WO2011066711A1 - Specific antigen of clonorchis sinensis and uses thereof - Google Patents

Abstract

The invention provides a specific antigen gene of Clonorchis sinensis, a separate protein encoded by the antigen gene, the carrier and host cell comprising the antigen gene, and the antibody specifically binding to the antigen of Clonorchis sinensis. The invention also provides the vaccine and kit comprising the said specific antigen or antibody, and the uses of the antigen, antibody, separate protein or vaccine for manufacturing the drugs for the treatment, diagnosis or prevention of clonorchiasis.

Description

 Specific antigen and use of Clonorchis sinensis

 The invention belongs to the field of bioengineering, and particularly relates to a clonorchiasis, in particular to a specific antigen and use of a clonorchiasis.

Background technique

 Clonorchiasis sinensis is a zoonotic disease caused by infection with C. sinensis [ C3⁄4mordiis sinensis, s]. The disease is mainly distributed in Asia, such as China, Japan, South Korea (which is the main human parasite in Korea), North Korea, Vietnam, Southeast Asia and other countries. It is estimated that 35 million people worldwide are infected. In China, except for Xinjiang, Inner Mongolia, Gansu, Qinghai, Tibet, Ningxia and other provinces and autonomous regions, there are no reports, and the remaining 25 provinces, municipalities, autonomous regions, Taiwan Province and Hong Kong Special Administrative Region have reported or reported cases of the disease. . Case reports of the disease are increasing in some non-endemic areas and developed countries (including North America and Western Europe) due to the flow of immigrants and the increasing frequency of global tourism and economic activities.

 According to the survey conducted by the Ministry of Health from June 2001 to the end of 2004 on the status of important human parasitic diseases in the country (excluding Taiwan, Hong Kong and Macao), the infection rate of Clonorchis sinensis was higher than that of the 1990 national parasitic disease distribution. The results increased by 75%, with the provinces, autonomous regions of Guangdong, Guangxi and Jilin rising by 182%, 164% and 630% respectively. It is estimated that there are more than 12 million people infected with clonorchiasis in China. Clonorchis sinensis is one of the few parasitic diseases in China that has shown an upward trend. The prevention and treatment of this disease is imminent.

In southern China (such as Guangdong, Guangxi) and some northern regions (such as the Korean residential areas in the three northeastern provinces), the local population has the habit of eating raw or uncooked freshwater fish, and the live cysts in the fish are ingested. The human body causes human infection. Although residents of these areas know that eating "fish" will infect the disease, eating habits are difficult to change. And with the improvement of living standards, people who have not eaten or eaten "fish" in the past have begun to eat "fish". Nowadays, the flavor of sashimi is eaten in non-popular areas, especially in large and medium-sized cities. . At the same time, due to the opening of the market, the fish in the endemic areas containing C. sinensis are transported to various places, so the number of people infected with C. sinensis in urban residents has an increasing trend. Adult Clonorchis sinensis parasitic in the hepatic bile duct of human or other terminal host, the secretion/metabolite of the worm and the mechanical stimulation of the worm itself can cause bile ducts, especially the secondary bile duct inflammation, moderate infection of the bile duct There may be localized expansion, bile duct epithelial hyperplasia, thickening of the wall, stenosis of the lumen, such as bacterial infection, can cause cholangitis and cholangitis, peripheral fibrous tissue hyperplasia, cirrhosis can occur in advanced stage. There is evidence that Clonorchis sinensis infection increases the risk of cholangiocarcinoma (CLG) in patients with clonorchiasis-associated hepatobiliary tumors, an important public health problem in the epidemic area of clonorchiasis.

 In order to quickly diagnose, timely treat and effectively control clonorchiasis, a specific, sensitive and simple method is needed to detect the infection of Clonorchis sinensis in the population.

 For the diagnosis of clonorchiasis patients, traditional fecal examination is still the main method for the diagnosis of clonorchiasis. However, this method has certain defects due to poor patient compliance and small susceptibility to C. sinensis.

 At present, immunodiagnostic methods (mainly ELISA) are widely used in on-site prevention and treatment work. However, there are still some problems: There are different degrees of false positives for healthy people, certain false negatives for clonorchiasis patients, and some cross-reactions for other parasites (schistosomiasis, paragonimiasis). Yong TS applied ELISA to detect serum antibodies in 48 patients with Clonorchis sinensis, only 75% of the positive reactions, but in 28 normal controls and 16 cases of Paragonimus, 7.1% and 37.5% of false positives, respectively.

Studies have shown that some parasite excretory secretory antigens (ESA) or recombinant ESA can be applied to the serological diagnosis of parasitic diseases. Kim SI used ESA to detect the dynamic response of serum antibodies in patients with active clonorchiasis, and found that the 30kDa and 7kDa bands were strongly positive in patients with serum, but the serum response was weaker after 6 months of treatment with praziquantel. , in the 7kDa zone, there is no cross-reaction with the serum of Hessian paragonimiasis, Yokogawa trematode, and clonorchiasis patients, the specificity is stronger than the 30kDa zone, and it is considered that the 7kDa antigen can be As a landmark diagnostic antigen for active clonorchiasis. Min-Ho CHOI compared and evaluated the diagnostic value of the ELISA method for excretory secretion antigen (ESA) detection antibody of C. sinensis and the ELISA method for detection of adult crude antigen (CA) antibodies. The specificity of the ELISA method for detecting antibodies by ESA is significant The ELISA method was higher than that of the antibody detected by CA (previous specificity was 93.1%, and the latter was 87.8%), indicating that ESA was used to detect serum antibody in Chinese patients with Clonorchis sinensis better than crude antigen. Therefore, compared with CA of Clonorchis sinensis, ESA has higher specificity and sensitivity as a diagnostic antigen for clonorchiasis. However, traditional insect-derived antigens are complex to prepare, have low quality controllability, and are limited in preparation, making it difficult to adapt to the needs of large-scale disease investigation.

 In addition, because the insect is parasitic on the specific part of the hepatic bile duct, it determines that the antigen and antibody levels in the host serum are low and difficult to detect (and the reason for the lack of good diagnostic reagents in the disease), but in the fecal sample. The existence of ESA. Yong TS et al. identified the C. hominis antigen that reacted with the IgE antibody and found that the 28 kDa antigen reacted most strongly with IgE, and the protein was also present in fecal excretion. Sirisinha S et al. used a monoclonal antibody ELISA (Monoclonal antibody ELISA, McAb-ELISA) to detect antigens in the fecal samples of squirrels in squirrels. The minimum amount of antigen that can be detected is 0.05 ng-0.1 ng. Studies have shown that the detection of specific antigens in patients' fecal samples can provide a basis for early diagnosis, diagnosis and evaluation of current patients.

 The nature of the diagnostic antibody or antigen determines the diagnostic effect of immunological methods such as detection of antigen or serum antibodies in fecal samples and serum. At the same time, since it is difficult to obtain a high-purity specific antigen or a large number of natural antigens, the molecular route of using molecular biology techniques to construct a specific recombinant antigen is the only way to find a suitable antigen.

Summary of the invention

 SUMMARY OF THE INVENTION The object of the present invention is to provide a specific antigen and use of Clonorchis sinensis which solves the technical problem of the specificity and sensitivity of diagnosing clonorchiasis in the prior art.

 The present invention provides a specific antigen of Clonorchis sinensis, the antigen gene having the nucleotide sequence shown in SEQ ID NO: 1, or homologous to the nucleotide sequence shown in SEQ ID NO: 1. More than 90% of the nucleotide sequence, or an antigenic nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 1 after partial substitution, deletion or addition.

