CN102071205B - Specific antigen of clonorchis sinensis and application thereof - Google Patents

Abstract

The invention provides a specific antigen of clonorchis sinensis, and also provides a specific antibody combining the specific antigen of the clonorchis sinensis, a separated protein which is coded by a nucleotide sequence of a specific antigenic gene of the clonorchis sinensis, a vector containing the specific antigenic gene of the clonorchis sinensis, a vaccine, a kit and application of the specific antigen of the clonorchis sinensis, the antibody, the vaccine and the kit in preparing a drug for treating, diagnosing or preventing the clonorchiosis. The Cs1 antibody of the clonorchis sinensis has higher immunogenicity, can be used for easily preparing a polyclonal/monoclonal antibody, and is further applied to the aspects such as antigen detection and the like. Moreover, the stronger immune response of an experimental animal (mouse) on the antigen is realized.

Description

The specific antigens of clonorchis sinensis and purposes

Technical field:

The invention belongs to bioengineering field, relate in particular to a kind of clonorchis sinensis, is a kind of specific antigens and purposes of clonorchis sinensis specifically.

Background technology:

Clonorchiasis sinensis (Clonorchiasis sinensis) is to infect caused Amphixenosis by clonorchis sinensis [Clonorchis sinensis, Cs].This disease mainly is distributed in the Asia, like countries such as China, Japan, Korea S's (being the main parasite of human of Korea S), Korea, Vietnam, South East Asia.Estimate that the whole world has 3,500 ten thousand people to infect.In China, except that province, autonomous regions such as Xinjiang, the Inner Mongol, Gansu, Qinghai, Tibet, Ningxia did not appear in the newspapers, all the other 25 provinces and Taiwan Province and the Hong Kong Special Administrative Region all had the popular report or the case report of this disease.Because it is the immigrant's flows, and the tourism of frequent day by day global range and economic activity, also more and more in some non-popular districts and this sick case report of developed country's (comprising North America and West Europe).

The sick on-site investigation result of human body important parasite who will carry out in the whole nation (except that Taiwan, Hong Kong, Macao) June calendar year 2001～end of the year 2004 according to the Ministry of Health shows; The clonorchis sinensis infection rate has risen 75% than the result of nineteen ninety national parasitosis distribution investigation, and wherein Guangdong, Guangxi, Jilin 3 provinces (district) have risen 182%, 164% and 630% respectively.Estimate that present national clonorchis sinensis the infected reaches more than 1,200 ten thousand people.Clonorchiasis sinensis is one of indivedual several parasitosis that present ascendant trend of China's minority, and these sick preventing and controlling are extremely urgent.

(like Guangdong, Guangxi) and part northern area in China south (like the Korean nationality residential district of northeast 3 provinces), local crowd has the custom of eating fresh water living or that the do not boil flesh of fish, and the bladder worm that lives in the flesh of fish human body that is ingested causes people's infection.Though these regional residents know that eating " sashimi (raw fish) " can infect this disease, food habits is difficult to change for the moment.And along with growth in the living standard, the crowd who did not eat in the past or seldom ate " sashimi (raw fish) " also begins to eat " sashimi (raw fish) ", and the local flavor eating method of sashimi is also very in vogue in non-popular district, particularly big and medium-sized cities now.Simultaneously because market access, the fish that popular district contains encysted metacercaria of clonorchis sinensis transports various places to, so town dweller's number of infecting clonorchis sinensis has the trend of increase.

The clonorchis sinensis adult parasitizes in people or other final host's the hepatic duct, and the mechanical stimulus of secretion/meta-bolites of polypide and polypide itself can cause bile duct; Particularly cause the inflammatory reaction of secondary bile duct, the limitation expansion can appear in the grade and moderate infection bile duct, and epithelial duct hyperplasia, tube wall thicken, luminal stenosis; As merge infectation of bacteria; Can cause cholangitis and cholangiohepatitis, proliferation of fibrous tissue on every side, liver cirrhosis can take place in late period.Evidence suggests that clonorchis sinensis infects can increase the risk that the patient suffers from cholangiocarcinoma (CLG), it is the important public health problem in the popular district of clonorchiasis sinensis that clonorchis sinensis infects dependency hepatic duct tumour.

For quick diagnosis, treatment in time with effectively control clonorchiasis sinensis, need the infection of clonorchis sinensis among special, sensitive, the easy method detection crowd.

For clonorchis sinensis patient's diagnosis, the traditional fecal inspection is still the main method of making a definite diagnosis clonorchiasis sinensis at present.But because patient's compliance is relatively poor, and the clonorchis sinensis worm's ovum is less is easy to omission, and this method has certain defective.

Used immune diagnostic method (mainly being the ELISA method) more widely in the preventing and controlling at the scene at present.But still have some problems: healthy subjects is had false positive in various degree, the clonorchiasis sinensis people is had certain false negative, other parasite (schistosomicide, paragonimus) is infected also has certain cross reaction.Yong TS uses the ELISA method and detects 48 routine clonorchis sinensis patients serum antibody, has only 75% positive reaction, but in 28 routine normal controls and 16 routine paragonimus cases, has occurred 7.1% and 37.5% false positive respectively.

