KR20120034173A - Composition for diagnosing clonorchiasis - Google Patents

Abstract

PURPOSE: A composition containing antigen proteins for diagnosing clonorchiosis is provided to enable early diagnosis and to improve diagnosis accuracy. CONSTITUTION: A composition for diagnosing clonorchiasis contains polypeptide venom 1 having an amino acid sequence of sequence number 6 as an active ingredient. A composition diagnosing clonorchiasis contains a gene encoding venom 1 protein as an active ingredient. A diagnosis reagent for diagnosing clonorchiasis contains a substrate solution containing a fixture coated with anti-human IgG antibody, labeled antigen conjugate, and coloring agent. The anti-human IgG antibody is goat-derived human IgG antibody.

Description

Composition for diagnosing clonorchiasis {Composition for diagnosing Clonorchiasis}

The present invention relates to an antigenic protein for serological diagnosis of hepatic flukes, and more specifically, Cstegu19.4, Cstegu20.6, 14-3-3, propionyl-CoA carboxylase, prohibitin, venom1, venom2, or venom3 polypeptides are effective. It relates to a composition for diagnosing hepatic trematodes as a component.

Typically, a form ganheupchungjeung (Clonorchiasis) of food borne Paragonimiasis is in southern China, South Korea, Taiwan, Vietnam is one of the urgent public health problem (The prevalence of intestinal parasitic infection in Korea , Korean Centers for Disease Control and Prevention and Korea Association of Health Promotion 2004; Lin, R., Li, X., Lan, C., Yu, S., Kawanaka, M., Southeast Asian J Trop Med Public Health 2005, 36 , 1114-1117; Lun, ZR, Gasser, RB, Lai, DH, Li, AX , et al . , Lancet Infect Dis 2005, 5 , 31-41). Hepatobiliary disease is a hepatobiliary disease caused by Clonorchis sinensis , a hepatic flute (Rim, HJ, J Helminthol 2005, 79, 269-281). After oral infection caused by consumption of raw fish infected with C. sinensis metacercariae, the young fluke grows in the host's digestive tract until adult insects and migrates to the intrahepatic bile duct. C. sinensis infection induces biliary epithelial proliferation, periductal fibrosis, and cystic changes in the bile duct (Min, HK, Arzneimittelforschung). 1984, 34 , 1151-1153), it can promote the development of bile duct cancer (Lee, J., H. Rim, HJ Park, UB, Korean Journal of Parasitology 1993, 31 , 21-30; Watanapa, P., Watanapa, WB, Br J Surg 2002, 89 , 962-970) epidemiologically and experimentally reported.

Hepatic onchocerciasis is diagnosed using a method of checking eggs through a microscope. However, the microscopic feces test method has the disadvantage that accurate test results can be obtained only through repeated tests in the case of infected persons with low body load, and in case of overlapping infections with intestinal trematodes, the shape and size of the eggs are similar, so differential diagnosis is not fast. Recently, the ELISA (enzyme-linked immunosorbent assay) method, which checks the degree of reaction with the hepatofluid antigen protein in the blood of an infected person, has also been used for diagnosis. However, the ELISA method is highly sensitive, but has a low specificity for cross-reaction with other parasites, so it is necessary to identify new specific antigens for hepatic flukes. To date, cystein protease (Na BK, Lee HJ, Cho SH, Lee HW, Cho JH, Kho WG, Lee JS, Lee JS, Song KJ, Park PH, Song CY, Kim TS, J Parasitol, 2002, 88:1000- 1006), glutathione-S-trasnferase (Kang SY, Ahn IY, Park CY, Chung YB, Hong ST, Kong Y, Cho SY, Hong SJ, Exp Parasitol. 2001, 97(4):186-195), superoxide dismutase (Li AH, Kong Y, Cho SH, Lee HW, Na BK, Pak JK, Kim TS., Parasitology, 2005, 130:687-697), egg protein, York ferritin, etc., showing antigenicity to hepatofluid infection serum. It was identified as a protein. In addition, tegument of hepatic fluke is a protein that is very important not only for the survival of the parasite itself, but also for the interaction with the host (ZW Zhou, XC Hu, Y Huang, H Hu, CL Ma, XX Chen, FY Hu, J Xu, FL Lu, ZD Wu, XB Yu, Parasitol Res, 2007, 101:737-742). The tegument protein is attracting attention as a target for vaccine development even in schistosomiasis, which has a high infection rate in the world among fluke infections (F. C. Cardoso, et al., Clin Exp Immunol, 2006, 144: 382-391). Therefore, by identifying new tegument proteins, it is expected to improve diagnostic methods for hepatic flukes and develop vaccines (ZW Zhou, HM Xiaa, XC Hu, Y Huang, YW Li, L Li, CL Ma, XX Chen, FY Hu, J Xu, FL. Lu, ZD Wu, XB Yu, Vaccine, 2008, 26: 1817-1825).

14-3-3 protein is a protein family that exists in the cytoplasm and is present in all eukaryotes such as amphibians, insects, plants, and yeast, and is found in a wide range (Aitken A, Collinge DB, van Heusden BP, Isobe T, Roseboom. PH, Rosenfeld G, Soll J., Trends Biochem Sci. 1992 Dec; 17(12):498-501). The 14-3-3 protein has well-preserved amino acid sequences between species and is known to have a wide variety of functions. Its functions are activators of tyrosin and tryptophan hydroxylase (Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R, Takahashi Y. Proc Natl Acad Sci US A. 1988 Oct;85(19):7084-8) ), protein kinase C activity regulation (Toker A, Ellis CA, Sellers LA, Aitken A. Eur J Biochem. 1990 Jul 31;191(2):421-9), besides, it is involved in signaling pathways and promotion of exocytosis ( Aitken A. Trends Biochem Sci. 1995 Mar;20(3):95-7).

Prohibitin is a multifunctional protein that is involved in the regulation of cell proliferation and cell necrosis, as well as cell development, aging, and tumor suppression. prohibitin acts as a chaperone in the mitochondria and maintains mitochondrial function. It is also known to regulate transcriptional activity by interacting with various transcription factors in the nucleus (Rachinsky A, Guerrero FD, Scoles GA. Vet Parasitol. 2008. 152(3-4):294-313). Therefore, targeting prohibitin with these various functions suggests that it can be the subject of research for the treatment of various disease symptoms (S. Mishra, LC Murphy, BLG Nyomba and LJ Murphy, 2005, Trends Mol. Med. 11: 192-197).

Venom allergen-like protein (VAL) is a protein belonging to SCP/TAPS fammily and has an SCP/TAPS domain of 120-170 amino acids in length. SCP/TAPS domain is Schitosoma japonicum , Schistosoma Schistosoma , a fluke that causes schistosomiasis with hepatica As a domain being studied at mansoni, 13 SCP/TAPS proteins (SmVAL1 ~ SmVAL13) have been identified, and 15 other SmVAL proteins have been predicted to exist (Cantacessi C, Campbell BE, Visser A, Geldhof P, Nolan MJ, Nisbet AJ, Matthews JB, Loukas A, Hofmann A, Otranto D, Sternberg PW, Gasser RB. Biotechnol Adv. 2009. 4:376-388). Although the specific function of the SmVAL protein has not been identified, it is attracting attention as a candidate for vaccine development because it is related to the regulation of immune function and invasion of the terminating host (Chalmers IW, McArdle AJ, Coulson RM, Wagner MA, Schmid R, Hirai. H, Hoffmann KF, BMC Genomics. 2008, 9:89).

The present invention solves the above problems, and it is conceived by the necessity of the above, and an object of the present invention is to provide a composition for diagnosing hepatic trematodes.

In order to achieve the above object, the present invention is SEQ ID NO: 1 (Cstegu19.4), SEQ ID NO: 2 (Cstegu20.6), SEQ ID NO: 3 (14-3-3), SEQ ID NO: 4 (propionyl-CoA carboxylase), sequence No. 5 (prohibitin), SEQ ID NO: 6 (venom1), SEQ ID NO: 7 (venom2) and SEQ ID NO: 8 (venom3) It provides a composition for diagnosing hepatic oncosis using a polypeptide selected from the group of polypeptides consisting of the amino acid sequence as an active ingredient.

The antigenic protein of the present invention includes not only the protein selected from SEQ ID NOs: 1 to 8, but also all mutant proteins that have the purpose of diagnosing hepatic oncosis, which is the object of the present invention, through mutations such as one or more substitutions, deletions, inversions, and translocations in these proteins. do.

In addition, the present invention provides a composition for diagnosing hepatic trematodes using the gene encoding the protein of the present invention as an active ingredient.

In one embodiment of the present invention, the gene sequence is modified through mutations such as one or more substitutions, deletions, inversions, translocations, etc., as well as sequences encoding antigenic proteins selected from SEQ ID NOs: 1 to 8 of the present invention. It is preferable that it is a gene encoding all modified proteins having the same activity, and SEQ ID NO: 9 (Cstegu19.4), SEQ ID NO: 10 (Cstegu20.6), SEQ ID NO: 11 (14-3-3), SEQ ID NO: 12 (propionyl -CoA carboxylase), SEQ ID NO: 13 (prohibitin), SEQ ID NO: 14 (venom1), SEQ ID NO: 15 (venom2) or SEQ ID NO: 16 (venom3) is more preferable, but in consideration of code design, SEQ ID NOs: 9 to 16 It is preferably a gene having at least 80% or more homology with the gene sequence selected from, but is not limited thereto.