The present invention also provides an antibody which specifically binds to a specific antigen of the above-mentioned Clonorchis sinensis. Further, the antibody is a monoclonal antibody.

 The present invention also provides an isolated protein, which is encoded by the nucleotide sequence of a specific antigen gene of the above-mentioned Clonorchis sinensis, or with the nucleus of a specific antigen gene of the above-mentioned Clonorchis sinensis. The nucleotide sequence of 90% or more of the nucleotide sequence is encoded, or is encoded by the nucleotide sequence of the specific antigen gene of the above-mentioned one of the above-mentioned C. sinensis after partial substitution, deletion or addition.

 Further, the amino acid sequence of the isolated protein is as shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted or added with one or several amino acids, and then SEQ ID NO: The amino acid sequence derived from the amino acid sequence shown in 2.

 The present invention also provides a vector comprising the above-described specific antigen gene of Clonorchis sinensis.

 The present invention also provides a host cell comprising the above vector, or the cell is transformed or transfected with a specific antigen gene of C. sinensis as described above.

 The present invention also provides a vaccine comprising the above-described specific antigen of Clonorchis sinensis, or the above-mentioned antibody, or the above-described isolated protein, or the above-described vector.

 The present invention also provides the use of the above specific antigen of Clonorchis sinensis for the preparation of a medicament for treating, diagnosing or preventing clonorchiasis.

 The invention also provides the use of the above antibody for the preparation of a medicament for the treatment, diagnosis or prevention of clonorchiasis.

 The present invention also provides the use of the above isolated protein for the preparation of a medicament for the treatment, diagnosis or prevention of clonorchiasis.

 The present invention also provides the use of the above vaccine for the preparation of a medicament for treating or preventing clonorchiasis.

The present invention also provides a kit comprising the above antigen, or the above antibody. The term "expression vector" as used herein refers to bacterial plasmids, yeast plasmids, and various other viral vectors commonly used in the art. Vectors suitable for use in the present invention include, but are not limited to, vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (such as Pichia vectors), A vector for expression in a mammalian cell (retrovirus, adenoviral vector, etc.). In a preferred embodiment, the expression vector is an E. coli expression vector. Those skilled in the art can construct a expression vector containing a specific sequence of a DNA sequence encoding the fusion protein, a suitable transcriptional and translational regulatory sequence, a promoter, and a selectable marker gene, using a series of techniques such as DNA recombination technology. Such vectors can be used to transform, transfect, and adapt a suitable host cell to obtain the desired fusion protein.

 The host cell of the present invention may be a prokaryotic cell or a eukaryotic cell such as a bacterial cell, a mammalian cell or the like. The host cell, after transformation or transfection of the gene sequence comprising the fusion protein of the present invention, constitutes an engineered cell or cell line and can be used to produce the desired fusion protein. Those skilled in the art will be able to appropriately select appropriate vectors, host cells, and how to efficiently transform or transfect vectors into host cells, including but not limited to: calcium chloride method, electroporation for bacterial cells , liposome encapsulation, electrofusion method for eukaryotic cells such as mammalian cells.

 The host cell of the present invention can be cultured and induced by a conventional method to express a desired fusion protein, including a fermentation process and a purification process. The protein expressed above can be secreted intracellularly, on the cell membrane, or secreted into the periplasm and extracellular. Separation and purification can be carried out using the physical, chemical and other biological properties of the fusion protein as needed. Methods include, but are not limited to, schizophage, centrifugation, salting out, molecular sieve chromatography, ion exchange chromatography, adsorption chromatography, reversed phase chromatography, and conventional variability, renaturation, and the like, all of which are well known to those skilled in the art.

In the present invention, the Cs1 antigen of Clonorchis sinensis is screened by immunoscreening by using the cDNA expression library of Clonorchis sinensis adults constructed by the present inventors in a mixed sera from the Guangxi Zhuang Autonomous Region. By comparison of homology, the Csl antigen of C. sinensis of the present invention is a novel specific antigen of Clonorchis sinensis which has not been disclosed so far. Through the expression and purification of Csl antigen of Clonorchis sinensis, a preliminary method for the diagnosis of clonorchiasis with high specificity and sensitivity has been established. It is easy to prepare multi/monoclonal antibodies, and the application of clonorchiasis can be applied. Different aspects such as prevention and treatment; The vaccine of the present invention has an effect of preventing infection of Clonorchis sinensis. The kit of the invention can also be used to detect clonorchiasis. DRAWINGS

 Figure 1 is a 1% gel electrophoresis pattern of the Csl 5 'RACE amplified cDNA fragment of Clonorchis sinensis antigen, M: DNA marker, 1-4: Amplified cDNA fragment (about 200 bp).

 Figure 2 is a 1% gel electrophoresis map of pBluescript SK-Cs1 phagemid EcoRI/XhoI double digestion, Ml: λΟΝΑ/ΗίηάΙΙΙ+ΕοοΚ I Marker, Μ2: ΦΧ 174 DNA/BsuRI (Haelll) Marker, 1-2: pBluescript SK-Csl phagemid EcoRI/XhoI double digestion results (at 160 bp, there is still an unclear band).

 Figure 3 is a 1% agarose gel electrophoresis of pET28a NcoI/HindIII double digestion and Csl-Ncol PCR amplification, M: DNA Marker; 1-2: pET28a NcoI/HindIII double digestion result; 3-4: Csl- NcoI PCR amplification results (about 610 bp).

 Figure 4 is a 1% agarose gel electrophoresis pattern of pET28a-CSl-NCOI plasmid Ncol/Hindlll, wherein: M: DNA Marker; 1: pET28a-CSl-NCOI plasmid Ncol/Hindlll double digestion results.

 Figure 5 is a 16% polyacrylamide gel electrophoresis pattern of pET28b-CSl BL21(DE3) recombinant protein expression, M : Protein Marker ( FERMENTAS MBI ) ; 1: pET28b-CSl BL21 (DE3) is not induced; 2: pET28b- CSl BL21 (DE3) induction; 3: induction of supernatant; 4: induction of precipitation.

 Figure 6 is a 16% polyacrylamide gel electrophoresis pattern of pET28a-CSl-NcoI BL21(DE3) recombinant protein expression, M: Protein Marker (FERMENTAS MBI); 1: pET28a-CSl-NcoI BL21 (DE3) is not induced; 2: pET28a-CSl-NcoI BL21 (DE3) induction; 3: induction supernatant;

4: Induced precipitation.

 Figure 7 is a 12% polyacrylamide gel electrophoresis pattern of pET28b-CS1 BLR(DE3) recombinant protein expression identification, M: Protein Marker ( FERMENTAS MBI ); 1: pET28b-CSl BLR (DE3) uninduced; 2: pET28b-CSl BLR (DE3) induction; 3: induction of supernatant; 4: induction of precipitation.

Figure 8 is a 12% polyacrylamide gel electrophoresis pattern of pET28a-CSl-NcoI BLR (DE3) recombinant protein expression identification, M: Protein Marker ( FERMENTAS MBI ); 1: pET28a-CSl-NcoI BLR (DE3) is not induced; 2 : pET28a-CSl-NcoI BLR (DE3) induction; 3: induction supernatant; 4: Induced precipitation.

 Figure 9 is a 12% polyacrylamide gel electrophoresis pattern of pET28a-CSlDS-NcoI B21(DE3) recombinant protein expression identification, wherein, M: Protein Marker ( FERMENTAS MBI ) ; 1: pET28a-CSlDS-NcoI BL21 (DE3) Induction; 2: pET28a-CSlDS-NcoI BL21 (DE3) induction; 3: induction of supernatant; 4: induction of precipitation.