Existing research data shows that the ESA of some parasitic excretory-secretory antigens (ESA) or reorganization can be applicable to the serodiagnosis of parasitosis.Kim SI comes the dynamic response of detected activity property clonorchiasis sinensis patients serum corresponding antibodies with ESA; Find that 30kDa and 7kDa district band is strong positive reaction with the patients serum; And a little less than reacting with the patients serum of praziquantel treatment after 6 months; Wherein 7kDa district band do not see with paragonimiasis westermani, metagonimiasis yokogawai, clonorchis sinensis patient recovery from illness after serum play cross reaction; Specificity is stronger than 30kDa district band, thinks that in view of the above 7kDa antigen can be used as the significant diagnostic antigen of reactivity clonorchiasis sinensis.Min-Ho CHOI detects ELISA method and the ELISA method of adult crude antigen (CA) detection antibody of antibody to the excretory-secretory antigen (ESA) of clonorchis sinensis diagnostic value compares and assesses.The specificity that ESA detects the ELISA method of antibody is significantly higher than the ELISA method (the former specificity is 93.1%, and the latter is 87.8%) that detects antibody with CA, shows that ESA is used to detect clonorchis sinensis patients serum antibody and is superior to crude antigen.Therefore, compare with the CA of clonorchis sinensis, ESA has higher specificity and susceptibility as the diagnostic antigen of clonorchiasis sinensis.Yet traditional worm source property antigen prepd is complicated, and quality controllability is lower, and preparation amount is limited, is difficult to adapt to the demand of looking into disease on a large scale.

In addition,, determined that antigen and the antibody horizontal in host's serum is lower, be difficult to detection (also being the reason that at present should disease lacks good diagnostic reagent), but the existence of ESA has been arranged in excrement appearance because this worm parasitizes this specific position of hepatic duct.Yong TS etc. has identified the clonorchis sinensis antigen with the IgE antibody response, finds that the antigen of 28kDa and IgE reaction are the strongest, and this albumen also is present in the excrement.(the antigen minimum quantity that can detect is 0.05ng-0.1ng to human monoclonal antibody ELISA such as Sirisinha S for Monoclonal antibody ELISA, the McAb-ELISA) antigen in the detection opisthorchis viverrini patient excrement appearance.Research shows, detects the specific antigen in patient's excrement appearance, can provide early diagnosis, existing disease patient to make a definite diagnosis and the foundation of efficacy assessment.

And diagnosis antibody or antigenic characteristic are determining the diagnosis effect of immunological method such as antigen or Serum Antibody Detection in excrement appearance and the serum.Owing to be difficult to obtain highly purified specific antigen or a large amount of natural antigens, so use Protocols in Molecular Biology, the technological line that makes up the specificity recombinant antigen is the only way of seeking suitable antigen simultaneously.

Summary of the invention:

The object of the present invention is to provide a kind of specific antigens and purposes of clonorchis sinensis, specificity and the susceptibility that described this antigen and purposes will solve in the prior art diagnosis clonorchiasis sinensis is the high-technology problem not.

The invention provides a kind of specific antigens of clonorchis sinensis, described antigen gene have nucleotide sequence shown in the SEQ ID NO:1, or with nucleotide sequence homology shown in the SEQ ID NO:1 reach 90% or more nucleotide sequence or its part through replacement, disappearance or add after have antigenic nucleotide sequence by the deutero-of nucleotide sequence institute shown in the SEQ ID NO:1.

The present invention also provides a kind of antibody, the specific antigens of a kind of clonorchis sinensis that its specific combination is above-mentioned.

Further, described antibody is monoclonal antibody.

The present invention also provides a kind of isolating protein, coded by the nucleotide sequence of the specific anti protogene of above-mentioned a kind of clonorchis sinensis, or with the nucleotide sequence of the specific anti protogene of above-mentioned a kind of clonorchis sinensis reach nucleotide sequence 90% or more coded, or by part through replacement, the nucleotide sequence of the specific anti protogene of above-mentioned a kind of clonorchis sinensis after disappearance or the interpolation is coded.

Further, described isolating proteinic aminoacid sequence shown in SEQ ID NO:2, or to the aminoacid sequence shown in the SEQ ID NO:2 through replacement, disappearance or add one or several amino acid after by the aminoacid sequence institute deutero-aminoacid sequence shown in the SEQ ID NO:2.

The present invention also provides a kind of carrier, and described carrier contains the specific anti protogene of above-mentioned a kind of clonorchis sinensis.

The present invention also provides a kind of host cell, and described cell contains above-mentioned carrier, and perhaps described cell is with the specific antigens gene transformation or the transfection of above-mentioned described a kind of clonorchis sinensis.

The present invention also provides a kind of vaccine, comprises specific antigens, or the above-mentioned antibody, or above-mentioned isolating protein, or above-mentioned carrier of above-mentioned a kind of clonorchis sinensis.

The specific antigens that the present invention also provides above-mentioned a kind of clonorchis sinensis is in preparation treatment, diagnosis or prevent the application in the medicine of clonorchiasis sinensis.

The present invention also provides the application in the medicine that above-mentioned antibody is treated in preparation, diagnosis perhaps prevents clonorchiasis sinensis.

The present invention also provides the application in the medicine that above-mentioned isolating protein is treated in preparation, diagnosis perhaps prevents clonorchiasis sinensis.

The present invention also provide above-mentioned vaccine in the preparation treatment, or prevent the application in the medicine of clonorchiasis sinensis.

The present invention also provides a kind of test kit, contains above-mentioned antigens, or above-mentioned antibody.

Term among this paper " expression vector " is meant bacterial plasmid, yeast plasmid and other various virus vector commonly used in this area.The carrier that is suitable among the present invention includes but not limited to: the carrier (retroviral vector, adenovirus carrier etc.) of in bacterium, expressing the carrier (prokaryotic expression carrier) of usefulness, in yeast, expressing the carrier (like pichia vector) of usefulness, in mammalian cell, expressing usefulness.In a preferred embodiment, said expression vector is a coli expression carrier.A series of technology such as those skilled in the art's DNA recombinant technology capable of using make up the dna sequence dna contain encoding fusion protein according to the invention, suitable expression vector of transcribing with particular element such as translational control sequence, promotor and selected markers.Above-mentioned carrier can be used to transform, the transfection proper host cell, so that obtain needed fusion rotein.