Hereinafter, the present invention will be described.

In the present invention, the present inventors have identified diagnostic candidates for hepatic flukes. In addition, using immunoblot analysis with serum obtained from patients, it was first discovered that Cstegu19.4 and Cstegu20.6 are antigens against hepatic flukes.

In addition, in the present invention, the present inventors constructed a two-dimensional proteome map of C. sinensis total lysate and identified diagnostic candidates for hepatic trematodes through an immuno-proteomic approach. In addition, using a 2-DE-based immunoblot analysis with serum obtained from a patient, the present inventors first discovered that 14-3-3, PCC, and prohibitin are antigens against hepatic flukes.

In addition, in the present invention, proteins of hepatic trematodes having amino acid sequences similar to SmVAL5, 11, and 13 were identified from cDAN libraries constructed from adult hepatic trematodes. It was discovered for the first time that it is an antigen for the first time.

In addition, the present invention provides a diagnostic reagent for hepatic trematodes, which contains antigens of adult hepatic fluke, cyst larva, or ova and detects hepatic fluke-specific IgG present in blood.

In one embodiment of the present invention, the present invention is a fixed body coated with an anti-human IgG antibody; Labeled antigen conjugate; And it provides a diagnostic reagent for hepatic onchocerciasis comprising a substrate solution containing a color developing agent.

Examples of the anti-human IgG antibody used in the diagnostic reagent for hepatic oncosis of the present invention include, but are not limited to, an anti-human IgG antibody derived from goat.

The antigen of the labeled antigen conjugate used in the diagnostic reagent for hepatic oncosis of the present invention is an antigen of an adult hepatic larva, cyst larva, or ova. As a preferred example, one antigenic polypeptide selected from the group consisting of SEQ ID NOs: 1 to 8 of the present invention A protein composed of all or part is used.

Examples of the labels of the labeled antigen conjugate used in the diagnostic reagent for liver flukes of the present invention include horseradish peroxidase (HRP), alkaline phosphatase, colloidal gold, fluorescein, and dye. ), and the like, but are not limited thereto. In the present invention, horseradish peroxidase (HRP) is used as a preferred label.

In addition, the reagent for diagnosing hepatic trematodes of the present invention includes a substrate solution that reacts with the labeled antigen conjugate to induce a color reaction, and preferably, tetramethylbenzidine, which reacts with the label of the labeled antigen conjugate to induce color development. A substrate solution consisting of a coloring agent and a buffer solution is used. Coloring agents such as tetramethylbenzidine in the substrate solution are decomposed by horseradish peroxidase (HRP), which is used as a preferred label for the labeled antigen conjugate, to induce a color development reaction, and the presence or absence of IgG and antibody by measuring the degree of color development by absorbance. Detect the amount.

The present invention discovered antigenic candidate proteins applicable to serological diagnosis of hepatic flukes through immunochemical analysis and mass spectrometry of hepatic flukes. Among them, Cstegu19.4, Cstegu20.6, 14-3-3, propionyl-CoA carboxylase, In the case of prohibitin, venom1, 2, or 3, the results of verifying that they exhibit antigenicity to the serum of patients with hepatic flukes infection through the expression of recombinant proteins show that immuno-proteomic analysis is useful for the development of diagnostic substances for parasitic infections. It is believed to show. In addition, the development of information and antibodies for hepatic trematodes specific protein improves the accuracy of diagnosis through immunological methods, and develops an early diagnosis method that can diagnose it early in the living body, thereby securing the basis for rapid and accurate diagnosis and treatment and establishment of preventive measures. You will be able to contribute.

1 shows the DNA sequence and amino acid sequence of (a) Cstegu19.4 (531 bp) and (b) Cstegu20.6 (567 bp).
2 shows a comparison of the amino acid sequences of tegumental antigens having similar sizes between different species. (1) Cstegu19.4, (2) Cstegu20.6, (3) S. japonicum 22.6 kDa (AAA29940), (4) S. mansoni 22.6 kDa (AAY57921), (5) S. mansoni 20.8 kDa (AAC79131), (6) C. sinensis 22.3 kDa (ABK60085), (6) C. sinensis 20.8 kDa (AB47326).
Figure 3 is a look at the gene expression pattern for each life stage of the liver fluke.
4 shows a restriction enzyme treatment to verify insert insertion after cloning. (a) As a result of treating pRSETb-Cstegu19.4 with KpnI/HindIII, the bands expected with the size of pRSET vector (2.9 kb) and Cstegu19.4 (528 bp) came out. (b) As a result of treatment of pRSETa-Cstegu20.6 with HindIII/BamHI, the predicted bands of pRSET vector (2.9 kb) and Cstegu20.6 (564 bp) came out.
Figure 5 is the expression and purification of the recombinant protein. (a) rCstegu19.4 and (b) rCstegu20.6 were induce protein expression in E. coli by addition of 0.2 mM IPTG for 5 hours, and then purified by Ni-NTA affinity chromatography. Proteins were separated on 12% SDS-PAGE and visualized by Comash staining. Arrows indicate recombinant proteins. Lane 1 : molecular size standard, Lane 2 : lysate of uninduced pRSET-Cstegu19.4 BL21(DE3)pLysS cells, Lane 3 : IPTG-induced lysate of pRSET-Cstegu20.6 BL21(DE3)pLysS cells, Lane 4 : Ni-NTA column flow-through, Lane 5-6 : 250 mM imidazole elution.
6 shows the antigenicity of the recombinant proteins (a) rCstegu19.4 and (b) rCstegu20.6 expressed in E. coli to the serum of patients with hepatic flukes. Each protein was tested in amounts of 1000, 500, 200, 100, and 50 ng, respectively.
7 shows a representative 2-DE image d of the total lysate protein of C. sinensis and a list of spots identified by LC-coupled ESI-MS/MS analysis.
8 shows the DNA and amino acid sequences of (a) 14-3-3 (741bp), (b) propionyl-CoA carboxylase (1605bp), and (c) prohibitin (831bp).
9 shows a restriction enzyme treatment to verify insert insertion after cloning of each gene. (a) As a result of treating pRSETa-14-3-3 with BamHI/HindIII, the bands expected with the size of pRSET vector (2.9 kb) and prohibitin (741 bp) came out. (b) As a result of treating pRSETa-14-3-3 with BamHI/KpnI, the bands expected with the size of pRSET vector (2.9 kb) and propionyl-CoA carboxylase (1605 bp) came out. (c) As a result of treatment of pRSETa-prohibitin with BamHI/HindIII, the bands expected to be pRSET vector (2.9 kb) and prohibitin (831 bp) appeared.
10 is the expression and purification of the recombinant protein. pRSETa-prohibitin was induce protein expression in E. coli by addition of 0.2 mM IPTG for 5 hours, and then purified by Ni-NTA affinity chromatography. Proteins were separated on 12% SDS-PAGE and visualized by Comash staining. Arrows indicate recombinant proteins. M: molecular size standard, Lane 1: supernatant of the lysate of IPTG-induced pRSET-prohibitin BL21(DE3)pLysS cells, Lane 2: Ni-NTA column flow-through, Lane 3: Ni-NTA column wash, Lane 4 -7: 250 mM imidazole elution. Lane 8: lysate of non-induced pRSET-prohibitin BL21(DE3)pLysS cells
11 shows the antigenicity of the recombinant protein 14-3-3 expressed in E. coli to the serum of patients with hepatic flukes. Each protein was tested in an amount of 500, 300, 100, and 50 ng, respectively (lane 1-4).
Proteins were separated on 12% SDS-PAGE and visualized by Comash staining (left picture). The antigenicity of the recombinant protein was confirmed with the serum of patients infected with hepatic flukes through immunotransferation (right picture).
12 shows the DNA sequence and amino acid sequence of (a) venom1, (b) venom2, and (c) venom3.
Figure 13 Schistosoma mansoni in venom allergn-like protein family and venom1, 2, indicates that a comparison of the amino acid sequence of the three. smVAL1(AAY43180), smVAL2(AAY43181), smVAL3(AAZ04923), smVAL4(AAY43182), smVAL5(ABB88846), smVAL6(AAY28955), smVAL7(AAZ04924), smVAL8(ABW98681), smVAL9(ABB88845), smVAL10(ABO09814), smVAL11(ABA54555), smVAL12(ABB88844), smVAL13(ABB88843)
14 shows a restriction enzyme treatment to verify insert insertion after cloning. (a) As a result of treating pRSETa-venom1 with BamHI/HindIII, the bands expected with the size of pRSET vector (2.9 kb) and venom1 (679 bp) came out. (b) As a result of treating pRSETa-venom2 with BamHI/HindIII, the bands expected with the size of pRSET vector (2.9 kb) and venom1 (420 bp) came out. (c) As a result of treating pRSETa-venom3 with BamHI/HindIII, the bands expected with the size of pRSET vector (2.9 kb) and venom1 (1185 bp) came out.
15 is the expression and purification of venom1 recombinant protein. In E. coli, protein expression was induced for 5 hours by the addition of 0.2 mM IPTG, and then purified by Ni-NTA affinity chromatography. Proteins were separated on 12% SDS-PAGE and visualized by Comash staining. Arrows indicate recombinant proteins. Lane 1 : molecular size standard, Lane 2 : supernatant of IPTG-induced pRSET-venom1 BL21(DE3)pLysS cells lysate, Lane 3 : Ni-NTA column flow-through, Lane 4 : Ni-NTA column washing solution, Lane 5-8 : 250 mM imidazole elution, Lane 9 : lysate of uninduced pRSET-venom1 BL21(DE3)pLysS cells, Lane 10: lyse of IPTG-induced pRSET-venom1 BL21(DE3)pLysS cells.
16 is the expression and purification of venom2 recombinant protein. In E. coli, protein expression was induced for 5 hours by the addition of 0.2 mM IPTG, and then purified by Ni-NTA affinity chromatography. Proteins were separated on 12% SDS-PAGE and visualized by Comash staining. Arrows indicate recombinant proteins. Lane 1 : molecular size standard, Lane 2 : supernatant of IPTG-induced pRSET-venom1 BL21(DE3)pLysS cells lysate, Lane 3 : Ni-NTA column flow-through, Lane 4 : Ni-NTA column washing solution, Lane 5-9 : 250 mM imidazole elution
Figure 17 is the expression and purification of venom3 recombinant protein. In E. coli, protein expression was induced for 5 hours by the addition of 0.2 mM IPTG, and then purified by Ni-NTA affinity chromatography. Proteins were separated on 12% SDS-PAGE and visualized by Comash staining. Arrows indicate recombinant proteins. Lane 1 : molecular size standard, Lane 2 : IPTG-induced lysate of pRSET BL21(DE3)pLysS cells. Lane 3 :
Lysis of uninduced pRSET-venom1 BL21(DE3)pLysS cells, Lane 4: Lysis of IPTG-induced pRSET-venom1 BL21(DE3)pLysS cells. Lane 5 : Supernatant of IPTG-induced pRSET-venom1 BL21(DE3)pLysS cells lysate, Lane 6 : Ni-NTA column flow-through, Lane 7 : Ni-NTA column wash, Lane 8-11 : 250 mM imidazole Elution.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example One: Liver flukes life - stage star tegument The presence or absence of