 Figure 10 is a 12% polyacrylamide gel electrophoresis map of pET28b-CSl BL21(DE3) insoluble protein purification, M: Protein marker; 1: effluent (F) pH 8.0; 2-4: Buffer C solution 1, 3 5 tubes (pH 6.3); 5-9: Buffer D solution 1-5 tubes (pH 5.9); 10-14: Buffer E solution 1-5 tubes (pH 4.5), of which, pET28b-CSl BL21 ( DE3) The insoluble protein is concentrated in the eluent at pH 4.5.

 Figure 11 is a 12% polyacrylamide gel electrophoresis map of pET28b-CSl BL21(DE3) soluble protein purification, M:Protein marker; 1: pET28b-CS 1 not induced; 2: pET28b-CS 1 IPTG induction 3: Ni-NTA Purified effluent (no imidazole); 4-7: 20 mM imidazole wash tube 1, 3, 5, 7; 8-9: 50 mM imidazole eluate 1, 2 tubes; 10-11: containing lOOmM imidazole wash Deliquolation 1, 2 tubes; 12-14: 250 mM imidazole eluate 1, 2, 3 tubes, wherein pET28b-CSl BL21 (DE3) soluble protein was concentrated in an eluate containing 50-250 mM imidazole.

 Figure 12 is a 12% polyacrylamide gel electrophoresis map of soluble and insoluble protein purified from pET28a-CSl-NcoI BL21(DE3), M: Protein marker; 1: effluent (F); 2-8: 250 mM imidazole soluble protein Eluent 1-7 tubes; 9-14: Insoluble protein Buffer E solution 1-6 tubes (pH 4.5), wherein pET28a-CSl-NcoI BL21 (DE3) soluble protein is concentrated in an eluent containing 250 mM imidazole in. The insoluble protein was concentrated in an eluent at pH 4.5.

Figure 13 is a purified protein of pET28b-CSl DS-NcoI BL21(DE3) 12°/. The polyacrylamide gel electrophoresis pattern, pET28b-CSlDS-NcoI BL21 (DE3) soluble protein was concentrated in an eluate containing 100-250 mM imidazole. Wherein, M:Protein marker; 1: Ni-NTA purification effluent; 2-3: 20 mM imidazole washing solution 2, 4 tubes; 4-6: 50 mM imidazole eluent 1-3 tubes; 7-9: lOOmM imidazole eluate 1-3 tube; 10-14: containing 250 mM imidazole eluate 1-5 tube. Figure 14 is a scatter plot of serum antibody detection of pET28b-CSl insoluble purified protein.

For ease of understanding, the present invention will be described in detail below by way of specific examples. It is to be understood that the description is not intended to be limiting of the scope of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in the light of the description herein.

detailed description

 The experimental methods described below, which are not specifically described, were carried out in accordance with the method described in the Guide to Molecular Cloning, 2002, Science Press.

Example 1 Construction of cDNA library of Clonorchis sinensis adults

 Five cats were infected by the method of gavage, which was collected from the Guangxi Zhuang Autonomous Region. After 40 days, the eggs of the fecal eggs were examined, and then the worms were collected and collected, and the adult worms were collected and washed with sterile physiological saline, and stored in liquid nitrogen.

 Total RNA of C. sinensis adults (1 gram of C. sinensis adultiasis, liquid nitrogen storage) was extracted using the TRIzol (GIBC0/BRL) kit.

 mRNA was purified from the extracted total RNA using an mRNA purification kit (mRNA Purification Kit, Amersham). See the product manual for specific steps.

 The cDNA library of Clonorchis sinensis adults was constructed by directional cloning using the ZAP-cDNA Synthesis Kit (Stratagene). Refer to the kit instructions for operation. The main processes include:

1. Synthesis of the first strand of cDNA, using a poly-T primer containing a Xho I endonuclease site, in order to prevent the first strand of cDNA from being destroyed by the restriction enzyme during the synthesis, 5-methyl dCTP was used. Instead of dCTP in dNTP, the synthesized DNA is hemimethylated, so that when Xho I digests cDNA, only the unmethylated site located in the poly-T primer can be cleaved;

 2. Synthesis of the second strand of CDNA, the RNA in the first strand synthesis product raRNA-DNA hybrid is digested by RNase H, and the resulting cDNA fragment is used as a primer to synthesize a second strand under the action of DNA polymerase I;

3. The ends of the synthesized double-stranded cDNA were filled in with Pfu DNA polymerase, and then an adaptor containing an EcoR I cleavage site was added to the 5' end of the double-stranded DNA, and the linker was phosphorylated. ;

4. Digested with restriction endonuclease Xho I, and digested double-stranded DNA was subjected to fractionation by Sepharose CL-2B gel column chromatography to remove free linker;

 5. Combine the isolated DNA fragments into a Uni-ZAP XR vector;

6. Finally, package the phage protein shell with the packaging extract (Gigapacklll Gold Packing Extract, Stratagene). After the construction of the cDNA library of Clonorchis sinensis adults, the library titer and the average length of the insert were further examined.

The calculated result showed that the titer of the cDNA library of Clonorchis sinensis adults was 1.43 X 10 ⁶ pfu/ml. 16 recombinant clones were randomly picked from the library for PCR amplification, and the PCR products were identified by 1% agarose gel electrophoresis. The clonal amplified fragment has a length ranging from 0.6 kb to 2 kb. The average insert length is 1. 1 kb.

 The recombinant insertion rate was 99.6%. Example 2 Immunoscreening of cDNA library

Phage infection:

 Take XLl-blue Mi? 200 μΐ and mix with appropriate amount of cDNA library phage solution (predetermined according to library titer, about 3000 plaques per plate), incubate at 37 ° C for 15 minutes, add 3 ml 48 ° C The top agar was mixed and immediately placed on the NZY medium plate. Incubate at room temperature for 10 minutes and incubate at 42 °C.

Induced expression of the fusion protein:

 After the appearance of plaques was observed (about 3.5 hours), the plates were covered with a nitrocellulose membrane (Hybond-C extra, Amersham, NC) soaked for 30 minutes with IPTG (15 mM/L), and incubated at 37 ° C for 3.5. Hour; remove the plate and cool at 4 ° C for 15 minutes, then mark the film and the plate with a needle. The membrane was removed and washed in TBST three times for 10 minutes each time, then immersed in the NC membrane and immersed in the blocking solution at room temperature, gently shaken, and blocked overnight.

 The NC membrane was taken out of the blocking solution, and the membrane was washed twice with TBST for 5 minutes each time. Add sera from patients with clonorchiasis (taken from Guangxi Zhuang Autonomous Region) (5ml/membrane) and shake gently for 3 hours at room temperature. The membrane was washed 3 times with TBST for 10 minutes each time. Add secondary antibody (5ml/membrane) ie goat anti-human alkaline phosphatase conjugate (AP-GAH, BIO-RAD, 1:3000 dilution). React for 1 hour at room temperature, wash membrane 3 times with TBST, 10 minutes each time. Wash the membrane twice with TBS for 5 minutes, and wash off the residual Tween20.

 The NC membrane was taken out of the TBS, and the excess solution was blotted with a Whatman 3MM filter paper, and placed in an AP buffer to soak the NC membrane for 3 minutes.

