easy vector (Promega). The nucleotide sequence analysis was performed using a dye terminator cycle sequencing kit (PD-Biosystem) (ABI) was used.

More specifically, the gene coding for ORF2, a capsid protein of swine hepatitis virus (swHEV), contains ATG as an expression initiation codon, inserts an Xho I restriction enzyme site on the Bam H restriction enzyme site and a terminal region, (TAG) located immediately in front of the I-acting site. PCR was performed using the starting and terminating sites as primers, and the amplified DNA was cloned into pGEMT-Easy vector (Promega). The sequences used as primers are shown in Table 1 below, and the gene sequences and amino acid sequences encoding the capsid proteins of the cloned pig E hepatitis virus are shown in SEQ ID NOS: 3 and 4, respectively.

Forward primer 5`-GGATCCATGGCGGTTTCACCGGCCCCCG-3`  SEQ ID NO: 1 Reverse primer 5`-CTCGAGCTACCAGGGAGCGCGGAAAGCG-3`  SEQ ID NO: 2

2. ORF2  Comprising a gene encoding a protein Lentivirus  vector( Lenti  pLEX HEV - ORF2 ) Produce.

To prepare a lentiviral vector for expressing the protein, a lentivirus vector (LentiORF pLEX-MCS, Thermo Scientific Open Biosystems, USA) for inserting a gene encoding the ORF2 protein of swine E virus (swHEV) was used .

After cloning the gene encoding the ORF2 protein was treated in turn with BamH and Xho I restriction enzymes, only the region corresponding to the capsid (ORF2) was purified from the gene region of the hepatitis virus (swHEV). The expression vector Lenti pLEX-MCS vector was also purified by treating the same restriction enzyme. A recombinant expression vector, Lenti pLEX HEV-ORF2 vector, was prepared by ligation of the gene encoding the purified capsid protein with Lenti pLEX-MCS. The structure of the prepared vector is shown in Fig.

[ Example  2] Pig E hepatitis virus ( swHEV )of Capsid  Protein ORF2 Expressing swHEV - ORF2 - LPK15  Cell line clone  One D7  And swHEV - ORF2-LPK15 clone1H4  Produce.

In order to prepare a cell line expressing the capsid protein of the hepatitis E virus, the recombinant lentivirus prepared in Example 1 was transfected into a PK-15 cell line (derived from pig kidney cells) The capsid protein (ORF2) gene of hepatitis E virus (swHEV) was inserted.

More specifically, the recombinant lentivirus was inserted into 293T cells using Lipofectamine Plus (Invitrogen, USA) at a ratio of 1: 1: 1 to the Lenti pLEX HEV-ORF2 vector, an external protein expression vector, Lt; / RTI > After that, the cell culture supernatant was filtered with a 0.45 μm membrane filter (Nalgene, USA), stored at -70 ° C., and then used. The recombinant lentivirus was transfected into PK-15 cell line, and the cells were cultured in DMEM supplemented with 10% fetal calf serum and 10% fetal bovine serum (DMEM) to prepare a capsular protein (ORF2) permanent expression cell line of swine hepatitis virus (swHEV) (Puromycin, InvivoGen, USA) and antibiotics (Antibioctic-antimycotic) for 2 weeks at 37 ℃. Cultured cells surviving the 2 weeks of puromycin treatment were subjected to limiting dilution of the culture at 10 ⁵ to 10 ⁶ on a 96-well culture plate and further cultured for 2 weeks to complete single cell cloning. (SwHEV-ORF2-LPK15 cell line clone 1D7, clone 1H4 (SEQ ID NO: 2)) was identified in a variety of ways to confirm the expression of the capsid protein (ORF2) ), And clone 1D7 of swHEV-ORF2-LPK15 cell line among the single cell line was deposited with Korea Research Institute of Bioscience and Biotechnology on Jul. 24, 2012 and received KCTC 12251BP.