PCR was performed using the primers used for cloning tegu2 and tegu4 as templates for each of the cDNA libraries of hepatic fluke adult, metacarpal, and ova. The reaction conditions were 95°C for 1 minute, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 2 minutes, and 72°C for 10 minutes. DNA amplified after the PCR reaction was confirmed using a 1% agarose gel containing ethidium bromide (EtBr, 0.5 mg/ml).

Example 2: Cstegu19.4 , Cstegu20 . 6 gene cloning and recombinant protein expression and purification

Clones of cDNAs encoding Cstegu19.4 and Cstegu20.6 were obtained from the cDNA library of adult C. sinensis. For Cstegu19.4, the gene-wide coding region is a forward primer (5'-GGG CAA GGT ACC ATG GAG CCA TTC TTA GAA G 3'; SEQ ID NO: 17) and reverse primer (5'-CCC GTT AAG CTT TCA GCT TGG TGT CTT CCA C 3'; SEQ ID NO: 18) is used, and Cstegu20.6 is a forward primer (5'-TTT ACA GGA TCC ATG GGC GAA CAA GGA TCG G -3'; SEQ ID NO: 19) and reverse primer (5'-GGG CTC AAG It was amplified using CTT TTA GTG GAC AGA TTG GGT C-3'; SEQ ID NO: 20). The underlined portion of the primer sequence represents a restriction enzyme. The amplified PCR product was inserted into a protein expression vector, pRSET (Invitrogen), cloned, and transformed into E. coli BL21 (DE3) pLysS. Transformed bacteria were cultured, and when the optical density was 0.7, IPTG (isopropyl-BD-thiogakactopyranoside) was added to a final concentration of 0.2 mM to induce overexpression of histidine-tagged proteins for 5 hours. The cells were collected and resuspensioned with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0), then lysosome (50mg/ml) and 1mM PMSF were added and reacted on ice for 30 minutes and sonicated to disrupt the cells. The crushed cells were centrifuged at 12,000 g for 30 minutes to obtain a supernant, Ni-NTA agarose (Qiagen) was mixed, and overnight at 4°C. After passing this through the column, the wash buffer (50mM NaH2PO4, 300mM NaCl, 50mM imidazole, pH 8.0) was passed. To obtain a protein, elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0) was added, fractionated, and put in a tube.

Example 3: one-dimensional electrophoresis and Immune blot analysis

To confirm the expressed recombinant proteins, they were separated by 1D SDS-PAGE (12% SDS-polyacrylamide gel) and transferred to Hybond-C pure NC membranes (Immobilon-P, 0.45 um; Millipore). The gels were run at 5 mA/gel for 15 minutes for initial transfer and then at 10 mA/gel for separation until the dye front reached the bottom of the gel. The blotted membranes were rinsed with TBS-T buffer (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20, pH 7.4), followed by blocking buffer (5% w/v skim milk in TBS-T buffer). Blocking was performed by overnight at 4°C. The membranes were incubated at room temperature for 2 hours with C. sinensis infected patient serum diluted 1:500 in blocking buffer and 1 hour with anti-polyhistidine antibody (Abcam), and then washed three times with TBS-T buffer for 10 minutes. Anti-human IgG peroxidase conjugate (Sigma) diluted 1:5000 with blocking buffer and anti-mouse IgG peroxidase conjugate (Sigma) diluted 1:5000 were used to incubate for an additional hour. After washing three times with TBS-T buffer, the films were treated with an ECL solution (GE Healthcare), and then exposed to an X-ray film.

The results of the above example are as follows.

EST library in Cstegu19 .4, Cstegu20 . 6 protein Sympathy

The total lengths of the sequenced cDNAs of Cstegu19.4 and Cstegu20. are 650 bp and 761 bp, respectively, of which ORF is 531 bp and 567 bp (Fig. 1). The predicted pI values are 6.15 and 5.28, respectively, and the molecular weights are 19.4 kDa and 20.6 kDa. As can be seen in Figure 2, when comparing the sequence of the tegument proteins similar in size to Cstegu19.4 and Cstegu20.6, homology of 22% to 36% was observed, and other trematodes, Schistosoma japonicum, and S. mansonium The homology ranged from 24% to 45% with the tegument protein sequence was observed.

The transcripts of Cstegu19.4 and Cstegu20.6 were expressed at the stage of adult and cystoid larvae, but not at the egg stage.

gene Cloning Department Production of recombinant protein

In order to determine the antigenicity of Cstegu19.4 and Cstegu20.6, the present inventors prepared recombinant proteins for each. Genes encoding Cstegu19.4 and Cstegu20.6 were amplified using specific oligonucleotide primers, and sub-cloned into the expression vector pRSET. After sub-cloning, the size of the inserted DNA was confirmed by treatment with restriction enzymes (Fig. 4) . The plasmid whose cloning was confirmed was transformed into BL21 pLysS, an expression strain, and then transferred to induce protein expression (FIG. 5). The expressed recombinant proteins were purified through Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. In addition, Western blot analysis was performed using the histidine tag expressed on the N-terminal of the recombinant proteins (FIG. 5). The molecular weight of the recombinant protein was determined to be about 24 kDa and 25 kDa, respectively, and slightly larger than the predicted size.

Recombinant protein ( rCstegu19 .4, rCstegu20 .6) antigenicity confirmation

In order to confirm the antigenicity of the recombinant proteins, the present inventors performed immunoblot analysis with serum obtained from patients with hepatic oncosis. Serum was diluted 1:500, and peroxidase-conjugated anti-human IgG was diluted 1:5000. As a result of immunoblot analysis, it was shown that the bands of recombinant proteins (24 kDa, 25 kDa) responded dose-dependently to the serum of patients with hepatic oncosis (FIG. 6).

Example 4: Liver flukes total lysate

Intermediate hosts (Chamfish) infected with hepatic trematode cyst larvae were collected and used in Jinju, Gyeongnam, in the area of the Seomjin River, an epidemic area of hepatic trematodes. The cyst larva was isolated from the secondary intermediate host using artificial digestion. The isolated cystoid larvae were orally infected with rabbits and reared for 8 weeks, and then adult worms were collected from the rabbit's liver. The collected adult insects were quenched with liquid nitrogen and then pulverized using a percussion mortar. The pulverized sample was added with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tirs-HCl) and incubated for 30 minutes while slowly shaking at room temperature. After centrifugation at 100,000 g for 30 minutes, the supernatant was taken as total lysate and stored at -70°C.