Host cell of the present invention can be a prokaryotic cell prokaryocyte, also can be eukaryotic cell, as, bacterial cell, mammalian cell etc.Host cell promptly constitutes through engineering approaches cell or cell strain after transforming or transfection contains the gene order of encoding fusion protein according to the invention, can be used for producing required fusion rotein.Those skilled in the art can select appropriate carriers, host cell rightly; And how know carrier high-efficiency ground is transformed or is transfected in the host cell; Method therefor includes but not limited to: Calcium Chloride Method, electroporation are used for bacterial cell, and liposome, electro fusion method are used for eukaryotic cells such as mammalian cell.

Host cell of the present invention can be cultivated through ordinary method, induced and express needed fusion rotein, comprises fermenting process and purifying process.Above-mentioned expressed proteins can be in cell, on the cytolemma or be secreted into cell pericentral siphon, extracellular.As required, the physics of fusion rotein capable of using, chemistry and other biological characteristics carries out separation and purification.Method includes but not limited to: split bacterium, and centrifugal, saltout, molecular sieve chromatography, ion-exchange chromatography, adsorption chromatography, reverse chromatograms, and conventional sex change, renaturation processing etc., these methods all are well-known to those skilled in the art.

The clonorchis sinensis adult cDNA expression library of the present invention through the inventor is made up with the clonorchis sinensis patient pooled serum that picks up from Guangxi Zhuang Autonomous Region, uses immunoscreening to sift out clonorchis sinensis Cs1 antigen.Through homology relatively, clonorchis sinensis Cs1 antigen of the present invention is the specific antigens that does not also have disclosed clonorchis sinensis novel up to now.Through the antigenic expression and purification of clonorchis sinensis Cs1, further set up the first one step process of clonorchiasis sinensis diagnosis of high specific and susceptibility; And be prone to the many/monoclonal antibody of preparation, can use the different aspects such as scientific research and control of clonorchiasis sinensis; Vaccine of the present invention has the effect that the prevention clonorchis sinensis infects.Test kit of the present invention can also be used to detecting clonorchiasis sinensis.

Description of drawings:

Fig. 1 is clonorchis sinensis antigens c s1 5 ' RACE amplification cDNA fragment 1% gel electrophoresis figure, M:DNA marker, 1-4: amplification cDNA fragment (about 200bp).

Fig. 2 is pBluescript SK_Cs1 phagemid EcoRI/XhoI double digestion 1% gel electrophoresis figure; M1: λ DNA/HindIII+EcoR I Marker; M2: Φ X174 DNA/BsuRI (HaeIII) Marker; 1-2:pBluescript SK_Cs1 phagemid EcoRI/XhoI double digestion result (, a unsharp band being arranged still) at the 160bp place.

Fig. 3 is the agarose gel electrophoresis of pET28a NcoI/HindIII double digestion and Cs1-NcoI pcr amplification 1%, M:DNA Marker; 1-2:pET28a NcoI/HindIII double digestion result; 3-4:Cs1-NcoI pcr amplification result (about 610bp).

Fig. 4 is the agarose gel electrophoresis figure of pET28a-CS1-NCOI plasmid NcoI/HindIII double digestion 1%, wherein, and M:DNA Marker; 1:pET28a-CS1-NCOI plasmid NcoI/HindIII double digestion result.

Fig. 5 is that pET28b-CS1 BL21 (DE3) expression of recombinant proteins is identified 16% polypropylene amine gel electrophoresis figure, M:Protein Marker (FERMENTAS MBI); 1:pET28b-CS1 BL21 (DE3) does not induce; 2:pET28b-CS1 BL21 (DE3) induces; 3: induce supernatant; 4: induced precipitation.

Fig. 6 is that pET28a-CS1-NcoI BL21 (DE3) expression of recombinant proteins is identified 16% polypropylene amine gel electrophoresis figure, M:Protein Marker (FERMENTAS MBI); 1:pET28a-CS1-NcoI BL21 (DE3) does not induce; 2:pET28a-CS1-NcoI BL21 (DE3) induces; 3: induce supernatant; 4: induced precipitation.

Fig. 7 is that pET28b-CS1 BLR (DE3) expression of recombinant proteins is identified 12% polypropylene amine gel electrophoresis figure, M:Protein Marker (FERMENTAS MBI); 1:pET28b-CS1 BLR (DE3) does not induce; 2:pET28b-CS1 BLR (DE3) induces; 3: induce supernatant; 4: induced precipitation.

Fig. 8 is that pET28a-CS1-NcoI BLR (DE3) expression of recombinant proteins is identified 12% polypropylene amine gel electrophoresis figure, M:Protein Marker (FERMENTAS MBI); 1:pET28a-CS1-NcoI BLR (DE3) does not induce; 2:pET28a-CS1-NcoI BLR (DE3) induces; 3: induce supernatant; 4: induced precipitation.

Fig. 9 is that pET28a-CS1DS-NcoI B21 (DE3) expression of recombinant proteins is identified 12% polypropylene amine gel electrophoresis figure,, wherein, M:Protein Marker (FERMENTAS MBI); 1:pET28a-CS1DS-NcoI BL21 (DE3) does not induce; 2:pET28a-CS1DS-NcoIBL21 (DE3) induces; 3: induce supernatant; 4: induced precipitation.

Figure 10 is pET28b-CS1 BL21 (DE3) insolubility protein purification 12% polyacrylamide gel electrophoresis figure, M:Protein marker; 1: effluent (F) pH8.0; 2-4: Buffer C liquid 1,3,5 pipes (pH6.3); 5-9:Buffer D liquid 1-5 manages (pH5.9); 10-14:Buffer E liquid 1-5 manages (pH4.5), and wherein, pET28b-CS1 BL21 (DE3) insolubility albumen concentrates in the elutriant of pH4.5.