[ Example  3] swHEV - ORF2 - LPK15 clone  One D7  And swHEV - ORF2 - LPK15 clone1H4 Of Pig E hepatitis virus ORF2  Confirmation of protein expression.

One. RT - PCR Using ORF2  Identification of gene expression encoding proteins.

In order to confirm the expression of the ORF2 protein at the gene level, a primer site in which a part (289 bp) of a gene encoding the porcine E hepatitis virus ORF2 protein was amplified was selected with reference to the thesis. Respectively.

Forward primer 5`-GCTCACGTCATCTGTCGCTGCTGG-3` SEQ ID NO: 5 Reverse primer 5`-GGGCTGAACCAAAATCCTGACATC-3` SEQ ID NO: 6

The swHEV-ORF2-LPK15 cell line (parent (before cloning of single cell line), clone 1D7, Clone 1H4) was cultured in a thermostat at 37 ° C for 5 days, harvested and total RNA was extracted, RT-PCR (Qiagen) was performed. At 50 캜, a reverse transcription step was performed for 30 minutes. After reverse transcription was inactivated at 95 ° C for 15 minutes, the reaction was repeated 35 times in one cycle at 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 30 seconds, and finally The reaction was carried out at 72 ° C for 10 minutes and amplified. The results are shown in FIG.

As shown in FIG. 2, it was confirmed that a gene encoding ORF2 protein was expressed in all cell lines.

This indicates that ORF2 was normally transduced in all of the above cell lines and that a gene encoding the ORF2 protein was expressed at the mRNA level.

2. Indirect fluorescent antibody method ( indirect immuno 여광체 assay , IFA ) ORF2  Confirmation of protein expression

The swHEV-ORF2-LPK15 cell line (clone 1D7 and clone 1H4) was cultured in a puromycin selective medium to confirm the immunological reactivity of the expressed protein, and on day 5, the expressing cells were fixed with 100% cold acetone for 10 minutes. Thereafter, a monoclonal antibody of the ORF2 protein of hepatitis E virus was reacted, and a recombinant pig E (FITC), which expresses a specific reaction using anti-mouse IgG (KPL, USA) conjugated with FITC (Fluorescein isothiocyanate Flash In the Can) The expression of the hepatitis virus (swHEV) ORF2 protein was confirmed and the results are shown in Fig.

As shown in FIG. 3, it was confirmed that the ORF2 protein was expressed in both swHEV-ORF2-LPK15 clone 1D7 (Accession No .: KCTC 12251BP) and clone 1H4, but swHEV-ORF2-LPK15 clone 1D7 (accession number: KCTC 12251BP) By showing strong fluorescence, it was confirmed that the ORF2 protein was released in a large amount.

3. Western Blotting  Used OFR  Confirmation of protein expression

The HEV-LPK15 cell line containing the gene coding for ORF2 was cultured for 5 days, and the cells were harvested and cultured in a RIPA (Rapid Immunofilter Paper Assay) buffer (1% SDS, 10 mM Tris (pH 7.5), 300 mM NaCl , 0.5% NP-40), disrupted by ultrasonic waves, and then heat-treated for 10 minutes. Electrophoresis was carried out using this as a sample. After electrophoresis, the gel was transferred to a nitrocellulose filter, treated with a blocking solution containing 10% skim milk, and reacted with the ORF2 protein-specific monoclonal antibody of hepatitis E virus. After washing the cellulose filter, an AP (Alkaline Phosphatase) conjugate (anti-mouse AP (KPL, USA) for monoclonal antibody antibody was reacted and washed.

The presence of ORF2 was confirmed by treatment with BCIP / NBT (bromochloroindolyl phosphate / nitrobluetetrazolium) as a coloring agent, and the results are shown in FIG.

As shown in FIG. 4, ORF2 was not detected in PK-15 derived from porcine kidney cells in which ORF2 was not used as a negative control. However, in both swHEV-ORF2-LPK15 clone 1D7 and swHEV-ORF2-LPK15 clone 1H4, ORF2 protein of about 56 to 58 KDa was identified.

