KR20100043714A - Capsid protein and the gene of swine hepatitis e virus - Google Patents

Abstract

PURPOSE: A capsid protein of swine hepatitis E virus(HEV) and a gene encoding the same are provided to develop diagnosis and vaccine for HEV. CONSTITUTION: A swine hepatitis E virus(HEV) ORF2 capsid protein contains an amino acid of sequence number 7. A gene encoding the swine HEV ORF2 capsid protein ahs a sequence of sequence number 6. A vector pswHEV-capsid contains a gene encoding the protein. A transformed microorganism is obtained by introducing the vector pswHEV-capsid to host cell of bacteria, fungi, or yeast. The host cell is E.coli. The transformed microorganism is deposit number KCTC18139P.

Description

Capsid Protein and the Gene of Swine Hepatitis E Virus of Porcine Hepatitis E Virus

The present invention relates to a capsid protein of swine hepatitis E virus, a gene encoding the same, and a vector containing the gene.

Swine hepatitis E virus (swHEV) is a non-enveloped virus that has a positive-strand, single-stranded RNA genome of about 7.5 kb and belongs to the Hepeviridae family and Hepevirus by lineage classification (Emerson et al ., Virus taxonomy VIIIth report of the ICTV, 851, 2004; Tam et al ., Virology, 185: 120, 1991; Reyes et al ., Gastroenterol Jpn, 26: 142, 1991).

The HEV gene consists of three open reading frames (ORFs), of which ORF1 encodes a nonstructural protein and ORF2 encodes a capsid protein. ORF3 is known to encode a protein whose function is not yet clearly identified (Mushahwar et al ., Hepatitis E virus: Molecular biology and diagnosis.Eur J Gastroenterol Hepatol, 8: 312, 1996; Reyes et al ., Hepatitis E virus (HEV): The novel agent responsible for enterically-transmitted non-A, non-B hepatitis.Gastroenterol Jpn 26: 142, 1991).

In the past, it was recognized that diseases caused by HEV occurred only in India, China and other developing countries (Velazzquez et al. , Epidemic transmission of enterically transmitted non-A, non-B hepatitis in Mexico, 1986-1987. 263: 3281, 1990). Recently, however, the number of patients with HEV is increasing in advanced countries such as the United States, Japan, and many other European countries. In addition, the survey of HEV antibodies present in the serum of the general population of Korea showed that about 18% of people possess HEV antibodies, which proves that HEV is also seriously infected by Korean people. (Choi et al ., Identification of swine hepatitis E virus (HEV) and prevalence of anti-HEV antibodies in swine and human populations in Korea.J Clin Microbiol, 41: 3602, 2003).

In 1997, hepatitis E virus was isolated from pigs in the United States for the first time, and genetic association with hepatitis E virus isolated from humans revealed that the two viruses possess very similar nucleic acid and amino acid sequences (Meng). et al. , A novel virus in swine is closely related to the human hepatitis E virus.Proc Natl Acad Sci USA 94: 9860, 1997).

Based on these data, experiments were conducted in which hepatitis E virus isolated from humans was infected with pigs and, conversely, hepatitis E virus isolated from pigs was infected with primates. Meng et al. , Genetic and experimental evidence for cross-species infection by swine hepatitis E virus, J Virol 72: 9714, 1998).

In addition, it was confirmed that antibodies to hepatitis E virus were very high in the sera of wild rats and mice as well as pets such as domestic animals such as pigs and cows, and most animals were already infected with HEV. It is recognized that hepatitis E virus is very likely to be transmitted to humans through animals (Arankalle et al. , Prevalence of anti-hepatitis E virus antibodies in different Indian animal species, J Viral Hepat 8: 223, 2001; Vitral et al ., Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil, Mem Inst Oswaldo Cruz 100: 117, 2005). It is estimated that the probability of infection among animals is particularly high through contact with pigs because the HEVs of pigs and humans are systematically close (Meng et al. , A novel virus in swine is closely related to the human hepatitis E virus.Proc Natl Acad Sci USA 94: 9860, 1997). Because it is.