Figure 11 is pET28b-CS1 BL21 (DE3) soluble proteins purifying 12% polyacrylamide gel electrophoresis figure, M:Protein marker; 1:pET28b-CS1 does not induce; 2:pET28b-CS1 IPTG induces 3:Ni-NTA purified stream fluid (not containing imidazoles); 4-7: contain 20mM imidazoles washing lotion tube 1,3,5,7; 8-9: contain 50mM imidazoles elutriant 1,2 pipes; 10-11: contain 100mM imidazoles elutriant 1,2 pipes; 12-14: contain 250mM imidazoles elutriant 1,2,3 pipes, wherein, pET28b-CS1 BL21 (DE3) soluble proteins concentrates in the elutriant that contains the 50-250mM imidazoles.

Figure 12 is pET28a-CS1-NcoI BL21 (DE3) solubility and insolubility protein purification 12% polyacrylamide gel electrophoresis figure, M:Protein marker; 1: effluent (F); 2-8: contain 250mM imidazoles soluble proteins elutriant 1-7 pipe; 9-14: insolubility protein B uffer E liquid 1-6 manages (pH4.5), and wherein, pET28a-CS1-NcoI BL21 (DE3) soluble proteins concentrates in the elutriant that contains the 250mM imidazoles.Insolubility albumen concentrates in the elutriant of pH4.5.

Figure 13 is pET28b-CS1DS-NcoI BL21 (DE3) soluble proteins purifying 12% polyacrylamide gel electrophoresis figure, and pET28b-CS1DS-NcoI BL21 (DE3) soluble proteins concentrates in the elutriant that contains the 100-250mM imidazoles.Wherein, M:Protein marker; 1:Ni-NTA purified stream fluid; 2-3: contain 20mM imidazoles washings 2,4 pipes; 4-6: contain 50mM imidazoles elutriant 1-3 pipe; 7-9: contain 100mM imidazoles elutriant 1-3 pipe; 10-14: contain 250mM imidazoles elutriant 1-5 pipe.

Figure 14 is that pET28b-CS1 insolubility purifying protein detects the serum antibody scatter diagram.

For the ease of understanding, below will the present invention be described in detail through concrete embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change have been obviously all concerning one of ordinary skill in the art, and these equivalent modifications all should belong to institute of the present invention restricted portion.

Embodiment

The following stated experimental technique does not specify, all according to " molecular cloning experiment guide ",, Science Press in 2002) said method carries out.

The structure in embodiment 1 clonorchis sinensis adult cDNA library

With the encysted metacercaria of clonorchis sinensis that picks up from Guangxi Zhuang Autonomous Region, infect 5 of cats through administration by gavage.After 40 days, the inspection faecal egg cuts open then and kills, and collects the clonorchis sinensis adult, and is clean with the normal saline flushing of sterilization, and liquid nitrogen is preserved.

Utilize total RNA of TRIzol (GIBCO/BRL company) test kit extracting clonorchis sinensis adult (1 gram clonorchis sinensis adult hematocrit, liquid nitrogen is preserved).

Adopt mRNA purification kit (mRNA Purification Kit, Amersham company), purified mRNA from the total RNA that extracts.Concrete steps are seen product description.

The directed cloning method is adopted in clonorchis sinensis adult cDNA library, utilizes ZAP-cDNASynthesis Kit (Stratagene company) to make up.The operation of reference reagent box specification sheets, main process comprises:

1.cDNA first chain is synthetic; Application contains the poly T primer of Xho I restriction enzyme site; In order to make the destruction of cDNA first chain unrestricted restriction endonuclease in building-up process, substitute the dCTP among the dNTP with 5-methyl dCTP, make synthetic DNA hemimethylation; Thereby when Xho I digested cdna, the non-site that methylates that only is positioned at poly T primer can be by cracking;

2.cDNA second chain is synthetic, digests the RNA in the first chain synthetic product mRNA-DNA heterozygote by RNase H, the cDNA fragment of generation is as primer synthetic second chain under the dna polymerase i effect;

3. with the end-filling of Pfu archaeal dna polymerase, add the joint (adaptor) that contains EcoR I restriction enzyme site at the 5 ' end of mending flat double-stranded DNA afterwards, and make the joint phosphorylation the double-stranded cDNA of synthetic;

4. with restriction enzyme Xho I digestion, postdigestive double-stranded DNA carries out fractional separation through the SepharoseCL-2B gel filtration chromatography, removes free joint;

5. the dna fragmentation after will separating merges concentrated, is connected on the Uni-ZAP XR carrier;

6. last with packing extract (GigapackIII Gold Packing Extract, Stratagene) packing phage protein coat.

After clonorchis sinensis adult cDNA library construction is accomplished, further library titre and insertion fragment mean length etc. is detected.

Detection computations result, the titre in clonorchis sinensis adult cDNA library is 1.43 * 10 ⁶Pfu/ml.

16 recombinant clones of picking carry out pcr amplification at random from the library, and 1% agarose gel electrophoresis is identified the PCR product.Recombinant clone expanding fragment length scope is at 0.6kb～2kb.Average insertion fragment length is 1.1kb.

Reorganization insertion rate is 99.6%.

The immunoscreening in embodiment 2 cDNA libraries

Phage-infect:

Getting XL1-blue MRF ' bacterium liquid 200 μ l mixes (definite in advance according to the library titre with an amount of cDNA library phagocytosis body fluid; About 3000 plaques/every plate); 37 ℃ of incubations added top-layer agar (top agar) mixing of 48 ℃ of 3ml after 15 minutes, were laid on immediately on the NZY culture medium flat plate.Solidified under the room temperature 10 minutes, and hatched in 42 ℃.

The abduction delivering of fusion rotein:

Observe plaque and back (about 3.5 hours) occurs, use and soak 30 minutes nitrocellulose filter through IPTG (15mM/L) (Hybond-C extra, Amersham NC) cover dull and stereotypedly, cultivate 3.5 hours in 37 ℃ again; Take out dull and stereotyped 4 ℃ of coolings 15 minutes, on film and plate, do marked with pin then.Film taken off place TBST to wash 3 times, each 10 minutes, be dipped in the NC film then and be immersed in the confining liquid, room temperature, slight vibration, sealing is spent the night.