NBT was diluted with Color Development Solution to a final concentration of 0.3 mg/ml, and the final concentration of BCIP was 0.15 mg/ml (BCIP should be added dropwise to the diluted NBT to prevent precipitation.), formulated into NBT-BCIP coloring solution. The NC membrane was immersed in a NBT-BCIP color developing solution, and a color reaction was carried out in the dark until the positive spots were clearly visible. The color reaction was terminated. Positive plaques were picked according to the corresponding positions of the positive spots on the NC membrane on the original culture plate, and then subjected to rescreening and three sieves to make the positive phage monoclonal. Example 3 Phage deletion cyclization into pBluescript SK-phagemid and extraction of phagemid

E. coli XLl-blue MRF' was cultured overnight in LB medium (containing 10 mM MgS04, 0.2% maltose, 15 g/ml Tet). The next day, the culture medium was transferred to a new LB medium, and cultured at 37 ° C, shaking at 200 rpm for about 2 hours. The culture solution was centrifuged for 10 minutes at 2 000 g, and the bacteria were precipitated and resuspended in 10 mM MgSO 4 to OD _{6 (X} ) = 1.0. 200 μl of E. coli XLl-blue MRF' resuspension, 50 μl of SM buffer containing positive phage, and Ιμΐ helper phage were added to the bacterial culture tube. The bacterial culture tubes were placed in a 37 ° C water bath for 15 minutes. Then, 3 ml of LB was added, and the mixture was shake-cultured at 37 ° C for 5 hours. The bacterial culture tube was taken out, heated at 65 ° C for 20 minutes, then 3 000 g, centrifuged for 15 minutes, and the supernatant was transferred to a new centrifuge tube and stored at 4 ° C.

SOLR was cultured in shaking in LB medium (containing 10 mM MgSO4, 50 μ§/πι1 Kan), then 2 000 g, centrifuged for 10 minutes, resuspended in 10 mM _MgS04 to OD _6GQ = 1.0, and 200 μl SOLR was added to a 1.5 ml Ep centrifuge tube. The supernatant supernatant prepared above was incubated at 37 ° C for 15 minutes, and then 25 μl was taken out and uniformly coated on LB (100 g/ml Amp) agar plates, and cultured at 37 ° C overnight.

 The colonies grown on the medium were SOLR colonies of pBluescript SKJ1 granules containing the cDNA insert of Clonorchis sinensis.

 pBluescript SK-phagemid was extracted using AxyPrep Plasmid DNA Mini Kit (Aisijin Biotechnology (Hangzhou) Co., Ltd.). Example 4 Isolation of Csl antigen gene

Using a mixed serum of Clonorchis sinensis (taken from Guangxi Zhuang Autonomous Region) to screen the cDNA library of adult Clonorchis sinensis (taken from Guangxi Zhuang Autonomous Region) (using the ZAP-cDNA Synthesis Kit of Stratagene) to obtain positive clones. The cyclization is removed to pBluescript SK_Csl. A partial mR A sequence of the C. sinensis Csl antigen gene was obtained by sequencing by Shanghai Invitrogen (as shown in SEQ ID NO: 6). Use Invitrogen Kit ( 5 ' RACE System for Rapid Amplification of cDNA Ends ), using the lower jaw [JSPI (GSPl: TGCGCACCATCCGCATCG;

 GSP2: GATGTGCTCGAGCCTGAAG, synthesized by Shanghai Invitrogen) Reverse-transcribed and amplified cDNA fragment inserted into pGEM-T Easy Vector (Promega:), sequenced (sequenced by Shanghai Invitrogen) to obtain the Csl antigen gene of Clonorchis sinensis The full-length sequence, as shown in SEQ ID NO: 1, has a total of 733 bases, the 23 position is the initiator (ATG), the position 617 is the terminator (TAG), and the deduced amino acid sequence of the Csl antigen is SEQ ID NO: 2. The Csl antigen contains 198 amino acids. Csl 5 'RACE amplified cDNA fragment 1% gel electrophoresis pattern shown in Figure 1. Example 5 Construction of Expression Vector

1. Construction of pET28b-Csl expression vector

 The pBluescript SK-Cs1 phagemid was partially digested with EcoRI/XhoI (NEB) restriction enzyme and reacted at 37 ° C for 15 minutes.

 The digested product was electrophoresed on a 1% agarose gel, and the separated fragment was cleaved under an ultraviolet lamp, and the target fragment was purified by EZNAUltra-Sep® Gel Extraction Kit (Omiga) (718 bp) o due to the Csl antigen gene in pBluescript The SK-interposer contains two Xhol cleavage sites, so under partial cleavage conditions, the insert presents three fragments (718, 559 and 159 bp), as shown in Figure 2.

 The pET28b expression vector was also digested with EcoRI/XhoI (NEB) restriction endonuclease and reacted overnight at 37 °C. The digested product was subjected to electrophoresis on a 1% agarose gel, and the separated fragment was cut under an ultraviolet lamp and purified by E.Z.N.A. Ultra-Sep® Gel Extraction Kit (Omiga).

 The target fragment and the vector fragment were ligated at a molar ratio of 8:1, and the ligated system contained T4 DNA ligase (NEB) lul, 10xT4 1igase buffer 2 ul, deionized water, and the target fragment and the vector fragment total volume was 20 ul at 1.5. The ml Eppendorf tube was ligated overnight at 16 ° C to construct a prokaryotic expression recombinant plasmid of pET28b-Csl.

Linked pET28b-CSl recombinant plasmid and newly solubilized E. coli DH5a competent cells (Tiangen) was mixed, ice-bathed for 30 minutes, heat-shocked at 42 °C for 1 minute, and then placed in an ice bath for 3 minutes. Under sterile conditions, 1 ml of SOC medium was added, and the mixture was shaken at 37 ° C, 200 rpm for 1 hour. Then, the bacterial solution was centrifuged at 3 500 rpm for 3 minutes, and a part of the supernatant was discarded, leaving about ΙΟΟμΙ to resuspend the cells. The resuspension was spread on an LB plate (containing 5 (^g/ml of Kan), and after inhalation, it was inverted and cultured at 37 ° C overnight.

 The transformed DH5a bacteria were cultured overnight in LB medium (containing 5 (^g/ml of Kan) at 200 rpm. The bacteria were collected by centrifugation at 3 500 rpm for 10 minutes. AxyPrep plasmid DNA mini kit was used [Love Sijin Biotechnology (Hangzhou) Co., Ltd. extracted the plasmid and sequenced it by Shanghai Invitrogen to confirm that the target fragment was inserted correctly.

 The extracted pET28b-CS1 recombinant plasmid was again transformed into E. coli BL21 (DE3) competent cells (NOvagen) by the same method as above. The amino acid sequence of this recombinant protein (pET28b-CS1 recombinant plasmid) is shown as SEQ ID NO: 3.

2. Construction of pET28a-CSl-NcoI expression vector

 Based on the sequence of the Csl gene, the following primers (synthesized by Shanghai Invitrogen) were designed: PF-CS1-NCOI:

 5, CATGCCATGGGGATGAAACCGCAACTTGTATAC introduces Ncol bit,

 PR-CS1-NCOI:

 5, CCC AAGCTTTG ATATGATTCTTCGTAGAAT bow | into the Hindlll site

PCR amplification was carried out using pBluescript SK-Cs1 phagemid as a template. The reaction system was 50 ul, wherein the template was 0.2 ul, the bidirectional primers were each lul, dNTP (赛百胜公司) lul, 10 X buffer 5ul, platinum Taq enzyme (Invitrogen) 0.5 ul, 50 mM magnesium ion 1.5 ul, deionized water 39.8 ul. The reaction conditions were pre-denaturation at 95 ° C for 5 minutes; denaturation at 95 ° C for 1 minute; annealing at 50 ° C for 1 minute; renaturation at 72 ° C for 1 minute; cycle 30 times, the last cycle of renaturation was extended to 7 minutes, and the sample was stored in 4° (. PCR product was electrophoresed on a 1% agarose gel (as shown in Figure 3), cut under UV light The fragment of the fragment was separated and purified by EZNAUltra-Sep® Gel Extraction Kit (Omiga).