HEV has no clinical symptoms when infected with swine, but acute hepatitis symptoms when infected with human pigs, and death, especially when infected with pregnant women (Madan et al ., Detection of hepatitis C and E genomes in sera). of patients with acute viral hepatitis and fulminant hepatitis by their simultaneous amplication in PVR.J Gastroenterol Hepatol 13: 125, 1998). Therefore, it is very urgent to establish a standardized diagnosis method to identify the domestic swine HEV infection status of swine HEV, which is recognized as a cause of new common infectious diseases.

Accordingly, the present inventors have made intensive efforts to isolate and secure the above-described pig hepatitis E virus protein and genes encoding the same, thereby separating the pig capsid protein and gene from pig feces to complete the present invention.

After all, it is an object of the present invention to provide a pig HEV ORF2 capsid protein of SEQ ID NO: 7 and a gene encoding the same.

Another object of the present invention is to provide a vector containing the HEV ORF2 gene and a transformed microorganism obtained by introducing the same into a host cell selected from the group consisting of bacteria, fungi and yeast.

Another object of the present invention to provide a HEV antibody produced using a capsid protein obtained from the transforming microorganism and a HEV preventive vaccine produced using the same.

In order to achieve the above object, the present invention provides a swine HEV ORF2 capsid protein of SEQ ID NO: 7.

The present invention also provides a gene encoding the pig HEV ORF2 capsid protein, wherein the gene may be characterized by having a nucleotide sequence of SEQ ID NO: 6.

The present invention also provides a vector containing the gene, wherein the vector may be pswHEV-Capsid.

The present invention also provides a transformed microorganism obtained by introducing the expression vector into a host cell selected from the group consisting of bacteria, fungi and yeasts.

In the present invention, the host cell may be characterized in that E. coli, the transforming microorganism may be characterized in that the accession number KCTC18139P.

The present invention also provides a HEV antibody produced using a capsid protein gene obtained from the transformed microorganism and a HEV preventive vaccine produced using the same.

The present invention has the effect of providing a capsid protein of pig hepatitis E virus, a gene encoding the same, and a vector containing the gene.

Pig capsid protein and gene of the present invention was secured by separating the swine HEV from pig feces, which is expressed recombinant protein can be used for monoclonal antibody production, hepatitis virus antibody detection diagnostics and preventive vaccine development.

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

In the present invention, the RNA of the swine hepatitis E virus (Swine hepatitis E virus) was extracted from the pig feces, and then the capsid protein gene was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) technique.

Gene cloning revealed that the porcine HEV capsid gene was composed of a total of 1,983 nucleotides and encodes a total of 660 amino acids. The cloned porcine HEV capsid gene was compared with the HEV capsid genes derived from humans and pigs, and proved to belong to genotype 3.

In addition, the pig HEV capsid gene cloned in the present invention had about 92% genetic homology with the HEV capsid gene isolated from humans. Porcine HEV capsid gene was expressed and purified by recombinant protein corresponding to 75 kDa using an E. coli expression system, and the protein properties were confirmed by Western blot.

The method of making the HEV antibody of the present invention is as follows. First, HEV capsid protein is used to express and purify in E. coli, and then immunize Balb / C mice by intraperitoneal injection 4 times. The serum of the immunized mice is collected to confirm that the antibody specific for HEV capsid protein is highly formed, and the spleen is extracted. B lymphocyte cells are isolated from the isolated spleen and fused with myeloma cell line (SP2 / 0 Ag14). Among the fused cells, clones are generated that produce antibodies that specifically respond to the HEV capsid protein. The monoclonal antibody produced from the selected clone is purified.

The purified HEV capsid protein is prepared as a vaccine by stirring with Incomplete Freund's adjuvant. The primary vaccine is given by subcutaneous injection into pigs using the prepared vaccine, and the second vaccine is performed in the same manner two weeks after the primary vaccine.