Example 5: two-dimensional electrophoresis and Immune blot analysis

Rehydration buffer (2 M thiourea/6 M urea, 4% w/v CHAPS, 65 mM DTT, 0.5% IPG buffer, 0.002% w/v Bromophenol blue). And Gorg et al . using IPG strip (Immobiline DryStrip, pH 4-7 nonlinear, 130 mm; GE Healthcare). al . Method (Gorg, A., Obermaier, C., Boguth, G., Weiss, W., Electrophoresis 1999, 20 , 712-717) using the IPGphor system (GE Healthcare). The development was automated by programming for 12 hours without current at 20°C, followed by 4 hours at 100 V, 1 hour at 500 V and 1000 V, and 1 hour at 5000 V, and 8 hours at 8000 V, for a total of 79 kVh. Focused using the old run. After IEF, the IPG strips were placed in equilibration buffer for 15 minutes (6 M urea, 2% w/v SDS, 30% v/v glycerol, 0.002% w/v bromophenol blue, 50 mM Tris-HCl, pH 8.8). At 10 mg/ml DTT was equilibrated, and then the DTT was replaced with 40 mg/ml iodoacetamide and incubated in the same buffer for another 15 minutes. After equilibration, the IPG strips were placed on a 12% SDS-polyacrylamide gel sealed with 0.5% w/v agarose. The gels were run at 5 mA/gel for 15 minutes for initial transfer and then run at 10 mA/gel for separation until the dye front reached the bottom of the gel. Spots separated on the 2-DE gel were transferred to Hybond-C pure NC membranes (Immobilon-P, 0.45 um; Millipore). The blotted membranes were rinsed with TBS-T buffer (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20, pH 7.4), followed by blocking buffer (5% w/v skim milk in TBS-T buffer). Blocked for 12 at 4°C. The membranes were incubated for 2 hours with serum obtained from each parasite-infected patient diluted 1:500 in blocking buffer, and then washed three times for 20 minutes with TBS-T buffer. It was further incubated for 1 hour using a goat anti-immune IgG antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) diluted 1:2000 with blocking buffer. After washing 5 times with TBS-T buffer, the films were treated with ECL solution (GE Healthcare) for 1 minute, and then exposed to the X-ray film for 1 minute.

Example 6: protein Visualization And image analysis

Protein spots by Mortz et al . It was visualized by silver staining (GE Healthcare) according to the method (Mortz E, Krogh TN, et al. Electrophoresis. 2007. 28(10):1607-1614). The abundance of protein spots on the 2-D electrophoresis gel was analyzed by scanning the stained gel with a PowerLook 1100 scanner (UMAX Technology). The intensity of each protein spot was measured using Progenesis software (Perkin-Elmer Life Sciences). For each sample, 2-D electrophoresis was performed at least 3 times for regeneration. Gel images were corrected with pi and Mr using the 2D SDS-PAGE standard (Bio-Rad).

Example 7: mass spectrometry LC - Couple ESI - MS / MS )

For LC-coupled ESI-MS/MS analysis, separation and analysis of trypsin-treated peptides were performed using reverse-phase Capillary HPLC coupled directly with a Finnigan LCQ ion trap mass spectrometer (LC MS/MS). All data was collected in central mode using "triple play" mode; Full mass scan in the mass range of 395-2,000 Da (m/z), determination of the charge state of ions for zoom scan, and then capture of the MS/MS spectra of the ions on the full MS/MS scan, frame at 55%. With a set collision energy. The sequence of the uninterpreted MS/MS spectrum was identified by correlation with the translated in silico peptide sequences from the EST database of C. sinensis using Mascot software.

Example 8: gene Cloning And recombinant protein expression and purification

Clones of cDNAs encoding 14-3-3, PCC, and prohibitin were obtained from the cDNA library of adult C. sinensis. In the case of 14-3-3, the entire coding region of the gene is a forward primer (5'-GAA TTT GGA TCC ATG GTT TCC GAC GAG TCG -3'; SEQ ID NO: 21) and reverse primer (5'-ACC TGG AAG CTT TCA CTG GTC AGT TTC ACG-3'; SEQ ID NO: 22), for PCC, forward primer (5'- GAA CTT GGA TCC ATG CTT CAT TTC GCT CGC -3'; SEQ ID NO: 23) and reverse primer (5'- GGG CAT GGT ACC TTA CAA AGG CAT ATT GGC -3'; SEQ ID NO: 24) was used to amplify, and in the case of prohibitin, a forward primer (5'- CAA TTT GGA TCC ATG GCG CAG CTA AAT GCG -3'; SEQ ID NO: 25) and reverse primer (5'- CCC AGG AAG Amplified using CTT TTA GGT CTG AAC TGA AGG -3'; SEQ ID NO: 26). Restriction enzymes used for cloning are shown underlined in the primer sequence. The amplified PCR product was inserted into a protein expression vector, pRSET (Invitrogen), cloned, and transformed into E. coli BL21 (DE3) pLysS. Transformed bacteria were cultured, and when the optical density was 0.7, IPTG (isopropyl-BD-thiogakactopyranoside) was added to a final concentration of 0.2 mM to induce overexpression of histidine-tagged proteins for 5 hours. The cells were collected and resuspensioned with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0), then lysosome (50mg/ml) and 1mM PMSF were added and reacted on ice for 30 minutes and sonicated to disrupt the cells. The crushed cells were centrifuged at 12,000 g for 30 minutes to obtain a supernant, Ni-NTA agarose (Qiagen) was mixed, and overnight at 4°C. After passing this through the column, the wash buffer (50mM NaH2PO4, 300mM NaCl, 50mM imidazole, pH 8.0) was passed. To obtain a protein, elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0) was added, fractionated, and put in a tube. Meanwhile, in order to separate the recombinant protein expressed in an insoluble form, the cells were resuspended in lysis buffer B (100mM NaH2PO4, 10mM Tris-Cl, 8M urea, pH 8.0) and then centrifuged to obtain a supernatant. Purification was carried out by Ni-NTA affinity chromatography according to the instructions (Qiagen). This was reacted with Ni-NTA agarose (Qiagen) at room temperature for an hour. After passing through the column, it was washed with buffer C (100mM NaH2PO4, 10mM Tris-Cl, 8M urea, pH6.3), and buffer D (100mM NaH2PO4, 10mM Tris-Cl, 8M urea, pH5.9) and buffer E (100mM) Protein was eluted with NaH2PO4, 10mM Tris-Cl, 8M urea, pH4.5).

Example 9: One-dimensional electrophoresis and Immune blot analysis

To confirm the expressed recombinant proteins, they were separated by 1D SDS-PAGE (12% SDS-polyacrylamide gel) and transferred to Hybond-C pure NC membranes (Immobilon-P, 0.45 um; Millipore). The gels were run at 5 mA/gel for 15 minutes for initial transfer and then at 10 mA/gel for separation until the dye front reached the bottom of the gel. The blotted membranes were rinsed with TBS-T buffer (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20, pH 7.4), followed by blocking buffer (5% w/v skim milk in TBS-T buffer). Blocking was performed by overnight at 4°C. The membranes were incubated at room temperature for 1 hour with C. sinensis infected patient serum diluted 1:500 in blocking buffer, anti-polyhistidine antibody (Abcam), and washed three times for 10 minutes with TBS-T buffer. Anti-human IgG peroxidase conjugate (Sigma) diluted 1:5000 with blocking buffer and anti-mouse IgG peroxidase conjugate (Sigma) diluted 1:5000 were used to incubate for an additional hour. After washing three times with TBS-T buffer, the films were treated with an ECL solution (GE Healthcare), and then exposed to an X-ray film.

The results of the above example are as follows.

EST library In 14-3-3, PCC , prohibitin Protein identification

In order to identify a serodiagnostic candidate for hepatic flukes, the present inventors analyzed Cs total lysate proteins in a 2-DE gel by immunoblotting the serum obtained from patients with hepatic flukes. The antisera detected more than 20 protein spots mainly located between 25-40 kDa from the 2-DE gel (FIG. 7). These antigenic spots were later identified as cysteine proteinase, glutathione-S-transferase, prohibitin, propionyl-CoA carboxylase, and prohibitin through mass spectrometry.

gene Cloning Department Production of recombinant protein

In order to determine the antigenicity of 14-3-3, PCC, and prohibitin proteins, the present inventors prepared recombinant proteins for each. Genes encoding 14-3-3, PCC, and prohibitin were amplified using specific oligonucleotide primers, and sub-cloned into the expression vector pRSET. After sub-cloning, the size of the inserted DNA was confirmed by treatment with restriction enzymes (Fig. 9) . The plasmid whose cloning was confirmed was transformed into BL21 pLysS, an expression strain, and transferred to induce protein expression. The expressed recombinant proteins were purified through Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. In addition, Western blot analysis was performed using the histidine tag expressed on the N-terminal of the recombinant proteins (FIG. 10). The molecular weight of the recombinant protein was determined to be about 31 kDa, 63 kDa, and 35 kDa, respectively, and slightly larger than the predicted size.

Confirmation of antigenicity of recombinant protein

In order to confirm the antigenicity of the recombinant proteins, the present inventors performed immunoblot analysis with serum obtained from patients with hepatic oncosis. Serum was diluted 1:500, and peroxidase-conjugated anti-human IgG was diluted 1:5000. As a result of immunoblot analysis of 14-3-3-, PCC and prohibitin, it was shown that a band of recombinant proteins (approximately 31 kDa) reacted dose-dependently to the serum of patients with hepatic oncosis (FIG. 11).