 The pET28a expression vector plasmid was digested with NcoI, Hindlll restriction endonuclease (NEB) overnight; and dephosphorylated using calf intestinal alkaline phosphatase (CIP, NEB). The enzyme product was electrophoresed on a 1% agarose gel (as shown in Figure 3), and the separated fragment was cut under an ultraviolet lamp and purified by E.Z.N.A. Ultra-Sep® Gel Extraction Kit (Omiga).

 The purified Csl-Ncol PCR fragment was also digested with NcoI and Hindlll restriction enzymes. And recover using the same method as above.

 The target fragment and the vector fragment were ligated at a molar ratio of 8:1, and the ligated system contained T4 DNA ligase (NEB) lul, 10xT4 1igase buffer 2 ul, deionized water, and the target fragment and the vector fragment total volume was 20 ul at 1.5. The ml Eppendorf tube was ligated overnight at 16 ° C to construct a prokaryotic expression recombinant plasmid of pET28b-Csl-NcoI.

 The ligated expression vector was transformed into DH5a, and after the plasmid was extracted, the inserted sequence was verified to be correct. And further transformed E.coli BL21 (DE3) competent cells. The amino acid sequence of this recombinant protein (pET28b-Csl-NcoI recombinant plasmid) is shown in SEQ ID NO: 4.

3. Construction of pET28a-CSlDS-NcoI expression vector

 The pET28a-CSl-NCOI plasmid was digested with Pci l, Hind III restriction endonuclease (NEB). There are four fragments in the gel electrophoresis: 76 bp, 526 bp, 2260 bp 3000 bp (as shown in Figure 4), and the 526 bp fragment was purified by EZNAUltra-Sep® Gel Extraction Kit (Omiga), and the pET28a expression vector plasmid (Nco I, Hind) Lll restriction endonuclease double enzyme digestion) ligation. The ligated expression vector was transformed into DH5a, the plasmid was extracted, and the inserted sequence was verified to be correct, and the E. coli BL21 (DE3) competent cells were further transformed. The amino acid sequence of the pET28a-CS1DS-NcoI recombinant protein is shown in SEQ ID NO: 5. Example 6 Expression, Identification and Purification of Recombinant Protein

The transformed expression host strain was inoculated into 4 ml of LB medium (Kan containing 50 μ _§ / ηι1), and cultured to OD ₆ with shaking at 37 ° C, 200 rpm. . When =0.6, take 2ml of bacteria solution and add 2μ1 1M The IPTG was induced, and the remaining 2 ml of the bacterial liquid was cultured without shaking with IPTG as a control at 37 ° C, 200 rpm for 4 hours. The cultured bacterial liquid was collected by centrifugation at 5 OOO rpm for 10 minutes at 4 ° C, and the supernatant was discarded. The cells were resuspended by adding 200 μl of 0.15 M PBS to the induction tube and the control tube, respectively. 5 μl of the resuspension was taken from the induction tube and the control tube, respectively, and 2 μSDS-PAGE loading buffer 5 μl was added, mixed, and then denatured by boiling at 100 ° C for 5 minutes. SDS-PAGE analysis was performed on 8 μl/well samples (concentrated gel was 5%, and the gel was 12% or 16%). The electrophoresis conditions were concentrated gel 80V and separation gel 100V. After electrophoresis, stain with staining solution for 4 hours, then decolorize with decolorizing solution until the protein bands are clearly visible. ' Result:

 pET28b-CSl BL21 (DE3) was induced at 37 °C under I mM IPTG conditions, and a distinct recombinant protein band was observed at 43 KD. The recombinant recombinant protein is mainly located in the precipitate, and a small amount is located in the supernatant. As shown in Figure 5.

 pET28a-CSl-NcoI BL21 (DE3) was induced at 37 °C under I mM IPTG conditions, and a distinct recombinant protein band was observed at 42 KD. The recombinant recombinant protein is mainly located in the precipitate, and a small amount is located in the supernatant. As shown in Figure 6.

pET28b-CS1, _P ET28a-CSl-NcoI A large amount of recombinant protein can also be induced in the E. coli BLR (DE3) expression host strain (NOvagen), as shown in Figs.

 pET28a-CSlDS-NcoI BL21 (DE3) was induced at 37 °C under I mM IPTG conditions, and a distinct recombinant protein band was observed at 42 KD. The recombinant recombinant protein is mainly located in the supernatant as shown in FIG. Purification of recombinant protein

The induced bacterial solution (500 ml) was centrifuged at 4000 rpm, 4 ° C for 10 minutes, and the supernatant was discarded. Add 5ml per gram of bacterial paste, add BugBuster® protein extractant (NOvagen) to the cells, and add 5Ku rLysozyme (per gram of bacterial paste, NOvagen) and 125u Benzonase nuclease (per gram of bacterial paste, NOvagen), after fully suspending the precipitate, it was shaken at room temperature for 30 min. It was then centrifuged at 9 OOOC for 10 minutes at 4 °C. Purification of soluble proteins

 A 1 ml portion of Ni-NTA Agarose (Qiagen) was packed and washed with a large amount of 0.15 M PBS to remove residual ethanol.

 The induced bacterial supernatant after centrifugation was added to the treated Ni-NTA Agarose, placed on a shaker, and shaken at room temperature for 60-90 minutes to fully bind the protein to Ni-NTAAgarose.

 After the oscillation was stopped, the Ni-NTA Agarose was allowed to settle, and the plug under the column was opened to collect the discharged liquid.

 The column was washed twice with a 4x bed of Washing Buffer and the effluent was collected.

 The column was then washed with 2-4 volumes of Elution Buffer containing different concentrations of imidazole and the eluate was collected separately.

 The collected liquid was subjected to SDS-PAGE electrophoresis detection to examine the effect of protein purification. Purification of insoluble proteins

 A 1 ml portion of Ni-NTA Agarose (Qiagen) was packed and washed with a large amount of 0.15 M PBS to remove residual ethanol. Then add 2 times the bed Buffer B balance.

 The induced bacterial pellet after centrifugation was added to Lysis Buffer B (pH 8.0) in a ratio of 5 ml per gram, and shaken at room temperature for 30-60 minutes. Then, it was centrifuged at 9 000 rpm, 4 ° C for 10 minutes. The retained supernatant was added to the treated Ni-NTA Agarose, placed on a shaker, and shaken at room temperature for 60 minutes to fully bind the protein to Ni-NTAAgarose.

 After the oscillation was stopped, the Ni-NTA Agarose was allowed to settle, and the plug under the column was opened to collect the discharged liquid.

 The column was then washed with 2-4 times the Buffer C, D, E of different pH values, and the eluate was collected separately.

The collected liquid was subjected to SDS-PAGE electrophoresis detection to examine the effect of protein purification. The results are shown in Figures 10, 11, 12 and 13. Example 7 Enzyme-linked immunosorbent assay for detection of antibodies

1. Indirect ELISA for detection of antibodies in serum

 The recombinant protein was coated with the ELISA plate (Nunc, 96-well plate), overnight at 4 ° C; 1% BSA was blocked at 37 ° C for 1 hour; respectively, 1 : 100 diluted clonorchiasis patients, normal human serum 100 ul, The reaction was carried out at 37 ° C for 1.5 hours; HRP-labeled goat anti-human IgG (Sigma, 1 : 10 000 dilution) was added, and the reaction was carried out at 37 ° C for 1 hour; the substrate TMB (Tiangen) was added for color development, and the microplate reader was 450 nm. Read the value. Preliminary evaluation of the effect of recombinant protein antigen detection antibody

 Randomly selected 10 serum samples from Clonorchis sinensis from Hengxian County, Guangxi, mixed into positive mixed serum, and randomly selected 10 normal human serum from Jingxi City, Guangxi, which was collected from the non-endemic area of clonorchiasis, was mixed as a negative mixture. serum. The soluble purified protein, insoluble protein, pET28a-CSl-NcoI soluble purified protein and insoluble purified protein of the recombinant antigen pET28b-CSl were coated at 4, 2, 4 and 4 ug/ml, respectively, and 100 ul per well. The antibodies in the serum were detected by the above indirect ELISA method.