HEV present in pig feces and serum is detected by the following ELISA method. In other words, the antibody collected from rabbit immunized with HEV capsid protein is used as the primary antibody and coated on ELISA plate. Pig fecal and serum samples are added to the ELISA plate to allow attachment to the primary antibody. Then, a monoclonal antibody specific for HEV capsid protein is added as a secondary antibody to attach to the capsid. Add horse radish peroxidase (HRP) -conjugated anti-mouse IgG antibody that recognizes a secondary antibody attached to the HEV capsid. After the addition of the TMB substrate and color development using an ELISA reader to measure the optical density (OD). Based on the measured OD values, HEVs present in feces and serum are diagnosed.

HEV antibodies present in pig serum are diagnosed by the following ELISA method. That is, the purified HEV capsid protein is coated on an ELISA plate, and then serum from pigs is added. HRP-conjugated anti-porcine IgG antibody is then added. After the addition of the TMB substrate and color development using an ELISA reader to measure the optical density (OD). Based on the measured OD values, the presence of anti-HEV antibodies can be used to verify HEV using porcine serum.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples are merely to illustrate the invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Hepatitis E Virus RNA Extraction Using Porcine Fecal Samples

To extract pig hepatitis E virus RNA, 1 g of pig feces collected from 13 farms nationwide were suspended with 10 ml of PBS, centrifuged at 3,000 rpm for 30 minutes, and the supernatant was collected in a 1.5 ml tube. Further, centrifugation was carried out for 10 minutes at a speed of 13,000 rpm to precipitate bacteria and other suspended matter. Then, the supernatant was separated and stored at 70 ℃.

Example 2. Swine HEV RNA Extraction

RNA was extracted using a QIAamp® Viral RNA Mini kit (QIAGEN, USA). That is, 140 μl of the feces isolated in Example 1 was added to 560 μl of AVL buffer containing carrier RNA, vortexed for 15 seconds, and then allowed to stand at room temperature for 10 minutes. Thereafter, 560 µl of 100% ethanol was added and vortexed for 15 seconds, and 630 µl of solution was added to the column, followed by centrifugation for 1 minute at a speed of 8,000 rpm. The column was transferred to a new tube and 630 μl of the remaining sample was placed in the column and centrifuged for 1 minute at 8,000 rpm. After adding 500 μl of AW1 buffer, the resultant was centrifuged at 8,000 rpm for 1 minute, a column was placed in a new tube, and the AW2 buffer was added and centrifuged at 14,000 rpm for 3 minutes. Then, the column was transferred to a new tube, 60 μl of AVE buffer was added thereto, and incubated at room temperature for 1 minute, followed by centrifugation for 1 minute at a speed of 8,000 rpm. After that, the column was discarded and RNA was eluted from the tube. RNA remaining after use was stored at -20 ℃.

Example 3. Swine HEV ORF2 Gene RT-PCR

3-1. cDNA production

CDNA was prepared using Sensiscript® Reverse Transcription kit (QIAGEN, USA). That is, since HEV has a severe secondary structure, the RNA extracted in Example 2 was subjected to a denaturation step at 65 ° C. for 5 minutes before being cDNA and immediately placed on ice. 10x buffer RT, dNTP Mix and RNase-free water provided in the kit and HEV ORF2 external reverse primer of SEQ ID NO: 1 for nest PCR were dissolved at room temperature and placed on ice. Thereafter, according to the composition of Table 1 to make a master mix and vortexing to mix, then added template RNA, and reacted for 2 hours at 37 ℃ to produce a cDNA.

cDNA Mix Composition Component Volume / reaction 10x buffer RT 2.0 μl dNTP Mix (5mM each dNTP) 2.0 μl Primer (10 μM) 2.0 μl RNase inhibitor (10 units / μl) 1.0 μl Sensiscript Reverse Transcriptase (1reaction / μl) 1.0 μl RNase-free water 2.0 μl Template RNA 10 μl Total volume 20 μl

3-2. Primary PCR

The cDNA prepared in 3-1) was used as a template, and the first PCR was performed using the HEV ORF external primer set shown in Table 2 below. The PCR Mix composition is shown in Table 3, and the PCR reaction was performed for 5 minutes at 94 ° C for denaturation of template DNA, denaturation at 94 ° C for 30 seconds, annealing at 48 ° C for 45 seconds, and extension for 2 minutes at 72 ° C for 35 cycles. Finally, the final PCR product was prepared by reacting for 10 minutes at 72 ° C. for sufficient extension.