The NC film is taken out from confining liquid, wash film 2 times, each 5 minutes with TBST.Add slight the vibration 3 hours under clonorchis sinensis patient's pooled serum (picking up from Guangxi Zhuang Autonomous Region) (5ml/ film) room temperature.Wash film 3 times with TBST again, each 10 minutes.Adding two anti-(5ml/ films) is to react 1 hour under goat-anti people alkaline phosphatase enzyme conjugates (AP-GAH, BIO-RAD, dilution in 1: the 3 000) room temperature, washes film 3 times with TBST, each 10 minutes, wash film 2 times with TBS, and each 5 minutes, the Tween20 that flush away is residual.

The NC film is taken out from TBS, blot excessive solution, put into AP buffer and soaked the NC film 3 minutes with the filter paper of Whatman 3MM.

To final concentration 0.3mg/ml, the BCIP final concentration is that (BCIP should dropwise add among the NBT that has diluted 0.15mg/ml, prevents that deposition from forming with Color Development Solution dilution NBT.), be mixed with NBT-BCIP colour developing liquid.The NC film is immersed in the NBT-BCIP colour developing liquid, and it is high-visible up to positive spots in the dark to carry out coupling reaction.The color development stopping reaction.

According to the corresponding position of positive spots on former culture plate on the NC film, the picking positive plaque, process is sieved again again, three sieves make the positive bacteriophage mono-clonalization.

Embodiment 3 phages deletion cyclisation is pBluescript SK_ phagemid and extracts phagemid

E.coli XL1-blue MRF ' is overnight cultures in LB substratum (containing 10mM MgSO4,0.2% SANMALT-S, 15 μ g/ml Tet).Next day, get nutrient solution 100 μ l and be transferred in the new LB substratum, 37 ℃, about 2 hours of 200rpm shaking culture.Nutrient solution is through 2000g, and centrifugal 10 minutes, precipitum, resuspended with 10mM MgSO4 to OD ₆₀₀=1.0.The resuspended liquid of adding 200 μ l E.coli XL1-blue MRF ', 50 μ l contain the SM buffer and the 1 μ l helper phage of positive bacteriophage in the microbial culture pipe.The microbial culture pipe was positioned in 37 ℃ of water-baths 15 minutes.Add 3ml LB then, in 37 ℃ of shaking culture 5 hours.Take out the microbial culture pipe, 65 ℃ of heating 20 minutes, 3000g then, centrifugal 15 minutes, supernatant is changed in the new centrifuge tube, place 4 ℃ of preservations.

The SOLR bacterium is shaking culture in LB substratum (containing 10mM MgSO4,50 μ g/ml Kan), and 2000g is centrifugal 10 minutes, resuspended to OD with 10mM MgSO4 then ₆₀₀=1.0, the supernatant that in 1.5ml Ep centrifuge tube, adds 200 μ l SOLR and above-mentioned preparation is preserved liquid 1 μ l, 37 ℃ of incubations 15 minutes, takes out 25 μ l then and evenly coats on LB (the containing 100 μ g/ml Amp) agar plate, is inverted overnight cultures for 37 ℃.

The bacterium colony of growing on the substratum is and contains the SOLR bacterium colony that clonorchis sinensis cDNA inserts segmental pBluescript SK_ phagemid.

Use AxyPrep DNA small volume of reagent box [love pursue progress biotechnology (Hangzhou) ltd] to extract pBluescript SK_ phagemid.

The separation of embodiment 4 Cs1 antigen genes

Use clonorchis sinensis patient's pooled serum (picking up from Guangxi Zhuang Autonomous Region) screening clonorchis sinensis adult (picking up from Guangxi Zhuang Autonomous Region) cDNA library (using the The ZAP-cDNA synthesis Synthesis Kit of Stratagene company to make up); The positive colony that obtains is pBluescript SK_Cs1 through the deletion cyclisation.By the order-checking of Shanghai Invitrogen company, obtain the part mRNA sequence (shown in SEQ ID NO:6) of clonorchis sinensis Cs1 antigen gene.Use the test kit (5 ' RACE System for RapidAmplification of cDNA Ends) of Invitrogen company, use following primer (GSP1:TGCGCACCATCCGCATCG; GSP2:GATGTGCTCGAGCCTGAAG, Invitrogen company in Shanghai is synthetic) the cDNA fragment of reverse transcription and amplification, insert pGEM-T Easy Vector (Promega company); Through order-checking (by the order-checking of Shanghai Invitrogen company); Obtain the full length sequence of clonorchis sinensis Cs1 antigen gene, shown in SEQ ID NO:1, totally 733 bases; 23 is initial son (ATG); 617 is terminator (TAG), and the Cs1 antigen aminoacid sequence of derivation is shown in SEQID NO:2, and Cs1 antigen contains 198 amino acid.Cs1 5 ' RACE amplification cDNA fragment 1% gel electrophoresis figure is as shown in Figure 1.

The structure of embodiment 5 expression vectors

1.pET28b-Cs1 the structure of expression vector

With pBluescript SK_Cs1 phagemid, make the part double digestion with EcoRI/XhoI (NEB company) restriction enzyme, 37 ℃ were reacted 15 minutes.

Enzyme is cut product at 1% agarose gel electrophoresis; The isolating purpose fragment of cutting under uv lamp is with E.Z.N.A.Ultra-Sep

Gel Extraction Kit (Omiga company) purifying and recovering purpose fragment (718bp).Because of inserting fragment in pBluescript SK-, the Cs1 antigen gene contains two XhoI restriction enzyme sites, thus under partially digested condition, insert fragment and present three fragments (718,559 and 159bp), as shown in Figure 2.

The pET28b expression vector uses EcoRI/XhoI (NEB company) restriction enzyme to make double digestion equally, and 37 ℃ of reactions are spent the night.Enzyme is cut product at 1% agarose gel electrophoresis; The isolating purpose fragment of cutting under uv lamp is with E.Z.N.A.Ultra-Sep

Gel ExtractionKit (Omiga company) purifying and recovering.