As a result, it was confirmed that the gene encoding the ORF2 protein was transduced in both the swHEV-ORF2-LPK15 clone 1D7 and swHEV-ORF2-LPK15 clone1H4 cell lines and that the transgene was expressed in animal cells.

Korea Biotechnology Research Institute KCTC12251BP 20120724

<110> Animal, plant and fisheries quarantine and inspection agency <120> Recombinant cell expressing capsid protein of swine hepatitis E          virus <130> P-1-29 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> it is foward primer for DNA sequence encoding ORF2 protein <400> 1 ggatccatgg cggtttcacc ggcccccg 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for DNA sequence encoding ORF2 protein <400> 2 ctcgagctac cagggagcgc ggaaagcg 28 <210> 3 <211> 1737 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence encoding ORF2 protein <400> 3 ggatccatgg cggtttcacc ggcccccgac acagctcccg taccagacgt tgactcgcgg 60 ggtgctatcc tgcgccggca gtacaacctt agcaccagtc cgcttacttc atctgtcgct 120 gctggtacga acctggttct ctatgccgcc cctctgaacc ctctcttgcc cctccaggat 180 ggcaccaaca cgcatattat ggctactgag gcgtctaatt acgctcagta tcgggttgta 240 cgagccacga tccgctatcg accactggtg ccaaacgctg ttggtggcta tgcaatctct 300 atttctttct ggcctcaaac tacgaccacc cccacctcag ttgacatgaa cagtattacc 360 tccaccgatg tccgcatttt ggttcagccc ggtattgcct ccgaattggt gatccctagt 420 gagcgcctcc attatcgcaa tcaaggctgg agatctgtag aaaccacagg ggtggccgag 480 gaggaagcta catccgggct ggtcatgctt tgtattcacg ggtctcctgt taactcctat 540 accaacacac catacaccgg tgccctgggg ctcttagatt ttgcattaga gcttgaattc 600 caggacgag catagactgc gcagaggtgc tgatgggact gcagagctta ccaccacagc agctacaagg 720 ttcatgaagg acttgcactt caccggcaca aatggcgttg gtgaggtggg tcgcggcata 780 gctctgacac tgtttaacct tgctgacacc cttcttggtg ggttaccgac agaattgatt 840 agttccgccg ggggacaact gttttactcc cgccctgtcg tctctgccaa cggcgagccg 900 acggttaagc tctacacatc tgttgaaaat gcgcagcagg acaagggcat aaccatccca 960 cacgacattg acctgggcga ctcacgtgtg gttattcagg attacgacaa tcagcacgag 1020 caagaccgac ctactccaag tccagccccc tctagacctt tctcagtcct acgcgccaat 1080 gacgttctgt ggctctccct caccgccgct gagtacgatc agactacata tgggtcgtcc 1140 accaacccca tgtacgtctc cgacacggtc actctagtca atgtggccac tggcgcacag 1200 gctgttgcac gctctcttga ttggagcaaa gtcactctgg acggccgccc cctgactacc 1260 attcagcagt atagcaagac attctacgtg ctcccgctca gggggaagct gtccttttgg 1320 gaggctggca ccactaaagc cggctaccca tacaattaca ataccaccgc ttcggatcag 1380 attctcattg agaacgcggc tggccacagg gttgctatct ctacctatac caccagcttg 1440 ggtgccggcc ctaccagcat atccgccgtt ggtgtgctag ccccacacag tgcactcgcc 1500 gtccttgagg atacggttga ctaccccgca cgtgctcaca ctttcgatga tttctgccct 1560 gaatgcagaa cacttggact tcagggttgt gcattccaga gcactattgc tgaactgcag 1620 cggcttaaaa tgaaggtagg gaaaacccgg gagagctaac tcatcccctt tgtgccccct 1680 tcatgacttc ccctcgttct ttttcttatt agcgctttcc gcgctccctg gctcgag 1737 <210> 4 <211> 577 <212> PRT <213> Artificial Sequence <220> <223> it is amino sequence of ORF2 protein <400> 4 Met Ala Val Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp   1 5 10 15 Ser Arg Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro              20 25 30 Leu Thr Ser Ser Val Ala Ala Gly Thr Asn Leu Val Leu Tyr Ala Ala          35 40 45 Pro Leu Asn Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile      50 55 60 Met Ala Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala  65 70 75 80 Thr Ile Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala                  85 90 95 Ile Ser Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val             100 105 110 Asp Met Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro         115 120 125 Gly Ile Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg     130 135 140 Asn Gln Gly Trp Arg Ser Val Glu Thr Thr Gly Val Ala Glu Glu Glu 145 150 155 160 Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn                 165 170 175 Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe             180 185 190 Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr         195 200 205 Arg Val Ser Arg Tyr Thr Ser Thr Ala Arg His Arg Leu Arg Arg Gly     210 215 220 Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met 225 230 235 240 Lys Asp Leu His Phe Thr Gly Thr Asn Gly Val Gly Glu Val Gly Arg                 245 250 255 Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly             260 265 270 Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser         275 280 285 Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr     290 295 300 Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp 305 310 315 320 Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln                 325 330 335 His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe             340 345 350 Ser Val Leu Arg Ala Asn Val Leu Trp Leu Ser Leu Thr Ala Ala         355 360 365 Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val     370 375 380 Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val 385 390 395 400 Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu                 405 410 415 Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg             420 425 430 Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro         435 440 445 Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala     450 455 460 Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala 465 470 475 480 Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala                 485 490 495 Leu Ala Val Leu Glu Asp Thr Val Asp Tyr Pro Ala Arg Ala His Thr             500 505 510 Phe Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys         515 520 525 Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val     530 535 540 Gly Lys Thr Arg Glu Ser *** Leu Ile Pro Phe Val Pro Pro Ser *** 545 550 555 560 Leu Pro Leu Val Leu Phe Leu Ile Ser Ala Phe Arg Ala Pro Trp Leu                 565 570 575 Glu     <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer sequence for identifying mRNA expressed by          DNA sequece coding ORF2 protein <400> 5 gctcacgtca tctgtcgctg ctgg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer sequence for identifying mRNA expressed by          DNA sequece coding ORF2 protein <400> 6 gggctgaacc aaaatcctga catc 24