Example 10: venom1 ,2,3 genes Cloning And recombinant protein expression and purification

Clones of cDNAs encoding Venom1, 2, and 3 were obtained from the cDNA library of adult C. sinensis. In the case of venom1, the entire coding region of the gene is a forward primer (5'-GGG AAA GGA TCC ATG TTG GAT ACT GAG CTG-3'; SEQ ID NO: 27) and reverse primer (5'-GGG CCC AAG CTT CTA TAT TCC ACT CAA CG-3'; SEQ ID NO: 28) was used, and Venom2 was a forward primer (5'-TTT AAA GGA TCC ATG CCC CCA GTG GTA TGG GA -3'; SEQ ID NO: 29) and reverse primer (5'- GGG CCC AAG CTT TTA CAA ACA AAT GCA TGG C-3'; SEQ ID NO: 30), venom3 is a forward primer (5'- GGT AAA GGA TCC ATG CCC AAC ATG GAT GAC -3'; SEQ ID NO: 31) and reverse primer (5'- GGA CCT AAG CTT CTA GTT CAT TCC TTG CTT CAC-3'; SEQ ID NO: 32) was used to amplify. The underlined portion of the primer sequence represents a restriction enzyme. The amplified PCR product was inserted into a protein expression vector, pRSET (Invitrogen), cloned, and transformed into E. coli BL21 (DE3) pLysS. Transformed bacteria were cultured, and when the optical density was 0.7, IPTG (isopropyl-BD-thiogakactopyranoside) was added to a final concentration of 0.2 mM to induce overexpression of histidine-tagged proteins for 5 hours. The cells were collected and resuspensioned with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0), then lysosome (50mg/ml) and 1mM PMSF were added and reacted on ice for 30 minutes and sonicated to disrupt the cells. The crushed cells were centrifuged at 12,000 g for 30 minutes to obtain a supernant, Ni-NTA agarose (Qiagen) was mixed, and overnight at 4°C. After passing this through the column, the wash buffer (50mM NaH2PO4, 300mM NaCl, 50mM imidazole, pH 8.0) was passed. To obtain a protein, elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0) was added, fractionated, and put in a tube.

Example 11: One-dimensional electrophoresis and Immune blot analysis

To confirm the expressed recombinant proteins, they were separated by 1D SDS-PAGE (12% SDS-polyacrylamide gel) and transferred to Hybond-C pure NC membranes (Immobilon-P, 0.45 um; Millipore). The gels were run at 5 mA/gel for 15 minutes for initial transfer and then at 10 mA/gel for separation until the dye front reached the bottom of the gel. The blotted membranes were rinsed with TBS-T buffer (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20, pH 7.4), followed by blocking buffer (5% w/v skim milk in TBS-T buffer). Blocking was performed by overnight at 4°C. The membranes were incubated at room temperature for 2 hours with C. sinensis infected patient serum diluted 1:500 in blocking buffer and 1 hour with anti-polyhistidine antibody (Abcam), and then washed three times with TBS-T buffer for 10 minutes. Anti-human IgG peroxidase conjugate (Sigma) diluted 1:5000 with blocking buffer and anti-mouse IgG peroxidase conjugate (Sigma) diluted 1:5000 were used to incubate for an additional hour. After washing three times with TBS-T buffer, the films were treated with an ECL solution (GE Healthcare), and then exposed to an X-ray film.

The results of the above example are as follows.

EST library in venom1 , 2, 3 protein Sympathy

The total lengths of the sequenced cDNAs of Venom1, 2, and 3 are 1153 bp, 633 bp, and 1482 bp, respectively, of which ORF is 679 bp, 420 bp, and 1185 bp (Fig. 12). The predicted pI values are 9.16, 8.48 and 6.57, respectively, and the molecular weights are 24.9 kDa, 15.5 kDa, and 43.5 kDa.

As can be seen in Figure 13, when compared with the amino acid sequences of the venom allergen-like protein family of Schistosoma mansoni, venom1 has smVAL8 and% homology, venom2 has smVAL11 and %, venom3 is smVAL11 and% homology. Was observed.

gene Cloning Department Generation of recombinant protein and identification of antigenicity

In order to determine the antigenicity of Venom1, 2, and 3, the present inventors prepared recombinant proteins for each. Genes encoding Venom1, 2, and 3 were amplified using specific oligonucleotide primers, and sub-cloned into the expression vector pRSET. After sub-cloning, the size of the inserted DNA was confirmed by treatment with restriction enzymes (Fig. 14) . The plasmid whose cloning was confirmed was transformed into BL21 pLysS, an expression strain, and then transferred to induce protein expression (Figs. 15, 16, 17). The expressed recombinant proteins were purified through Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. In addition, Western blot analysis was performed using the histidine tag expressed on the N-terminal of the recombinant proteins. The molecular weight of the recombinant protein was determined to be about 29 kDa, 20 kDa, and 48 kDa, respectively, and slightly larger than the predicted size. In order to confirm the antigenicity of the recombinant proteins, the present inventors performed immunoblot analysis with serum obtained from patients with hepatic oncosis. Serum was diluted 1:500, and peroxidase-conjugated anti-human IgG was diluted 1:5000. As a result of immunoblot analysis, it was shown that the bands of the recombinant proteins (29 kDa, 20 kDa, 48 kDa) responded to the serum of patients with hepatic oncosis.