The purpose fragment is connected by 8: 1 mol ratios with carrier segments; Contain T4DNA ligase enzyme (NEB company) 1ul in the linked system; 10 * T4 ligase buffer 2ul; Deionized water, purpose fragment and carrier segments amount to volume be 20ul in 1.5ml Eppendorf pipe 16 ℃ be connected and spend the night, be built into the prokaryotic expression recombinant plasmid of pET28b-Cs1.

Get the pET28b-CS1 recombinant plasmid of connection and mix with the E.coli DH5 α competent cell that has just dissolved (day root company), ice bath 30 minutes was in 42 ℃ of heat shocks 1 minute; Place 3min through ice bath again, aseptic condition adds the SOC substratum of 1ml, 37 ℃ down; 200rpm, shaking culture 1 hour.Then, with bacterium liquid in 3 500rpm, centrifugal 3 minutes, discard the part supernatant, stay the resuspended thalline of about 100 μ l.Resuspended liquid is coated on the LB flat board (Kan that contains 50 μ g/ml), waited to absorb the back and be inverted overnight cultures in 37 ℃.

The DH5a bacterium that transforms is at LB substratum (Kan that contains 50 μ g/ml), 200rpm, and shaking culture is spent the night.Bacterium liquid in 3500rpm, centrifugal 10 minutes, is collected bacterium.Use AxyPrep DNA small volume of reagent box [love pursue progress biotechnology (Hangzhou) ltd] to extract plasmid, and by the order-checking of Shanghai Invitrogen company, confirm that the purpose fragment inserts correct.

The pET28b-CS1 recombinant plasmid of getting extraction is Transformed E .coli BL21 (DE3) competent cell (NOvagen company) once more, and method is the same.The aminoacid sequence of this recombinant protein (pET28b-CS1 recombinant plasmid) is as shown in SEQ ID NO:3.

2.pET28a-CS1-NcoI the structure of expression vector

According to the sequence of Cs1 gene, design following primer (synthetic) by Shanghai Invitrogen company:

PF-CS1-NCOI:5 ' CATGCCATGGGGATGAAACCGCAACTTGTATAC introduces the NcoI site

PR-CS1-NCOI:5 ' CCCAAGCTTTGATATGATTCTTCGTAGAAT introduces the HindIII site

With pBluescript SK_Cs1 phagemid is template, carries out pcr amplification.Reaction system 50ul, template 0.2ul wherein, each 1ul of two-way primer, dNTP (matching hundred victory companies) 1ul, 10 * buffer 5ul, platinum Taq enzyme (Invitrogen company) 0.5ul, 50mM mg ion 1.5ul, deionized water 39.8ul.Reaction conditions is 95 ℃ of preparatory sex change 5 minutes; 95 ℃ of sex change 1 minute; Anneal 50 ℃ 1 minute; 72 ℃ of renaturation 1 minute; Circulate 30 times, last circulation renaturation extends to 7 minutes, and sample retention is in 4 ℃.The PCR product is at 1% agarose gel electrophoresis (as shown in Figure 3); The isolating purpose fragment of cutting under uv lamp is with E.Z.N.A.Ultra-Sep Gel Extraction Kit (Omiga company) purifying and recovering.

PET28a expression vector plasmid is cut and is spent the night with NcoI, HindIII restriction enzyme (NEB company) enzyme; Use calf intestines alkaline phosphorus enzymes (CIP, NEB company) dephosphorylation.The enzyme product is at 1% agarose gel electrophoresis (as shown in Figure 3); The isolating purpose fragment of cutting under uv lamp is with E.Z.N.A.Ultra-Sep

Gel Extraction Kit (Omiga company) purifying and recovering.

The Cs1-NcoI PCR fragment of purifying and recovering is used NcoI, HindIII restriction enzymes double zyme cutting equally.And use and above-mentionedly reclaim with quadrat method.

The purpose fragment is connected by 8: 1 mol ratios with carrier segments; Contain T4DNA ligase enzyme (NEB company) 1ul in the linked system; 10 * T4 ligase buffer 2ul; Deionized water, purpose fragment and carrier segments amount to volume be 20ul in 1.5ml Eppendorf pipe 16 ℃ be connected and spend the night, be built into the prokaryotic expression recombinant plasmid of pET28b-Cs1-NcoI.

The expression vector that connects is converted into DH5a, and behind the extraction plasmid, the checking insertion sequence is errorless.And further Transformed E .coli BL21 (DE3) competent cell.The aminoacid sequence of this recombinant protein (pET28b-Cs1-NcoI recombinant plasmid) is as shown in SEQ ID NO:4.

3.pET28a-CS1DS-NcoI the structure of expression vector

Cut the pET28a-CS1-NCOI plasmid with Pci I, Hind III restriction enzyme (NEB company) enzyme.Gel electrophoresis has four fragment: 76bp, 526bp, 2260bp, 3000bp (as shown in Figure 4); With E.Z.N.A.Ultra-Sep

Gel ExtractionKit (Omiga company) rubber tapping purifying 526bp fragment, be connected with pET28a expression vector plasmid (NcoI, Hind III restriction enzymes double zyme cutting).The expression vector that connects is converted into DH5a, extracts plasmid, after the checking insertion sequence is errorless, and further Transformed E .coliBL21 (DE3) competent cell.The aminoacid sequence of pET28a-CS 1DS-NcoI recombinant protein is shown in SEQ ID NO:5.