 The results are as follows:

 The results showed that the four types of purified recombinant proteins can better distinguish between mixed positive and negative blood.

2. Further evaluation of the effect of pET28b-CSl insoluble protein purification antibody detection

35 sera of Clonorchis sinensis collected from Hengxian County, Guangxi, 36 normal human serum from Jingxi City, Guangxi, and 15 cases of sera from Schistosoma japonicum and 9 cases of Weiss The serum of Paragonimus patients was tested for antibodies in serum by the above indirect ELISA method. The insoluble purified protein of the recombinant antigen pET28b-CS1 was coated at a concentration of 10 ug/ml, 100 ul per well.

 The ELISA results are as follows:

 The mean OD of 36 normal human serum = 0.057, SD = 0.0234 (remove a discrete far value). Taking the mean value of 2.1 times as the criterion, the OD value is greater than 0.120. Of the 36 normal human serum negative for fecal test, 2 were greater than 0.120. The specificity of the ELISA method for detecting antibodies with pET28b-CSl insoluble protein was =34/36 X 100% = 94.4%.

 The mean OD of 35 fecal positive samples was 0.362, SD=0.234, and 30 of them had serum OD values greater than 0.120. The sensitivity of the established ELISA method = 30/35 X 100% = 85.7%.

 The total coincidence rate of this ELISA method = (34 + 30) I (36 + 35 ) X 100% = 90.1%.

The serum OD values of 15 patients with schistosomiasis and 9 patients with Paragonimus faecalis were less than 0.120, and none of them showed false positives. The ELISA method is highly specific and does not cross-react with schistosomiasis and paragonimiasi antibodies.

 Indirect ELISA results of pET28b-CSl insoluble purified protein for detection of specific antibodies in serum

 OD! Positive threshold Sample Positive Negative

 (X soil SD) (negative mean 2.1 times)

 Serum of Clonorchis sinensis patients 0.362 ± 0.234 0. 120 35 30 5

 Normal human serum 0.057 ±0.023 0. 120 36 2 34

 Serum of Schistosomiasis patients 0.059 ±0.029 0. 120 15 0 15

Serum of Paragonimus sinensis patients 0.040 ±0.014 0. 120 9 0 9

 A scatter plot of pET28b-CSl insoluble purified protein for the detection of specific antibodies in various sera is shown in Figure 14. Example 8 Detection of immunogenicity of Csl antigen of Clonorchis sinensis

Six Balb/c mice were immunized with pET28b-CSl insoluble and soluble purified protein, respectively. Each mouse was inoculated with 30 ug of antigen, and the first inoculation was performed with Freund's complete adjuvant (Sigma), subcutaneously; every 3 weeks, intensively with Freund's incomplete adjuvant (Sigma), intraperitoneal injection, and booster immunization 4 times. Take the fractional tail blood, indirect ELISA method to detect the mouse immune serum (pET28b-CSl insoluble and soluble purified protein 2ug/ml concentration, lOOul coated ELISA plate, The secondary antibody was Sigma's HRP-labeled goat anti-mouse IgG, 1: 5 000 dilution).

 Result:

 Balb/c mice were immunized with pET28b-CSl insoluble protein, and purified with pET28b-CSl insoluble protein 2ug/ml, lOOul coated with enzyme plate, and detected by indirect ELISA. The average OD value was 0.710 and the SD was 0.033. The OD values of normal mice and PBS blanks were 0.055 and 0.055, respectively.

 Balb/c mice were immunized with pET28b-CSl insoluble protein, and purified with pET28b-CSl soluble protein 2ug/ml, lOOul coated with enzyme plate, and indirect ELISA. The average OD value was 0.677 and the SD was 0.017. The OD values of normal mice and PBS blanks were 0.077 and 0.056, respectively.

 Balb/c mice were immunized with pET28b-CSl soluble purified protein, purified with pET28b-CSl soluble protein 2ug/mr concentration, lOOul coated with enzyme plate, and detected by indirect ELISA. The mean OD was 0.641 and the SD was 0.020. The OD values of normal mice and PBS blanks were 0.077 and 0.056, respectively.

 Balb/c mice were immunized with pET28b-CSl soluble purified protein, and the concentration of insoluble protein 2ug/ml was purified by pET28b-CSl, lOOul coated with enzyme plate, and indirect ELISA. The average OD value was 0.685 and the SD was 0.021. The OD values of normal mice and PBS blanks were 0.055 and 0.055, respectively.

 The specific results are shown in the following table:

 pET28b-CSl recombinant protein purification effect indirect ELISA test results

 Soluble pure insoluble pure soluble soluble pure insoluble pure

 Immune antigen

 Proteinized protein

 1 0. 628 0. 702 0. 712 0. 742

 2 0. 611 0. 661 0. 654 0. 694

 3 0. 642 0. 664 0. 669 0. 690

 Immune mouse number

 4 0. 634 0. 656 0. 674 0. 654

 5 0. 672 0. 683 0. 698 0. 735

6 0. 661 0. 693 0. 704 0. 744 Normal mouse/PBS blank

 0. 077 0. 056 0. 055 0. 055

 Control

 ELISA detection of coated anti-soluble protein, insoluble, purified protein

 The original 2ug/ml 2u?/ml experimental results show that both pET28b-CSl soluble purified protein and insoluble purified protein have strong immunogenicity and easy preparation of multi/monoclonal antibodies. Experimental mice produced a strong immune response to both groups of antigens. Example 9 Comparison of homology between Csl antigens of Clonorchis sinensis

 Using Blastp to compare the homology of the nr library in GenBank, the results are as follows:

 Search database NOn-redundant protein sequences (nr) using Blastp (protein-protein BLAST)

 Score E

 Sequences producing significant alignments: (Bits) Value emb|CAX64066.1 | conserved Plasmodium protein, unkNOwn functio... 54.3 7e-06 reflXP_001349168.11 hypothetical protein [Plasmodium falcipar... 53.9 9e-06 ref]YP_001321224.11 triple helix Repeat-containing collagen [... 51.2 5e-05 ref]ZP_05227486.1| RfbE [Mycobacterium intracellulars ATCC 13... 47.4 8e-04 reflXP~001137791.1| PREDICTED: hypothetical protein [Pan trog... 45.4 0.003 gb|AAH96211 .1| PRB3 protein [Homo sapiens] 44.7 0.005 sp|Q04118.2|PRB3_HUMAN RecName: Full=Basic salivary proline-r... 43.5 0.011 reflNP_006240.4| proline-rich protein BstNI subfamily 3 precu... 43.5 0.012 gb |EAW96230.11 proline-rich protein BstNI subfamily 3 [Homo s... 43.1 0.015 reflXP_001613963.11 ubiquitin-conj ugating enzyme domain conta... 41.2 0.060 gb|ACF35339.11 fiilensin [Cavia porcellus] 39.3 0.23 emb|CAA30728. 1| proline-rich protein Gl [Homo sapiens] 39.3 0.24

reflXP_001776976.11 predicted protein [Physcomitrella patens ... 38.9 0.30

reflYP— 896770.11 exosporium protein [Bacillus thuringiensis s... 38.9 0.32 gb|ABU49875.11 mucin [Schistosoma mansoni] 38.1 0.43