<110> Republic Of Korea (Korea Center for Disease Control and Prevention) <120> A composition for diagnosing Clonorchiasis <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 175 <212> PRT <213> C. sinensis <400> 1 Met Glu Pro Phe Leu Glu Ala Phe Phe Ser Ile Asp Thr Asp His Thr 1 5 10 15 Glu Arg Ile Thr Ile Arg Glu Leu Gln Asp Tyr Val Arg Arg Asn Asn 20 25 30 Ile Asp Pro Ser Met Ile Lys Arg Trp Gln Val Leu Phe Asp Ala Asp 35 40 45 Asp Ser Gly Val Ile Thr Leu Asp Glu Phe Cys Lys Thr Leu Gly Ile 50 55 60 Arg Pro Ser Glu Ala Arg Ala Tyr Asn Ala Asn Met Val Arg Ala Ser 65 70 75 80 Arg Gly Pro Ser Leu Pro Arg Glu Val Asp Val Ile Thr Ala Thr Leu 85 90 95 Pro Leu Asp Gln Gln Val Asp Ile Val Asn Glu Val Met Arg Leu Thr 100 105 110 Arg Asn Glu Pro Phe Asp Glu Asn Leu Val Ser Lys Gln Leu Lys Gln 115 120 125 Phe Leu Asp Arg Gln Tyr Gly Arg Met Trp His Val Val Ile Thr Lys 130 135 140 Gly Ser Ser Trp Cys Ser Phe Ser Tyr Glu Pro Lys Thr Ser Leu Phe 145 150 155 160 Phe Gln Leu Arg Lys Tyr Thr Tyr Leu Val Trp Lys Thr Pro Ser 165 170 175 <210> 2 <211> 188 <212> PRT <213> C. sinensis <400> 2 Met Gly Glu Gln Gly Ser Asp Met Glu Lys Met Ile Glu Met Phe Leu 1 5 10 15 Gly Met Asp Lys Asn Asp Asp Gly Phe Val Asp Leu Ser Glu Leu Arg 20 25 30 Thr Ala Cys Gln Glu Lys Lys Leu Asp Met Lys Gln Val Asn Gly Trp 35 40 45 Leu Ser Arg Tyr Asp Thr Asn Lys Asp Gly Lys Ile Ser Leu Asp Glu 50 55 60 Phe Cys Asp Gly Leu Gly Leu Gly Lys Gln Glu Met Ile Val Glu Lys 65 70 75 80 Glu Glu Arg Asp Ile Ser Asn Thr Lys Val Cys Pro Thr Ile Ala His 85 90 95 Glu Ile Lys Pro Leu Asp Thr Thr Met Ser Ile Ala Lys Gln Ala His 100 105 110 Ile Thr Asp Lys Phe Ile Glu Leu Ala Lys Glu Val Ser Ser Asp Pro 115 120 125 His Lys Met Asn Gln Val Ala Ala Lys Met Lys Arg Phe Leu Asp Glu 130 135 140 Gln Tyr Gly Arg Val Trp Gln Val Ile Val Leu Ala Gly Ser Tyr Trp 145 150 155 160 Ile Asn Tyr Ser His Ala Pro Phe Leu Ser Met Gln Phe Gln Tyr Gly 165 170 175 Pro Tyr Ile Cys Ile Val Trp Arg Thr Thr Ile Asn 180 185 <210> 3 <211> 247 <212> PRT <213> C. sinensis <400> 3 Met Val Ser Asp Glu Ser Trp Val Val Arg Glu Asn Ile Ser Asp Pro 1 5 10 15 Glu Ala Leu Leu His Ile Ala Lys Leu Ala Glu His Ala Glu Arg Phe 20 25 30 Thr Asp Met Ala Ala Ala Met Lys Lys Phe Thr Glu Leu Lys Lys Pro 35 40 45 Leu Ser Asn Asp Glu Arg Asn Leu Phe Ser Val Ala Tyr Lys Asn Val 50 55 60 Val Gly Ala Arg Arg Ser Ala Trp Arg Val Ile Thr Ser Ile Lys Asn 65 70 75 80 Lys Asp Ser Glu Asp Glu Lys Ser Pro Thr Asn Glu Leu Arg Arg Lys 85 90 95 Ile Glu Asn Glu Leu Glu Gln Val Cys Lys Glu Val Leu Asn Ile Leu 100 105 110 Glu Lys Asn Ser Leu Pro Ser Glu Thr Thr Asp Asp Gly Leu Val Phe 115 120 125 Tyr His Lys Met Lys Gly Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Gln 130 135 140 Thr Gly Asp Lys Arg Asn Glu Ser Val Gln Lys Ser His Gln Ala Tyr 145 150 155 160 Glu Glu Ala Thr Glu Lys Ala Lys Gln Ser Leu Ser Glu Thr His Pro 165 170 175 Ile Arg Leu Gly Leu Ala Leu Asn Tyr Ser Val Phe Tyr Tyr Glu Ile 180 185 190 Glu Asn Asn Pro Asp Lys Ala Cys Glu Leu Ala Lys Ser Ala Phe Asp 195 200 205 Asn Ala Ile Ser Arg Leu Asp Gln Ile Lys Asp Glu Ser Tyr Lys Asp 210 215 220 Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr 225 230 235 240 Ser Glu Arg Glu Thr Asp Gln 245 <210> 4 <211> 535 <212> PRT <213> C. sinensis <400> 4 Met Leu His Phe Ala Arg Asn Ser Leu Arg Leu Leu Ser Val Ala Arg 1 5 10 15 Thr Pro Asn Leu Arg Phe Ile Ser Gly Ala Ala His Thr Leu Glu Val 20 25 30 Arg His Lys Ile Glu Glu Thr Arg Glu Lys Ile Val Leu Gly Gly Gly 35 40 45 Gln Lys Arg Ile Asp Ala Gln His Lys Arg Gly Lys Leu Thr Ala Arg 50 55 60 Glu Arg Ile Asp Leu Leu Ala Asp Pro Asp Thr Phe Val Glu Tyr Asp 65 70 75 80 Ala Phe Met Glu His Asp Cys His Asp Phe Asp Met Gln Arg Gln Arg 85 90 95 Val Thr Gly Asp Ser Val Val Thr Gly His Cys Gln Val Asn Gly Lys 100 105 110 Thr Val Tyr Leu Phe Ser Gln Asp Phe Thr Val Phe Gly Gly Ser Leu 115 120 125 Ser Leu Val His Ala Arg Lys Ile Cys Lys Val Leu Asp Gln Ala Met 130 135 140 Leu Val Gly Ala Pro Val Ile Gly Leu Asn Asp Ser Gly Gly Ala Arg 145 150 155 160 Ile Gln Glu Gly Val Ala Ser Leu Ala Gly Tyr Ala Asp Ile Phe Gln 165 170 175 Arg Asn Val Asn Ala Ser Gly Val Ile Pro Gln Ile Ser Leu Ile Met 180 185 190 Gly Pro Cys Ala Gly Gly Ala Val Tyr Ser Pro Ala Leu Thr Asp Phe 195 200 205 Ile Phe Met Val Gln Asp Thr Ser Tyr Met Phe Ile Thr Gly Pro Asp 210 215 220 Val Val Lys Ser Val Thr Asn Glu Asp Val Thr Gln Asp Glu Leu Gly 225 230 235 240 Gly Ala Lys Thr His Cys Ser Val Ser Gly Val Ala His Arg Ala Tyr 245 250 255 Gln Asn Asp Val Glu Ala Ile Leu Ser Val Arg Asp Phe Leu Thr Tyr 260 265 270 Leu Pro Ser Ser Asn Arg Gln Lys Cys Ala Pro Leu Arg Glu Cys His 275 280 285 Asp Pro Val Asp Arg Leu Val Pro Thr Leu Asn Thr Ile Val Pro Leu 290 295 300 Glu Pro Thr Ser Ala Tyr Asp Met Tyr Glu Val Ile Tyr Ala Ile Val 305 310 315 320 Asp Glu Arg Glu Phe Phe Glu Ile Met Pro Ser Tyr Ala Lys Asn Leu 325 330 335 Ile Val Gly Phe Ala Arg Leu Gly Gly Arg Thr Val Gly Val Val Ala 340 345 350 Asn Gln Pro Arg Val Ser Ala Gly Cys Leu Asp Ile Asn Cys Ser Val 355 360 365 Lys Gly Ala Arg Phe Val Arg Phe Cys Asp Ala Phe Asn Ile Pro Leu 370 375 380 Ile Thr Phe Val Asp Val Pro Gly Phe Leu Pro Gly Thr Ser Gln Glu 385 390 395 400 Tyr Gly Gly Ile Ile Arg His Gly Ala Lys Leu Leu Phe Ala Phe Ala 405 410 415 Glu Ala Thr Val Pro Lys Leu Thr Val Thr Thr Arg Lys Ser Tyr Gly 420 425 430 Gly Ala Tyr Cys Val Met Ser Ser Lys His Leu Arg Gly Asp Ile Asn 435 440 445 Tyr Ala Trp Pro Ser Ala Glu Val Ala Val Met Gly Ala Lys Gly Ala 450 455 460 Val Gln Ile Ile Phe Arg Gly Lys Glu Asp Gln Ala Gln Ala Glu Glu 465 470 475 480 Asp Tyr Ile Arg Thr Phe Ala Asn Pro Phe Pro Ala Ala Val Arg Gly 485 490 495 Tyr Met Asp Asp Ile Leu Asp Pro Pro Leu Thr Arg Lys Arg Leu Cys 500 505 510 His Asp Leu Glu Met Leu Gln Thr Lys Ser Leu Thr Asn Pro Trp Lys 515 520 525 Lys His Ala Asn Met Pro Leu 530 535 <210> 5 <211> 277 <212> PRT <213> C. sinensis <400> 5 Met Ala Gln Leu Asn Ala Met Phe Gly Arg Leu Val Lys Leu Gly Val 1 5 10 15 Gly Ile Val Ala Ala Gly Ser Ile Leu Pro Met Val Leu Tyr Asn Val 20 25 30 Asp Gly Gly His Arg Ala Val Ile Phe Asp Arg Phe Lys Gly Val His 35 40 45 Pro Glu Val Val Gly Glu Gly Thr His Phe Ile Ile Pro Trp Val Gln 50 55 60 Lys Pro Ile Ile Phe Asp Ile Arg Ser Lys Pro Arg Asn Ile Pro Val 65 70 75 80 Met Thr Gly Ser Lys Asp Leu Gln Thr Val Asn Ile Thr Leu Arg Ile 85 90 95 Leu Phe Arg Pro Glu Ser Ser Leu Leu Pro Lys Ile Tyr Gln Asn Leu 100 105 110 Gly Phe Asp Tyr Glu Glu Arg Val Leu Pro Ser Ile Thr Thr Glu Val 115 120 125 Leu Lys Gly Val Val Ala Gln Phe Asp Ala Ser Glu Leu Ile Thr Gln 130 135 140 Arg Glu Leu Val Ser Gln Arg Val Asn Asp Asp Leu Thr Glu Arg Ala 145 150 155 160 Ser Ser Phe Gly Ile Leu Leu Asp Asp Ile Ala Leu Thr Gln Ile Ser 165 170 175 Phe Gly Arg Glu Phe Ser Glu Ala Val Glu Ala Lys Gln Val Ala Gln 180 185 190 Gln Glu Ala Glu Arg Ala Arg Tyr Leu Val Glu Lys Ala Glu Gln Gln 195 200 205 Lys Leu Ala Ala Val Ile Thr Ala Gly Gly Asp Ser Glu Ala Ala Thr 210 215 220 Leu Leu Ala Lys Ala Phe Gly Ser Ser Gly Glu Gly Leu Ile Glu Leu 225 230 235 240 