Claims (12)

delete Wherein the gene coding for the ORF2 (open reading frame 2) protein which is a capsid protein of the porcine E-type virus comprises the nucleotide sequence shown in SEQ ID NO: 3. Wherein the ORF2 protein, which is the capsid protein of the pig E-type virus, is composed of the amino acid sequence shown in SEQ ID NO: 4. delete delete Wherein the recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector is swHEV-ORF2-LPK15 (accession number: KCTC1225BP) cells. Wherein the recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector comprises the nucleotide sequence shown in SEQ ID NO: 3. Wherein the recombinant animal cell expressing the ORF2 protein of the porcine E type virus transduced by the lentiviral vector comprises the amino acid sequence shown in SEQ ID NO: 4. 9. A vaccine composition for preventing porcine hepatitis E comprising the ORF2 protein expressed by a recombinant animal cell according to any one of claims 6 to 8. 9. A kit for the diagnosis of porcine hepatitis E virus using ORF2 protein expressed by a recombinant animal cell according to any one of claims 6 to 8. Characterized in that the ORF2 protein expressed by the recombinant animal cells according to any one of claims 6 to 8 is used to detect a porcine E virus in an infected or infected cell through an antigen- A method for detecting hepatitis E virus. The method of claim 11, wherein the antigen-antibody reaction is selected from the group consisting of tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay Characterized in that the method is carried out using at least one method selected from the group consisting of Assay, Complement Fixation Assay, Fluorescence-activated Cell Sorter (FACS) and Protein Chip Assay A method for detecting hepatitis virus.

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