Gb|ACE88754.1| polymorphic mucin truncated splice variant IC6... 38.1 0.52 gb|ACE88790.1 j polymorphic mucin variant C7/2/25r2.1 [Schisto... 38.1 0.55

Gbj ACE88791.1| polymorphic mucin variant C7/2/25r2.2 [Schisto... 38.1 0.55

Gb|ACE88792.1| polymorphic mucin variant C7/2/25r2.3 [Schisto... 37.7 0.57

Gb|ACE88793.1 j polymorphic mucin variant C7/2/25r2.4 [Schisto... 37.7 0.58

Gb|ACE88794.1 j polymorphic mucin variant C7/2/25r2.5 [Schisto... 37.7 0.59

Gb|ACE88814.1| polymorphic mucin variant IC2/2/25r2.2 [Schist... 37.4 0.73

Gb|ACE8883 l.lj polymorphic mucin variant IC6/2/25r2.3 [Schist... 37.4 0.77

reffNP— 570367.11 collagen alpha 1 (I) chain precursor [Synecho... 37.4 0.84

Sp|P10T63.3|PRB4— HUMAN RecName: Full=Basic salivary proline-r... 37.4 0.94 gb|ACE88795.11 polymorphic mucin variant C7/2/30r2 [Schistose. 37.0 0.98 gb|ACE88808.11 polymorphic mucin variant IC10 /2/30r2.2 [Schis... 37.0 1.0

Gb|ACE88767.11 polymorphic mucin truncated splice variant IC9... 37.0 1.0

Gb|ACE88806.1 j polymorphic mucin variant IC 10/2/25r2 [Schisto... 37.0 1.1

Gb|ABU49878.11 mucin [Schistosoma mansoni] 37.0 1.1

gbjACE88807.il polymorphic mucin variant IC10/2/30r2.1 [Schis... 37.0 1.2

GbjACE88813.il polymorphic mucin variant ICl l/2/30r2.2 [Schis... 36.6 1.3

Gbj ACE88821.1 | polymorphic mucin variant IC3/2/30r2 [Schistos... 36.6 1.3

Gb|ACE88758.1 | polymorphic mucin truncated splice variant IC7... 36.6 1.4 i -

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 Sbjct 770 NGDD PNGDDKP-NGDDKPNGDDKPNGDDKPNGDD PNGDDKPNG — DDKPNSDDKQSS 825

Query 117 DPE— SDGAVADDAPPSQV PQGDPESDGAVADDAPPSQVKPQGDPESDGA 165

 D + D +DD S K D D +DD S D + G+

Sbjct 826 DDKRNSDDKQNSDDKQNSDDKQNSD— DKQNSDDKQNSDDKQNSDDNNGGS 874

 Score = 33.9 bits (76), Expect = 8.4, Method: Composition-based stats.

 Identities = 21/64 (32%), Positives = 26/64 (40%), Gaps = 12/64 (18%)

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 + + DG DD P KP GD D P KP GD + +G D P K

Sbjct 745 ETKEDGQKGDDKPNGDDKPNGD DKPNGDDKPNGDDKPNG — DDKPNGDDK 792

 Query 177 PQGE 180

 P G+

 Sbjct 793 PNGD 796

Using TBlastn to compare the homology of nr and nt libraries in GenBank, the results are as follows:

Search database Nucleotide collection (nr/nt) using Tblastn (search translated nucleotide using a protein query)

 Score E

 Sequences producing significant alignments: (Bits) Value

 Gb|BC096210.3| Homo sapiens proline-rich protein BstNI subfam... 52.4 9e-05 gb|BC096209.3| Homo sapiens proline-rich protein BstNI subfam... 52.4 9e-05 gb|BC096211.1 | Homo sapiens Proline-rich protein BstNI subfam... 52.0 le-04 gb|AC010176.121 Homo sapiens 12 BAC RP11-711 1 (Roswell Park ... 52.0 le-04 ref|N _006249.4| Homo sapiens proline-rich protein BstNI Subf... 50.8 3e-04 gb|CP000724.11 Alkaliphilus metalliredigens QYMF, complete ge... 50.4 3e-04 emb|X07881.11 Human gene PRB3L for proline-rich protein Gl 49.3 8e-04 reflNG Ol 3305.11 Homo sapiens proline -rich protein BstNI subf... 48.5 0.002 emb|X07637.11 Human PRB3 gene (PRB3S) for G 1 protein, exon 3 48.1 0.002 reflXM_001137791.1| PREDICTED: Pan troglodytes hypothetical L... 45.8 0.009 gb|AC 193211.4| Pan troglodytes BAC clone CH251-646021 from ch... 43.5 0.043 gb|AC 193209.31 Pan troglodytes BAC clone CH251-562J12 from ch... 43.5 0.043 gb|AC 192834.31 Pan troglodytes BAC clone CH251-398J20 from ch... 43.5 0.049 Gb|CP001337.1 | Chloroflexus aggregans DSM 9485, complete geNOme 43.1 0.066 dbj|AK098523.11 Homo sapiens cDNA FLJ25657 fis, clone TST00324 40.0 0.50 ref|NM_l 2051 1.1 | Arabidopsis thaliana KTFl (KOW DOMAIN-CONTAI... 39.7 0.66 gb|AC213901.5| MACACA MULATTA BAC clone CH250-175D19 from ch •...37.7 2.3

 Gb|AC206545.3| Pongo abelii BAC clone CH276-487H8 from chromo... 37.7 2.5 emb|X07715.1 | Human gene PRB4 for proline-rich protein Po, al... 37.4 3.1

 Gb| AF247176.11 AF247176 Synthetic construct plasmid pSB3278 RP... 37.4 3.1 gb|AC140118.11| Homo sapiens 12 BAC RP13-507P19 (Roswell Park... 36.6 4.9 gb|EU827599.1| Cavia porcellus fiilensin (BFSP1) mRNA , comple... 36.6 5.1 and the first case: conserved Plasmodium protein, unkNOwn function [Plasmodium falciparum 3D7] has an amino acid identity of only 33%. As shown below: gb|BC096210.3| Homo sapiens proline-rich protein BstNI subfamily 3, mRNA (cDNA clone MGC: 116863

IMAGE: 40004741), complete cds

 Length=1058

 GENE ID: 5544 PRB3 | proline-rich protein BstNI subfamily 3 [Homo sapiens]

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Z+ = aureij

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 £+ = 3UIEJJ

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 8£9 Hddd00S0N000dddO I6S fqS

Ή 0+ O

 ^61 yiIIJ0D3⁄4dN3⁄4W3⁄4IHO 6LI ^i

06S acraO<raddd00S0N000dddOH< IOdHddd00S0N000dcIdOHd"HOd«dddOC)S0NOOe)d llf fqs

 +d3⁄4 dd + o do+d3⁄4 dd + o do+<ra dd + o

 8.I OcraA0sddvaovAVoas3dao0d3⁄4A0SddvaavAVoasadao r3⁄4A0SdrvaavAvoas3 6ii ^snf) oi ddOHcraojaddd00si)Noo0dd0oad3⁄4Ocrawd00si) 000dcicioaci3⁄4o<raddd00s0No \zz pfqs dO+cT dd + Ό dO+d3⁄4 dd + O d 33⁄4d 0 + +