3-3. Nested PCR

The product of the primary PCR prepared in 3-2) was used as a template, and nested PCR was performed using the HEV ORF internal primer set in Table 2 below. The PCR composition is shown in Table 3 below, and the PCR reaction was performed for 5 minutes at 94 ° C. for denaturation of the template DNA, denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension for 2 minutes at 72 ° C. for 35 cycles. Repeating, the final reaction was carried out for 10 minutes at 72 ℃ for sufficient extension to produce a nested PCR product, and then electrophoresed on a 1% agarose gel, to confirm the DNA band of 2kbp size (Fig. 1). Figure 1 shows the pig hepatitis E virus nested PCR results, lane M is a 1kb DNA ladder, lane 1 is a nested PCR product of HEV capsid gene.

PCR Primer set division Primer Sequence (5 '→ 3') SEQ ID NO: 2 HEV ORF2 external forward primer CGG GTG GAA TGA ATA ACA TGT CT SEQ ID NO: 3 HEV ORF2 external reverse primer CGC GRA AAG CAG AAA WAA GAA SEQ ID NO: 4 HEV ORF2 internal forward primer CAT GTC TTT TGC ATC GCC CAT G SEQ ID NO: 5 HEV ORF2 internal reverse primer AGA GRC TRC GAA GGG GGC ACA

PCR Mix Composition Material Quantity 2x GC buffer I 25 μl D. W. 7.5 μl dNTP Mixture (2.5mM each) 8 μl Forward primer (10 μM) 2 μl Reverse primer (10 μM) 2 μl LA Taq polymerase (5unit / μl) 0.5 μl Template 5 μl

Example 4. Swine HEV ORF2 Gene Cloning

The swine HEV ORF2 gene was cloned using T & A Cloning Kit (RBC ™, Taiwan) and HIT Competent Cells ™ -DH5α (RBC ™, Taiwan). That is, the nested PCR product prepared in Example 3-3) was electrophoresed on a 1% agarose gel to cut a band of about 2kbp expected as the HEV ORF2 portion, using a Gel Extraction kit (Dokdo, Korea) After purification, the DNA was reacted with a Litigation Mix composition solution as shown in Table 4 at 22 ° C. for 15 minutes, and transformed into a Competent Cell. The transformed cells were plated on LB plates containing 50 μg / ml ampicillin, 20 μl 50 mg / ml X-gal and 100 μl 100 mM IPTG, and incubated at 37 ° C. for 14-16 hours. White colonies were selected from the LB plate and re-incubated for 14-16 hours in LB broth containing 50 ㎍ / ml ampicillin.

Ligation Mix Material Quantity 10X Ligation Buffer A 1 μl 10X Ligation Buffer B 1 μl T & A Cloning Vector (25ng / μl) 2 μl PCR product 1 μl T4 DNA Ligase (3units / μl) 1 μl Deionized Water 4 μl

After the incubation was completed, plasmid DNA was extracted using a plasmid mini prep kit (GeneAll Co., Korea), and electrophoresis was performed on an agarose gel, and the results were confirmed (FIG. 2). As a result, as shown in Figure 2, it can be seen that there is a difference in size between the clone inserted insert and the clone not inserted. In other words, the plasmid DNA extracted from the colony, which was estimated to be cloned from the HEV ORF2 gene, was about 2kbp larger than the control plasmid DNA extracted from the blue colony where the insert DNA was not cloned. DNA sequencing of the plasmid DNA into which the HEV capsid gene was inserted was found to have the Capsid protein gene of porcine hepatitis E virus consisting of 1,983 nucleic acids of SEQ ID NO: 6.

The capsid protein gene of the cloned porcine hepatitis E virus ranged from 5,197 nucleotides to 7,179 nucleotides based on US-2 (AF060669), a human HEV prototype. The cloned porcine HEV capsid gene possessed ATG start codon and TAA stop codon, and it can be seen that it expresses 660 amino acids of SEQ ID NO.