Arg Arg Ile Glu Ala Ala Glu Asp Ile Ala Tyr Gln Leu Ser Lys Asn 245 250 255 Arg Asn Val Thr Tyr Leu Pro Glu Gly Gln His Thr Leu Leu Asn Leu 260 265 270 Pro Ser Val Gln Thr 275 <210> 6 <211> 226 <212> PRT <213> C. sinensis <400> 6 Met Leu Asp Thr Glu Leu Asn Thr Gln Ala Ile Ala Leu His Asn Gln 1 5 10 15 Phe Arg Glu Lys His Gly Ser Pro Pro Leu Val Tyr Asp Ala Lys Leu 20 25 30 Ala Gln Thr Ala Gln Asn Trp Ala Glu Gln Leu Ala Gln Thr Lys Cys 35 40 45 Met Arg His Ser Asp Met Glu Thr Tyr Gly Glu Asn Leu Ala Tyr Lys 50 55 60 Gly Ala Trp Glu Asn Ala Thr Ile Thr Gly Glu Glu Ala Thr Lys Ser 65 70 75 80 Trp Tyr Ala Gln Gly Asp Tyr His Asp Phe Asn Glu Ser Phe Thr Tyr 85 90 95 Glu Thr Ser Tyr Phe Ser Gln Leu Ile Trp Lys Gly Ser Lys Asn Val 100 105 110 Gly Phe Gly Arg Ala Val Ser Glu Asp Gly Glu Ala Ala Tyr Ile Val 115 120 125 Ala His Tyr Phe Pro Lys Gly Asn Ile Arg Ser Val Phe Ser Asn Asn 130 135 140 Val Pro Lys Leu Cys Ser Ala Pro Ser Ala Asn Thr Thr Gly Thr Pro 145 150 155 160 Val Ser Thr Pro Asn Met Arg Tyr Thr Lys Leu Glu Thr Lys Lys Glu 165 170 175 Leu Lys Glu Arg Glu Lys Ala Glu Lys Lys Ala Arg Glu Arg Ala Glu 180 185 190 Lys Glu Arg Lys Glu Arg Glu Lys Gln Leu Lys Lys Glu Gln Lys Glu 195 200 205 Arg Glu Lys Gln Ala Lys Lys Asp Lys Leu Lys Ser Lys Ser Leu Ser 210 215 220 Gly Ile 225 <210> 7 <211> 140 <212> PRT <213> C. sinensis <400> 7 Met Pro Pro Val Val Trp Asp Asn Glu Leu Ala Glu Lys Ala Gln Lys 1 5 10 15 Trp Ala Ser Lys Cys Gln Ala Gly His Asp Ser Asn Ser Glu Arg Lys 20 25 30 Thr Lys Lys Phe Asp Leu Val Gly Gln Asn Trp Ala Gly Gly Tyr Asp 35 40 45 Leu Gln Gly Ala Phe Asn Ala Trp Phe Asp Glu Tyr Arg Asn Tyr Asn 50 55 60 Tyr Ala Asn Arg Ser Cys Thr Gly Val Cys Gly His Tyr Thr Gln Ile 65 70 75 80 Val Trp Asn Lys Thr Thr His Ile Gly Cys Gly Phe Ala Gln Cys Pro 85 90 95 Ser Gln Pro Trp Arg His Ala Phe Val Cys Asn Tyr Gly Pro Ala Gly 100 105 110 Asn Met Arg Met Arg Thr Leu Asn Gly Ala Ile Ile Val Leu Pro Pro 115 120 125 Tyr Glu Glu Ser Ser Thr Cys Pro Gly His Leu Lys 130 135 140 <210> 8 <211> 395 <212> PRT <213> C. sinensis <400> 8 Met Pro Asn Met Asp Asp Phe Ile Glu Glu Cys Leu Arg Glu His Asn 1 5 10 15 Ala Lys Arg Glu Leu His Gly Ala Pro Ala Leu Lys His Ser Arg Ala 20 25 30 Leu Asp Lys Thr Ala Gln Asp Trp Ala Glu Ala Leu Ile Ser Glu Pro 35 40 45 Ser Ile Lys Asn Ser Pro Leu Ser Ser Arg Gly Glu Val Gly Glu Ser 50 55 60 Ile Ser Met Arg Thr Ser Ser Ala Ser His Val Asp Ile Gln Gly Asn 65 70 75 80 Glu Val Val Asn Gln Trp Tyr Ala Asp Ile Lys Asn Tyr Asn Phe Ala 85 90 95 Glu Gly Lys Gly Pro Ala Gly Asn Phe Thr Gln Leu Val Trp Lys Ala 100 105 110 Thr Arg Glu Val Gly Phe Gly Lys Ala Arg Ser Ser Gly Lys Cys Ile 115 120 125 Val Val Ala His Tyr Arg Pro Pro Gly Asn Val Arg Gly His Tyr Ala 130 135 140 Glu Asn Val Gly Thr Pro Thr Gly Glu Gln Ala Ala Ser Val Ala Ser 145 150 155 160 Ala Thr Asp Thr Gly Asn Leu Asp Pro Asn Ala Lys Arg Thr Val Val 165 170 175 Thr Glu Glu Val Thr Ser Pro Glu Gly Lys Arg Tyr Thr Val His Arg 180 185 190 Glu Val Ile Glu Thr Thr Glu Pro Asp Gly His Val Arg Arg Cys Val 195 200 205 Asn Glu Thr Phe Gln Asp Ser Pro Asp Gln Ala Thr Ala Gly Gly Lys 210 215 220 His Gly Ala Ser Ser Glu Ala Ala His Gly Glu Asn Phe Ala Asp Ala 225 230 235 240 Val Thr Arg Ala His Asn Val Tyr Arg Lys Arg His Gly Val Ala Asp 245 250 255 Leu Gln Leu Asp Pro Glu Ile Ser His Met Ala Gln Asp Trp Ala Glu 260 265 270 Gln Leu Val Asn Arg Ala His Leu Ser Asn Ser Gly Phe Thr Tyr Gln 275 280 285 Gly Val Arg Leu Gly Glu Asn Val Leu Cys Arg Trp Ser Asn Thr Ala 290 295 300 Ala Thr Val Ser Ala Gln Asp Val Val Asp His Trp Tyr Gln Glu Ser 305 310 315 320 Ser Lys Tyr Lys Phe Asn Ser Glu Pro Lys Ser Ile Gln Gly Ile Gly 325 330 335 Gly Phe Thr Gln Met Val Trp Asn Gly Ser Gln Arg Ile Gly Val Gly 340 345 350 Ile Ala Ser Gln Ala Lys Lys Asp Phe Tyr Asn Gln Pro Ser Gln Ser 355 360 365 Lys Val Ile Val Val Cys Phe Tyr Tyr Pro Pro Gly Asn Val Thr Gly 370 375 380 Gln Phe Arg Ala Asn Val Lys Gln Gly Met Asn 385 390 395 <210> 9 <211> 525 <212> DNA <213> C. sinensis <400> 9 atggagccat tcttagaagc cttttttagc attgacacgg accacacaga gaggatcact 60 atacgggagc tgcaagacta tgtgaggcga aataatattg atccgtcaat gattaagcga 120 tggcaagttt tattcgacgc cgacgattcc ggagtgatta cactggatga attttgcaag 180 acgcttggaa ttcgtccctc tgaagcccgg gcttacaacg caaatatggt tcgagctagt 240 cgtggtcctt cgctgccacg cgaggttgac gttattactg caactctgcc tttggaccaa 300 caggttgata ttgtcaatga ggtgatgcga ctgacgcgca atgaaccctt tgatgagaat 360 ctggtaagca aacaactgaa gcaatttctc gaccgtcaat acggacgaat gtggcatgtg 420 gtgataacaa aaggatccag ctggtgcagt ttctcttacg agccgaagac ctcacttttc 480 ttccaactgc gcaaatacac ctaccttgtg tggaagacac caagc 525 <210> 10 <211> 564 <212> DNA <213> C. sinensis <400> 10 atgggcgaac aaggatcgga catggaaaaa atgattgaaa tgtttttggg aatggacaaa 60 aacgatgatg gattcgtgga tttaagtgaa ttgcgaaccg cctgtcagga gaagaagttg 120 gatatgaagc aagtgaatgg ttggttgtca agatatgata caaacaaaga cggaaaaatc 180 agcctggatg agttttgcga tggtcttggt cttggaaagc aagaaatgat tgtggagaag 240 gaggaacgcg atatttcaaa caccaaggtt tgtccaacta tcgctcacga gatcaaacca 300 ctggacacaa caatgtctat agccaaacaa gcgcacatta cagacaaatt tatcgagttg 360 gccaaggaag tgtccagtga tccacataag atgaaccaag tggctgcgaa gatgaagcgc 420 ttcttggatg aacagtatgg aagagtgtgg caggtgatcg tgttggctgg aagttactgg 480 attaattact ctcacgctcc tttcctatcg atgcaattcc aatatggacc atacatttgt 540 attgtatggc gtaccaccat caac 564 <210> 11 <211> 741 <212> DNA <213> C. sinensis <400> 11 atggtttccg acgagtcgtg ggttgttcgt gaaaatattt ccgatccgga ggctttgttg 60 catatcgcga agttggctga acatgccgag cgcttcactg atatggcagc cgctatgaaa 120 aagttcacgg aactgaaaaa accgcttagc aacgatgaac ggaatctttt ttccgtagct 180 tacaaaaatg tggtgggcgc tcgccggtcg gcctggcgtg taatcacaag catcaagaac 240 aaggattctg aggatgaaaa gtcaccaacg aacgaacttc gtcgaaagat agagaacgag 300 cttgagcaag tctgcaagga ggttttgaac atcctggaga aaaattctct gccatctgag 360 actacagacg acggactggt tttttatcac aaaatgaaag gagattatta tcgttatttg 420 gctgaagtac aaacgggaga caagcgcaat gaatccgttc agaaatccca tcaggcctat 480 gaagaagcca cggagaaagc caaacaatct ctcagtgaga ctcatcctat tagattggga 540 ctagccctaa actactctgt attctactac gaaatcgaga ataatccgga caaggcatgc 600 gaattggcca agtctgcctt cgataacgcc ataagccggc tcgatcaaat caaggatgag 660 tcctacaaag acagtacctt gattatgcag ctacttcggg acaatttgac gctgtggacg 720 tcggagcgtg aaactgacca g 741 <210> 12 <211> 1605 <212> DNA <213> C. sinensis <400> 12 atgcttcatt tcgctcgcaa ttctttacgg cttttgtcag tggctcgtac acctaatctc 60 cgtttcatct cgggggcagc acacaccctt gaggtccgcc acaaaatcga ggaaactcgc 120 gaaaagatcg tacttggggg tggtcagaag cgtatcgatg cgcagcacaa gcgtggcaag 180 ctgacggcgc gcgaacgtat cgacctgttg gctgatcctg acacatttgt ggagtacgac 240 gccttcatgg aacatgattg tcatgacttt gacatgcaga gacagcgggt cacgggggac 300 agtgttgtta ccggtcactg ccaggtcaat ggaaaaactg tctacctatt cagtcaagac 360 tttaccgtat ttggtggaag tttgagtctg gttcatgctc gaaagatctg caaggtcttg 420 gatcaagcta tgctcgtcgg agctcctgtg attggtttga atgactctgg tggtgcacgc 480 atccaagagg gcgtagcgtc gctggccggc tatgcagata tcttccagcg gaacgtgaac 540 gcctccggtg tcattccaca aatctctctc atcatgggcc cctgtgctgg tggtgctgtt 