 8u <ia!Di)diAi)ScWvaavAVoas3<iaoi)craAC)ScidvaavAV{as3da:)3£) Adss0VE>avaav 6S

(%0) 9EI/0 = sd3⁄4o '(%6ε) 9El/ = ssAijisoj '(ο _/ο0ε ) _{9 1?} = ssijijuspi •jsnfpB xujBiu iBuo;i;sodui03: poqjsj^ 'ΟΖ Ό = sdxg '(pOl) sjiq = sjoog oz.1 dAoM3⁄4ADOoodJOScniDODcWT a dDooyoddOSdOOODddd ayWdOD 9ΐε pfqs

 J Ή+0 d+ dd O dDd dd a o

 98i JM3⁄4iArai30— i)d3⁄4A0ScWva£)VAVDas3dac)C»craAi)S<icivaavAV{) ε^ι

Hi 0HDdd0Sd3003ddHAVaAVdOODy0dd0Sd9903ddJAVI dO0O«0dJOScI0O0Dddd CLV\ 96 fqS

 Dod dd a D dOd dd a o dDd dd a zn as3dcro. craAiScHvaavAV€)as3iaoC)d3⁄4AC)SddvaavAvocis3dao0craA0Sdcivaav £8

 Z- = aurej

601/S = sdEQ '(%££) 60l/9£ = saAtjisoj '(o/ _ol£ ) ₆ oi/w = saijijuapi •jsnfpe uieui |Buoiiisoduio3: ροφ3)Λΐ ' 10Ό = loadxg '(SOI) si;qs ^ = aioos

 Od+ dd oo dOd+ dd ao do 6L\ oC)<raAC)Sddva£)VAV£)as3daoi)<raAC)SdcivaavAVi)(iS3da£>C) 9ti su dD003ddd ayWdooo3⁄4OddosdDoooddHwa dooo3⁄4OdJOSdOD03ddJAa doooaod it pfqs d dd a D doa dd ao dod dd Aod eei d3⁄4Ai)ScidvaavAvoas3da£f)d3⁄4At)ScidvaavA £)as3ciaof)<raAi)S<HvaavAvoas3<i 9L

(%0) Ol/0 = sdBQ '(o _/oSi ;) oi/a = saAiiisoj \%iz) Wl/Se = saijijuapi •IsnfpB xiajBiu [Buoi}isodtuo3: poipsj^ 'S0— ³ 6 = sd g '( ^i) s;iq γις = aioos

ZZll00/0l0ZN3/X3d ΐΐ .990/ΤΤ0ί ΟΛ\ G

 Sbjct 717 G 719

 Score = 43.1 bits ( 100), Expect = 0.055, Method: Compositional matrix adjust.

 Identities = 28/86 (32%), Positives = 31/86 (36%), Gaps = 0/86 (0%)

 Frame = -2

 Query 76 PESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQV P 135

 P G D PP P G P G D PP P G P G D PP +P

Sbjct 391 PGRGGGPWDWFPPCGGCPSGFPGRGGGPWDWFPPCGGGPSGFPGRGGGPWDWLPPCGGRP 212

Query 136 QGDPESDGAVADDAPPSQVKPQGDPE 161

 G P G PP +P G P+

Sbjct 211 SGFPGGGGVRWGWFPPCGRRPSGFPD 134

 Score = 43.1 bits ( 100), Expect = 0.062, Method: Compositional matrix adjust.

 Identities = 40/126 (31%), Positives = 51/126 (40%), Gaps = 0/126 (0%)

 Frame = +3

 Query 69 PV GECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDA 128

 P K E P G + PP KP+G P G + PP KP+G P G +

Sbjct 198 PGKPEGRPPQGGNQSQGPPPRPG PEGPPPQGGNQSQGPPP PGKPEGQPPQGGNQSQGP 377

Query 129 PPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQGEIRMRKFRC 188

 PP KP+G P G + PP KP+G P G + PP KP+G +

Sbjct 378 PPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPHPGKPEGPPPQGGNQS 557

Query 189 QFIILR 194

 Q

Sbjct 558 QGPPPR 575

 Score = 40.4 bits (93), Expect = 0.36, Method: Compositional matrix adjust.

 Identities = 34/106 (32%), Positives = 44/106 (41%), Gaps = 0/106 (0%)

 Frame = +3

 Query 59 ADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQV PQGDP 118

 + + G P K E P G + PP KP+G P G + PP KP+G P

Sbjct 420 GNQSQGPPPRPG PEGPPPQGGNQSQGPPPHPGKPEGPPPQGGNQSQGPPPRPG PEGPP 599

Query 119 ESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQV PQGDPESDG 164

 G + PP KP+G P G + PP KP+G P G

Sbjct 600 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGSPSQGG 737

Score = 38.1 bits (87), Expect = 2.1, Method: Compositional matrix adjust.

 Identities = 34/100 (34%), Positives = 43/100 (43%), Gaps = 0/100 (0%)

 Frame = +3

 Query 80 GAVADDAPPSQVKPQGDPESDGAVADDAPPSQV PQGDPESDGAVADDAPPSQV PQGDP 139

 G PP KP+G P G + PP KP+G P G + PP KP+G P

Sbjct 168 GNQPQRTPPPPG PEGRPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGQP 347

Query 140 ESDGAVADDAPPSQV PQGDPESDGAVAGDAPPSQV PQG 179

 G + PP KP+G P G + PP KP+G

Sbjct 348 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEG 467 By comparison of homology, the Csl antigen gene of C. sinensis is a novel specific antigen of C. sinensis.

Claims

Claim

 A specific antigen of Clonorchis sinensis, characterized in that the antigen gene has the nucleotide sequence shown in SEQ ID NO: 1, or is homologous to the nucleotide sequence shown in SEQ ID NO: 1. A nucleotide sequence having an antigenicity of 90% or more, or an antigenic nucleotide sequence derived from the nucleotide sequence shown by SEQ ID NO: 1 after partial substitution, deletion or addition.

 An antibody which specifically binds to a specific antigen of C. sinensis according to claim 1.

 3. An antibody according to claim 2, wherein the antibody is a monoclonal antibody.

An isolated protein characterized by: a nucleotide sequence encoding a specific antigen gene of Clonorchis sinensis according to claim 1, or a Chinese branch according to claim 1. Specificity of a Clostridium sinensis according to claim 1 in which the nucleotide sequence of the specific antigen gene of S. cerevisiae is encoded by more than 90% of the nucleotide sequence, or is partially substituted, deleted or added. The nucleotide sequence of the antigen gene is encoded.

 The isolated protein according to claim 4, wherein the amino acid sequence is as shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted or added one or more The amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 after the amino acid.

 A vector comprising: the vector comprising the specific antigen gene of C. sinensis according to claim 1.

 7. A host cell, characterized in that: the cell comprises the vector of claim 6, or the cell comprises the specific antigen gene of C. sinensis according to claim 1. Transformation or transfection.

 A vaccine comprising: a specific antigen of Clonorchis sinensis according to claim 1, or the antibody of claim 2, or the isolated protein of claim 4, or a right The carrier of claim 6.

9. The use of a specific antigen of Clonorchis sinensis according to claim 1 for the preparation of a medicament for the treatment, diagnosis or prevention of clonorchiasis. Use of the antibody of claim 2 for the manufacture of a medicament for the treatment, diagnosis or prevention of clonorchiasis.

Use of the isolated protein of claim 4 for the manufacture of a medicament for the treatment, diagnosis or prevention of clonorchiasis.

Use of the vaccine of claim 8 for the manufacture of a medicament for the treatment or prevention of clonorchiasis.

A kit comprising the antigen according to claim 1 or the antibody according to claim 2.