The cloned porcine HEV capsid gene was analyzed by comparison with the human-derived and porcine-derived HEV capsid genes reported in GenBank. As shown in Table 5 below, HE-JA10 (AB089824) and HEV- which are human hepatitis E viruses A 92% agreement with US2 (AF060669) was found.

Human Swine Isolates HE-JA10 HEV-US2 HEV-US1 JMY-HAW swUS1 Arkell swJ570 Identity (%) 92 92 91 91 91 89 88

The name of the plasmid carrying the swine HEV ORF2 gene cloned through the present invention was named pswHEV-capsid.

In addition, the present inventors identified the E. coli containing pswHEV-capsid as described above, and the E. coli DH5 to the Korean Collection for Type Cultures (KCTC) dated September 22, 2008 @ / pswHEV-capsid was deposited with an accession number of KCTC18139P.

Example 5 Phylogenetic Tree Analysis

The swine HEV ORF2 gene sequence cloned in Example 4 was analyzed using Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Phylogenetic trees were created using the neighbor joining method. Bootstrap was analyzed 1,000 times and the genetic distance was Kimura two-parameter method. Genbank accession numbers of the genes used to prepare the phylogenetic tree are as shown in Table 6 below, and the phylogenetic analysis was performed using the HEV capsid protein gene portion of these nucleotide sequences. As a result, as shown in Figure 3, it was found that the capsid protein gene (KOR-HW) of swine HEV belongs to genotype 3.

Human HEV Pig HEV AB108537 (CCC220, China), AB074917 (JKK-Sap, Japan), AB074915 (JAK-Sai, Japan), AB080575 (HE-JI4, Japan), AB097812 (HE-JA1, Japan), AB091395 (JSN-Sap-FH , Japan), AB099347 (HE-JK4, Japan), AJ272108 (T1, China), AB091394 (JJT-KAN, Japan), AP003430 (JRA1, Japan), AB089824 (HE-JA10, Japan), AB074920 (JMY-HAW , Japan), AB074918 (JKN-Sap, Japan), AF082843 (swUS1, US), AF060669 (US2, US), AF060668 (US1, US), M74506 (M1, Mexico), X98292 (I1 (FHF), India) , AY204877 (T3, Chad (N'Djamena)), AY230202 (Morocco, Morocco), L08816 (C3 (CHT-87), China), D11093 (C4 (Uigh179), China), D11092 (C1 (CHT-88) , China), L25595 (C2 (KS2-87), China), AF444002 (Sar55, Bangladeshi), M80581 (P1, Bangladeshi), AF185822 (P2 [Abb-2B], Pakistan), X99441 (I2 [Mad-93] , India), AF051830 (NP1 (TK15 / 92), Nepal), AF076239 (I3, India), AF459438 (Yam-67, India), M73218 (B1 (Bur-82), Burma), D10330 (B2 (Bur86) , Burma) AF455784 (Kyrgyz, Kyrgyzstan), AY115488 (Arkell, Canada), AB073912 (swJ570, Japan), AB097811 (swJ13-1, Japan)

Example 6 Amplification of the HEV ORF2 Gene Cloning in an E. Coli Protein Expression Vector

Since there is already a start codon and an end codon in the protein expression vector, PCR performed for amplification of the HEV ORF2 gene for insertion was amplified except for the start codon ATG and the end codon UAA of the gene encoding ORF2 (FIG. 4). The templete used at this time is swine HEV ORF2 cloned in TA vector and Primer is as follows.

[SEQ ID NO: 8] Forward primer: 5'-CGC CCT AGG GCT GTT CTG TT-3 '

[SEQ ID NO: 9] Reverse Primer: 5'-AGA CTC CCG GGT TTT ACC TAC C-3 'PCR

In addition, PCR Mix composition is shown in Table 7, PCR reaction was reacted for 5 minutes at 94 ℃ for denaturation of template DNA, denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension for 2 minutes at 72 ℃ The procedure was repeated 35 cycles, and the last reaction was carried out for 10 minutes at 72 ° C for sufficient extension.