Embodiment 6 Recombinant Protein Expression are identified and purifying

Transform good expressive host bacterium and be inoculated in the LB substratum (Kan that contains 50 μ g/ml) of 4ml, in 37 ℃, 200rpm, shaking culture is to OD ₆₀₀=0.6 o'clock, the IPTG that taking-up 2ml bacterium liquid adds 2 μ l 1M induced, and remaining 2ml bacterium liquid does not add IPTG as contrast, and 37 ℃, 200rpm, shaking culture 4 hours.Cultured bacterium liquid in 4 ℃, was collected thalline, supernatant discarded in centrifugal 10 minutes through 5000rpm.The resuspended thalline of PBS that in inducing pipe and control tube, adds 200 μ l 0.15M respectively.From induce pipe and control tube, take out the resuspended liquid of 5 μ l respectively, add 2 * SDS-PAGE sample loading buffer, 5 μ l, boil sex change in 5 minutes in 100 ℃ behind the mixing.Carry out SDS-PAGE with 8 μ l/ hole samples and analyze (concentrating glue is 5%, and separation gel is 12% or 16%).Deposition condition is for concentrating glue 80V, separation gel 100V.After electrophoresis finishes,, with the destainer decolouring, high-visible again up to protein band with staining fluid dyeing 4 hours.

The result:

PET28b-CS1 BL21 (DE3) under the IPTG of 1mM condition 37 ℃ induce visible significantly recombinant protein band at the 43KD place.This reorganization recombinant protein mainly is arranged in deposition, is positioned at supernatant on a small quantity.As shown in Figure 5.

PET28a-CS1-NcoI BL21 (DE3) under the IPTG of 1mM condition 37 ℃ induce visible significantly recombinant protein band at the 42KD place.This reorganization recombinant protein mainly is arranged in deposition, is positioned at supernatant on a small quantity.As shown in Figure 6.

PET28b-CS1, pET28a-CS1-NcoI also can produce a large amount of recombinant proteins through inducing in E.coli BLR (DE3) expressive host bacterium (NOvagen company), like Fig. 7, shown in 8.

PET28a-CS1DS-NcoI BL21 (DE3) under the IPTG of 1mM condition 37 ℃ induce visible significantly recombinant protein band at the 42KD place.This reorganization recombinant protein mainly is arranged in supernatant, and is as shown in Figure 9.

The purifying of recombinant protein

Bacterium liquid (500ml) after inducing 4 ℃, centrifugal 10 minutes, is abandoned most supernatant through 4000rpm.Stick with paste the ratio that adds 5ml in every gram bacterium; In thalline, add BugBuster

albumen extractant (NOvagen company); And (every gram bacterium is stuck with paste to add 5Ku rLysozyme; NOvagen company) and 125u Benzonase nucleicacidase (every gram bacterium is stuck with paste; NOvagen company), the post precipitation that fully suspends, room temperature vibration 30min.Then in 4 ℃, centrifugal 10 minutes of 9 000rpm.

The purifying of soluble proteins

Get Ni-NTAAgarose (Qiagen company) the dress post of 1ml, add the PBS flushing of a large amount of 0.15M, remove residual ethanol.

The bacterium supernatant of inducing after centrifugal is joined among the Ni-NTAAgarose that has handled well, place on the shaking table, room temperature vibration 60-90 minute makes albumen fully combine with Ni-NTAAgarose.

After the failure of oscillations, treat Ni-NTA Agarose sedimentation, open the stopper below the pillar, collect effusive liquid.

Washing Buffer with 4 times of bed bodies washes post twice, collects effusive liquid.

With the Elution Buffer washing pillar that contains the different concns imidazoles of 2-4 times of bed body, collect elutriant respectively then.

The liquid of collecting is carried out the analysis of SDS-PAGE electrophoresis detection, detect the effect of protein purification.

The proteic purifying of insolubility

Get Ni-NTA Agarose (Qiagen company) the dress post of 1ml, add the PBS flushing of a large amount of 0.15M, remove residual ethanol.The Buffer B balance that adds 2 times of bed bodies then.

With inducing the bacterial precipitation thing after centrifugal, add Lysis BufferB (pH8.0) mixing, room temperature vibration 30-60 minute in the ratio of every gram 5ml.Then, through 9 000rpm, 4 ℃, centrifugal 10 minutes.Go bail among the Ni-NTA Agarose that stays supernatant to join to have handled well, place on the shaking table, room temperature vibration 60 minutes makes albumen fully combine with Ni-NTA Agarose.

After the failure of oscillations, treat Ni-NTA Agarose sedimentation, open the stopper below the pillar, collect effusive liquid.

Then with 2-4 doubly Buffer C, D, the E of the different pH values of bed body wash pillar, collect elutriant respectively.

The liquid of collecting is carried out the analysis of SDS-PAGE electrophoresis detection, detect the effect of protein purification.The result is like Figure 10,11, shown in 12 and 13.

Embodiment 7 EUSAs detect antibody

1. indirect ELISA method detects the antibody in the serum

With recombinant protein coated elisa plate (Nunc company, 96 orifice plates), 4 ℃ are spent the night; 1%BSA37 ℃ was sealed 1 hour; The clonorchis sinensis patient, the normal human serum 100ul that add dilution in 1: 100 respectively, 37 ℃ were reacted 1.5 hours; Add the HRP mark goat anti-human igg (Sigma company, dilution in 1: 10000) of 100 μ l, 37 ℃ were reacted 1 hour; Add substrate TMB (day root company) colour developing, ELIASA 450nm reading numerical values.

Recombinant protein antigen detects the preliminary assessment of antibody effect

The clonorchis sinensis patients serum that picked at random is picked up from the Heng County, Guangxi is mixed into positive pooled serum for 10 parts, randomly draws 10 parts of negative pooled serums of normal human serum mixing that pick up from Jingxi, Guangxi city in the non-popular district of clonorchiasis sinensis.The concentration that encapsulates of the solubility purifying protein of the solubility purifying protein of recombinant antigen pET28b-CS1, insolubility purifying protein, pET28a-CS1-NcoI, insolubility purifying protein is respectively 4,2,4 and 4ug/ml, every hole 100ul.Detect the antibody in the serum with above-mentioned indirect ELISA method.