600 tactcccccg ctttgacaga ttttattttc atggttcaag atacctcata tatgttcatc 660 actggtccag atgtagtcaa gtcggtgaca aatgaggacg tcacccaaga tgaactgggt 720 ggtgcgaaga ctcattgcag tgtctctggc gtagcccata gagcctacca gaacgacgtg 780 gaggccatac tcagtgttcg tgacttcttg acctacttgc cgtcgtccaa taggcaaaaa 840 tgcgccccac tgcgtgaatg tcacgatcca gtggaccgac tggttccaac tttaaatacg 900 attgtcccac tggagcctac cagtgcctac gatatgtacg aggtcatcta tgcaatcgtg 960 gatgagcgcg agtttttcga aatcatgcct tcgtacgcga agaacctcat cgtcggattc 1020 gctcgccttg gaggccgcac agttggtgtg gttgctaacc aaccccgtgt atcagccggc 1080 tgcctggata tcaactgttc ggtgaagggt gctcgattcg ttcgtttctg tgatgcattc 1140 aacatcccgc tcatcacatt tgtcgatgtt cccggttttc tgccaggaac tagtcaggaa 1200 tacggtggga ttattcgaca tggagcgaag cttctgttcg cctttgctga ggctactgtg 1260 cctaaactga ctgtgaccac gcgtaaatcg tacggcggag cctactgtgt gatgagtagc 1320 aagcacctcc gtggggatat caattatgct tggccctcag cggaggtggc tgttatgggt 1380 gctaagggtg cggtccaaat catttttcgt ggaaaagagg accaggctca ggccgaagaa 1440 gactacattc gaacatttgc aaacccattc ccagctgctg ttcgaggtta tatggatgat 1500 atcctagatc ctccacttac acggaaacgc ctttgtcatg acctggaaat gctgcagacc 1560 aagtcgctga cgaacccgtg gaagaaacat gccaatatgc ctttg 1605 <210> 13 <211> 831 <212> DNA <213> C. sinensis <400> 13 atggcgcagc taaatgcgat gttcggtcgc ttggtcaagt tgggagttgg tattgttgcg 60 gcgggaagta ttctgccaat ggtgctttac aatgttgatg gtgggcatcg tgccgtgatc 120 tttgatcggt tcaagggcgt gcatcctgaa gttgtcggtg aagggacaca cttcatcata 180 ccttgggtac agaaaccgat catatttgat atccgctcaa agccccgaaa cattccggtt 240 atgacgggat ctaaagatct gcagactgtt aacatcactc tgcgaattct cttccggcca 300 gagtcttcat tgttgcccaa gatttatcag aaccttggtt tcgactacga ggaacgcgtt 360 ctaccgtcaa taacaaccga agtgctcaag ggtgttgtag cacagtttga tgccagtgag 420 ctaatcacac agcgagaact cgtgagccag cgggttaatg atgatctaac cgaacgtgct 480 tcttcctttg ggatattgct ggacgatatt gctttaacac aaatctcgtt cggtcgagaa 540 ttctctgaag ccgtggaagc taaacaagtc gcacaacaag aagccgagcg cgcacgctac 600 ctagttgaaa aagcagaaca gcaaaagctg gctgctgtca ttacggctgg gggcgattcg 660 gaagcagcta cacttttggc caaagcattc gggtcgtccg gggaaggtct aatcgagctc 720 agacgaatcg aagcagccga ggatattgca tatcagctgt cgaaaaaccg gaacgttact 780 tacctccccg aaggccagca tacgctactc aatttgcctt cagttcagac c 831 <210> 14 <211> 678 <212> DNA <213> C. sinensis <400> 14 atgttggata ctgagctgaa tactcaagca attgctcttc acaatcaatt ccgtgagaaa 60 cacggaagcc cacctctagt ttacgatgca aaattggccc agacagccca gaattgggca 120 gaacaactgg cacagaccaa atgcatgcgg cacagcgata tggaaaccta cggagaaaac 180 ttggcctaca aaggagcgtg ggaaaatgct actatcaccg gagaagaagc tacaaagtct 240 tggtatgcgc aaggggatta tcatgacttc aatgaaagtt tcacctacga gacgagctac 300 tttagtcagc ttatctggaa aggaagtaaa aatgttggat ttggtcgcgc ggtttcagag 360 gacggagaag cggcatatat cgtagcacac tactttccca aaggcaacat ccggagcgtc 420 ttctccaaca atgttccaaa actctgctct gctccctcgg ccaacacaac tggaactcct 480 gtatccactc cgaacatgcg gtacacaaaa ctggaaacca agaaggagct caaagagcga 540 gaaaaagcgg agaaaaaggc ccgtgagcgc gctgagaagg aacgaaaaga acgtgaaaaa 600 caactgaaga aggaacagaa agagcgggaa aagcaggcga aaaaagacaa actaaagtcc 660 aaatcgttga gtggaata 678 <210> 15 <211> 420 <212> DNA <213> C. sinensis <400> 15 atgcccccag tggtatggga caatgaactg gccgaaaaag cccaaaaatg ggcgagcaag 60 tgccaagcag gccacgactc caattcagaa cgcaaaacca agaaatttga tttggttggg 120 caaaactggg caggaggtta cgacctgcag ggtgctttca atgcatggtt tgatgaatat 180 agaaactaca actacgcgaa tagaagttgt acgggtgttt gcggacatta cactcagatt 240 gtatggaaca aaactacaca cataggctgc ggttttgccc agtgccccag tcaaccatgg 300 cgccatgcat ttgtttgtaa ctatgggcca gctgggaaca tgaggatgag gacactaaat 360 ggagctatca tagtgctgcc accgtacgag gaatcgtcca cgtgtccagg ccacttgaag 420 420 <210> 16 <211> 1185 <212> DNA <213> C. sinensis <400> 16 atgcccaaca tggatgactt tatcgaagaa tgccttcgtg aacataatgc caaacgtgaa 60 ttacacggtg ctccagcttt gaaacatagt agagcacttg acaaaactgc ccaggattgg 120 gcggaagctc tgatctctga gccgtccatc aaaaacagcc ccttatctag ccgtggcgag 180 gttggtgaga gcatcagcat gcgaactagc tcggcaagtc atgtggatat tcaaggaaac 240 gaagtagtaa atcaatggta cgccgacatc aagaattaca actttgctga aggcaaaggt 300 ccagcgggta atttcactca attggtctgg aaagcaacac gtgaggtcgg attcggaaag 360 gcacggtctt ctgggaaatg tattgtcgtt gcgcattacc gaccaccggg caatgttaga 420 ggacactatg cagagaatgt cggtaccccg actggcgaac aagcggcctc ggtagccagt 480 gctacggaca cagggaatct ggaccctaat gctaaacgga cagtggttac ggaagaagtc 540 acctcaccag aagggaagcg ttacactgtt caccgggagg tcattgaaac cacagaaccc 600 gatggtcatg tcagaagatg tgtcaacgag acgttccagg attcacccga tcaggccact 660 gccggtggta aacatggcgc aagttccgaa gcagcccacg gagaaaattt cgctgatgca 720 gtgacccgag cgcacaacgt ctaccggaaa cggcatggtg ttgcggatct gcaattagac 780 ccggagatta gtcatatggc gcaggactgg gctgaacagt tggtaaatcg agcccacttg 840 tcgaacagcg ggttcaccta tcaaggagta cgtcttggag aaaacgtcct ttgtcgttgg 900 tcgaacacgg ctgccaccgt ttctgcccaa gatgtcgttg atcactggta tcaagaaagt 960 tccaagtaca agtttaattc ggaacccaaa agtattcaag gcattggcgg attcacccag 1020 atggtatgga acggatcaca acgcatcggg gttgggatcg cttcacaggc aaagaaggat 1080 ttttacaacc agcccagcca atcaaaagtc attgttgtct gcttctatta tccacccgga 1140 aacgtcaccg gacaattcag agcaaatgtg aagcaaggaa tgaac 1185 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gggcaaggta ccatggagcc attcttagaa g 31 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cccgttaagc tttcagcttg gtgtcttcca c 31 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tttacaggat ccatgggcga acaaggatcg g 31 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gggctcaagc ttttagtgga cagattgggt c 31 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gaatttggat ccatggtttc cgacgagtcg 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 acctggaagc tttcactggt cagtttcacg 30 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaacttggat ccatgcttca tttcgctcgc 30 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gggcatggta ccttacaaag gcatattggc 30 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 caatttggat ccatggcgca gctaaatgcg 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 cccaggaagc ttttaggtct gaactgaagg 30 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gggaaaggat ccatgttgga tactgagctg 30 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gggcccaagc ttctatattc cactcaacg 29 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 tttaaaggat ccatgccccc agtggtatgg ga 32 <210> 30 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gggcccaagc ttttacaaac aaatgcatgg c 31 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 ggtaaaggat ccatgcccaa catggatgac 30 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 ggacctaagc ttctagttca ttccttgctt cac 33

Claims (3)

Composition for diagnosing hepatomegaly as an active ingredient the polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 (venom1). A composition for diagnosing hepatic pneumonia, wherein the gene encoding the protein of claim 1 is used as an active ingredient. The method of claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 14 (venom1) characterized in that the composition for diagnosing hepatic pneumoconiosis.

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