PCR Mix Composition Material Quantity 10x buffer 5 μl D. W. 35.5 μl dNTP Mixture (2.5mM each) 4 μl Forward primer (10 μM) 2 μl Reverse primer (10 μM) 2 μl Ex Taq polymerase (5unit / μl) 0.5 μl Template plasmid DNA 1 μl

Example 7. Cloning to an E. coli Protein Expression Vector

The QIAexpressionist ™ (QIAGEN, USA) was used to clone the HEV ORF2 protein gene. That is, the pQE-30-UA vector provided in the kit and the swine HEV ORF2 protein gene, which is the PCR product of Example 3, were ligation. After mixing the ligation mix and reacted for 2 hours at 16 ℃, the composition of the ligation mix is shown in Table 8.

pQE-30 UA Ligation mix composition Component Volume / reaction pQE-30 UA vector (50 ng / μl) 1 μl PCR product 2 μl Distilled water 2 μl 2x Ligation Master Mix 5 μl Total volume 10 μl

Meanwhile, 100 μl of competent M15 cell or SG13009 cell was dissolved in ice, and the ligation mix was mixed with competent cell for 20 minutes on ice and reacted for 90 seconds at 42 ° C. heating block, and 500 μl of Psi broth was added. The reaction was stirred for 60 to 90 minutes in a shaking shaking incubator. After that, the plate was plated on an LB-agar plate containing 25 µg / ml kanamycin and 100 µg / ml ampicillin and incubated at 37 ° C. for 14-16 hours.

In addition, the colony was selected and cultured, and protein expression was induced with 1 mM IPTG. The cultured E. coli was centrifuged at 13,000 rpm for 5 minutes to dissolve pellets in SDS buffer, and electrophoresis was performed on SDS-PAGE (FIG. 5). Plasmids were extracted from E. coli expressing the expected size of the protein on SDS-PAGE, and the DNA sequencing was again confirmed to be the capsid gene of swine HEV. In other words, 6 out of 1 to 11 samples expressed about 75 kD of HEV capsid protein, so DNA sequencing of the sample confirmed that they possessed pig HEV capsid gene.

Example 8. Swine HEV ORF2 Protein Expression and Purification

The QIAexpressionist ™ (QIAGEN, USA) was used to express and purify the HEV ORF2 recombinant protein.

In other words, clones with confirmed protein expression were incubated in LB broth containing 25 μg / ml kanamycin and 100 μg / ml ampicillin, induced protein expression with 1 mM IPTG, followed by further growth for 4-5 hours, followed by centrifugation. The solution was discarded and the E. coli pellets were stored at -20 ° C. In addition, the E. coli cells were lysed with lysis buffer (6M GuHCl), centrifuged to obtain supernatant, and shaken with Ni-NTA agarose at 4: 1 for 1 hour at room temperature. Since the protein was purified using a protein purification column, washing buffer, elution buffer and the like provided in the kit (Fig. 6). That is, the protein expression was checked again first, and the capsid protein was confirmed to be water-soluble. 6 is a sample before induction by IPTG and 2 is a sample after induction. 75kD protein was generated after induction, and 3 times were soluble and 4 times were insoluble samples, indicating that most capsid proteins were insoluble. 5 to 12 was the elution step, the elution concentration was increased to 12 times.

The purified protein as described above was finally confirmed that the HEV capsid protein by SDS-PAGE and Western blotting (Fig. 7). The primary antibody used in Western blot was diluted to 1,000 times the serum of pigs already in HEV, and the secondary antibody was diluted to 10,000 times the anti-porcine IgG attached to Horseradish peroxidase (HRP). Color development was carried out for 20 minutes with DAB (3,3'-diaminobenzidine) HRP staining solution.

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the pig hepatitis E virus nested PCR results, lane M is a 1kb DNA ladder, lane 1 is a nested PCR product of HEV capsid gene.

Figure 2 shows the result of cloning the pig hepatitis E virus capsid gene, lane M is 1kb DNA ladder, lane 1 is HEV capsid gene cloned in TA vector, lane 2 is negative control (blue colony).

Figure 3 shows the results of Phylogenetic analysis of the HEV capsid gene, the underlined KOR-HW is the capsid protein gene of the swine HEV isolated in the present invention.

Figure 4 shows the PCR amplification result of the HEV ORF2 gene to be cloned in E. coli protein expression vector, lane M is 1kb DNA ladder lane 1 is the product of HEV ORF2 PCR product.

Figure 5 shows the results of confirming the pig HEV capsid protein expression colony, lane M is a protein marker induction lanes 1 to 11 induction by IPTG.

Figure 6 shows the results of the expression and purification of the protein, lane M is a protein ladder lanes 1 to 12 are samples obtained by protein purification step.

Figure 7 shows the western blotting results of Swine HEV ORF2 protein, lane M is a protein marker, lane 1 is a negative control, lane 2 is a recombinant HEV capsid protein.

<110> Konkuk University Industrial Cooperation Corp. <120> Capsid Protein and the gene of Swine hepatitis E virus <130> P08-E267 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 external reverse primer <400> 1 cgcgraaagc agaaawaaga a 21 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 external forward primer <400> 2 cgggtggaat gaataacatg tct 23 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 external reverse primer <400> 3 cgcgraaagc agaaawaaga a 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 internal forward primer <400> 4 catgtctttt gcatcgccca tg 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 internal reverse primer <400> 5 agagrctrcg aagggggcac a 21 <210> 6 <211> 1983 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 capsid DNA <400> 6 atgcgcccta gggctgttct gttgctgctc ctcgtgtttc tgcctatgct gcccgcgcca 60 ccggccggcc agccgtctgg ccgtcgtcgt gggcggcgca gcggcggtac cagcggtggt 120 ttctggggtg acagggttga ttctcagccc ttcgccatcc cctatattca tccaaccaac 180 cccttcgccg ccgatgtcgt ttcacaatcc ggggctggaa ctcgccctcg acagccgccc 240 cgcccccttg gctccgcttg gcgcgaccag tcccagcgcc cccccgctgc cccccgtcgt 300 cgatctgccc cagctggggc tgcgccgttg accgctatat caccggcccc tgacacagcc 360 cctgtacctg atgtagattc acgtggcgct attctgcgtc gccagtataa tttgtccacg 420 tctccgctta cgtcttctgt tgcttctggt actaatctgg tcctctatgc cgctccgctg 480 aaccctcttt tgcctcttca ggatggtacc aacactcata ttatggctac tgaggcatcc 540 aattatgccc agtatcgggt tgtccgagct acaatccgtt atcgcccgct ggtgccaaat 600 gctgttggtg gttatgctat ctctatctca ttctggcctc aaactacaac tactcctacc 660 tctgttgata tgaattctat tacttccact gatgttagga ttttggtcca gcctggtatt 720 gcctccgagt tagtcatccc tagtgagcgc cttcattacc gtaatcaagg ctggcgctct 780 gttgagacca cgggtgtggc tgaggaggag gctacttccg gtctggtaat gctttgcatc 840 catggctctc ctgttaattc ttatactaat acaccttaca ctggtgcact ggggcttctt 900 gattttgcat tagagcttga gtttagaaat ttaacacccg ggaacaccaa cacccgtgtt 960 tcccggtata ccagcacagc ccgccaccgg ctgcgccgcg gtgccgacgg taccgctgag 1020 cttactgcca ctgcagccac acgtttcatg aaagatctgc actttactgg cacgaatggc 1080 gttggtgagg tgggccgtgg tattgctctg acactgttta atctagctga tacgcttctc 1140 ggtggtttac cgacagaatt gatttcgtcg gctgggggtc agatgtttta ctcccgccct 1200 gttgtctctg ccaatggcga gccgacagaa aagctatata catctgtaga gaatgcgcag 1260 caagataagg gcattaccat cccacacgat atagatttgg gtgactctcg tgtggttatt 1320 caggattatg ataaccagca cgagcaagac cgacctactc cgtcacctgc cccctcccgc 1380 cctttttcag ttcttcgtgc caatgatgtt ctgtggcttt ccctcactgc cgctgggtac 1440 gaccagacta cgtatgggtc gtctaccaac cctatgtatg tctccgatac tgtcacattt 1500 gttaatgtgg ccactggtgc tcaagctgtt gcccgctctc ttgattggtc caaagttact 1560 ctggatggtc gccccctcac taccattcag cagtactcta agacatttta tgttctcccg 1620 ctccgcggga agttgtcctt ctgggaggct ggcacaacta aggccggcta cccgtacaat 1680 tataacacta ctgctagtga ccaaattttg attgggaacg cggccggtca ccgtgtcgct 1740 atttctactt atactactag ccttggtgcc ggccctacct cgatctccgc ggtcggtgta 1800 ttagccccac acacggccct tgctgttctt gaggatacta ttgattaccc cgcttgcgct 1860 catacctttg atgatttctg cccggagtgc cgcaccttgg gtctgcaggg ttgtgcattc 1920 cagtctactg ttgctgagct tcagcgcctt aagatgaagg taggtaaaac ccgggagtct 1980 taa 1983 <210> 7 <211> 660 <212> PRT <213> Artificial Sequence <220> <223> HEV ORF2 capsid protein <400> 7 Met Arg Pro Arg Ala Val Leu Leu Leu Leu Leu Val Phe Leu Pro Met   1 5 10 15 Leu Pro Ala Pro Pro Ala Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg              20 25 30 Arg Ser Gly Gly Thr Ser Gly Gly Phe Trp Gly Asp Arg Val Asp Ser          35 40 45 Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Ala      50 55 60 Asp Val Val Ser Gln Ser Gly Ala Gly Thr Arg Pro Arg Gln Pro Pro  65 70 75 80 Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln Ser Gln Arg Pro Pro Ala                  85 90 95 Ala Pro Arg Arg Arg Ser Ala Pro Ala Gly Ala Ala Pro Leu Thr Ala             100 105 110 Ile Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg         115 120 125 Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr     130 135 140 Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu 145 150 155 160 Asn Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala                 165 170 175 Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala Thr Ile             180 185 190 Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser         195 200 205 Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met     210 215 220 Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile 225 230 235 240 Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln                 245 250 255 Gly Trp Arg Ser Val Glu Thr Thr Gly Val Ala Glu Glu Glu Ala Thr             260 265 270 Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr         275 280 285 Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu     290 295 300 Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val 305 310 315 320 Ser Arg Tyr Thr Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp                 325 330 335 Gly Thr Ala Glu Leu Thr Ala Thr Ala Ala Thr Arg Phe Met Lys Asp             340 345 350 Leu His Phe Thr Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile         355 360 365 Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro     370 375 380 Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Met Phe Tyr Ser Arg Pro 385 390 395 400 Val Val Ser Ala Asn Gly Glu Pro Thr Glu Lys Leu Tyr Thr Ser Val                 405 410 415 Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp             420 425 430 Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu         435 440 445 Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val     450 455 460 Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Gly Tyr 465 470 475 480 Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp                 485 490 495 Thr Val Thr Phe Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg             500 505 510 Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr         515 520 525 Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys     530 535 540 Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn 545 550 555 560 Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Gly Asn Ala Ala Gly                 565 570 575 His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro             580 585 590 Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Thr Ala Leu Ala         595 600 605 Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Cys Ala His Thr Phe Asp     610 615 620 Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe 625 630 635 640 Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys                 645 650 655 Thr Arg Glu Ser             660 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 8 cgccctaggg ctgttctgtt 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 9 agactcccgg gttttaccta cc 22  

Claims (10)

Porcine HEV ORF2 capsid protein of SEQ ID NO: 7. The gene encoding the pig HEV ORF2 capsid protein of claim 1. The gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO. A vector containing the gene of claim 2. 5. The cloning vector according to claim 4, wherein said vector is pswHEV-capsid. A transformed microorganism obtained by introducing a vector of claim 4 or 5 into a host cell selected from the group consisting of bacteria, fungi and yeasts. The transforming microorganism according to claim 6, wherein the host cell is Escherichia coli. The transforming microorganism according to claim 7, wherein the transforming microorganism is Accession No. KCTC18139P. The HEV antibody according to any one of claims 6 to 8, produced using a capsid protein obtained from the transgenic microorganism. HEV vaccine produced using the HEV antibody of claim 9.

Images