The result is following:

Envelope antigen	Mix positive serum OD value	Mix negative serum OD value	The ratio of yin and yang attribute OD value
				PET28b-CS1 insolubility purifying protein	0.496	0.066	7.57
PET28b-CS1 solubility purifying protein	0.532	0.142	3.76
				PET28a-CS1-NcoI insolubility purifying protein	0.496	0.090	5.54
PET28a-CS1-NcoI solubility purifying protein	0.499	0.117	4.28

The result shows that 4 types of purified recombinant albumen all can be distinguished preferably and mixes positive serum and negative serum.

2.pET28b-CS1 the insolubility purifying protein detects the further evaluation of antibody effect

To picking up from 35 parts of clonorchis sinensis patients serums of Heng County, Guangxi; Pick up from 36 portions of normal human serums in Jingxi, Guangxi city in the non-popular district of clonorchiasis sinensis; 15 routine schistosomicide human serums and 9 routine pulmonary distomiasis human serums detect the antibody in the serum with above-mentioned indirect ELISA method.The concentration that encapsulates of the insolubility purifying protein of recombinant antigen pET28b-CS1 is respectively 10ug/ml, every hole 100ul.

ELISA result is following:

The 36 parts of average OD value of normal human serum=0.057, SD=0.0234 (removing a discrete numerical value far away).2.1 times with average is judgement criteria, and the OD value is positive greater than 0.120.In the normal human serum of 36 parts of excrement inspection feminine genders, 2 examples are arranged greater than 0.120.Detect the specificity=34/36 * 100%=94.4% of the ELISA method of antibody with pET28b-CS1 insolubility purifying protein.

The 35 parts of average OD value of excrement inspection positive serum=0.362, SD=0.234, wherein 30 routine serum OD values are greater than 0.120.Susceptibility=30/35 * the 100%=85.7% of the ELISA method of setting up.

Total coincidence rate of this ELISA method=(34+30)/(36+35) * 100%=90.1%.

The OD value of 15 routine schistosomicide human serums and 9 routine pulmonary distomiasis human serums is all less than 0.120, and none presents false positive.This ELISA method specificity is higher, with schistosomicide, lung fluke antibody no cross reaction.

PET28b-CS1 insolubility purifying protein detects the scatter diagram of the specific antibody in all kinds of serum and sees Figure 14.

PET28b-CS1 insolubility purifying protein detects specific antibody indirect ELISA result in the serum

Embodiment 8 clonorchis sinensis Cs1 antigen immune originality detect

Select pET28b-CS1 insolubility and solubility purifying protein for use, respectively 6 Balb/c mouse of immunity.Every mouse antigen inoculation 30ug, Fu Shi Freund's complete adjuvant (Sigma company), subcutaneous injection are used in inoculation first; Every at a distance from 3 weeks, use freund 's incomplete adjuvant (Sigma company) to strengthen, abdominal injection, reinforced immunological is 4 times altogether.Get decimal tail blood, indirect ELISA method detects mouse immune serum (respectively with pET28b-CS1 insolubility and solubility purifying protein 2ug/ml concentration, 100ul coated elisa plate, two anti-ly are the HRP mark sheep anti-mouse igg of Sigma company, dilute in 1: 5 000).

The result:

With pET28b-CS1 insolubility purifying protein immunity Balb/c mouse, with pET28b-CS1 insolubility purifying protein 2ug/ml concentration, the 100ul coated elisa plate, indirect ELISA method detects.Average OD value is 0.710, and SD is 0.033; The OD value of normal mouse and PBS blank is respectively 0.055,0.055.

With pET28b-CS1 insolubility purifying protein immunity Balb/c mouse, with pET28b-CS1 solubility purifying protein 2ug/ml concentration, the 100ul coated elisa plate, indirect ELISA method detects.Average OD value is 0.677, and SD is 0.017; The OD value of normal mouse and PBS blank is respectively 0.077,0.056.

With pET28b-CS1 solubility purifying protein immunity Balb/c mouse, with pET28b-CS1 solubility purifying protein 2ug/ml concentration, the 100ul coated elisa plate, indirect ELISA method detects.Average OD value is 0.641, and SD is 0.020; The OD value of normal mouse and PBS blank is respectively 0.077,0.056.

With pET28b-CS1 solubility purifying protein immunity Balb/c mouse, with pET28b-CS1 insolubility purifying protein 2ug/ml concentration, the 100ul coated elisa plate, indirect ELISA method detects.Average OD value is 0.685, and SD is 0.021; The OD value of normal mouse and PBS blank is respectively 0.055,0.055.

Concrete outcome is as shown in the table:

PET28b-CS1 reorganization purifying protein immune effect indirect ELISA detected result

Experimental result shows, no matter is pET28b-CS1 solubility purifying protein, or the insolubility purifying protein, and all the very strong immunogenicity of tool is prone to the many/monoclonal antibody of preparation.Experiment mice all can produce the intensive immunne response to 2 groups of recombinant antigens.

Embodiment 9 clonorchis sinensis Cs1 antigen homologys relatively

Use Blastp that homology is carried out relatively in nr storehouse among the GenBank, the result is following:

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

ref|XP_001349168.1|hypothetical protein[Plasmodium falcipar... 53.9 9e-06

ref|YP_001321224.1|triple helix repeat-containing collagen[... 51.2 5e-05

ref|ZP_05227486.1|RfbE[Mycobacterium intracellulare ATCC 13... 47.4 8e-04

ref|XP_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

ref|NP_006240.4|proline-rich protein BstNI subfamily 3 precu... 43.5 0.012

gb|EAW96230.1|proline-rich protein BstNI subfamily 3 [Homo s... 43.1 0.015

ref|XP_001613963.1|ubiquitin-conjugating enzyme domain conta... 41.2 0.060

gb|ACF35339.1|fiilensin[Cavia porcellus] 39.3 0.23

emb|CAA30728.1|proline-rich protein G1[Homo sapiens] 39.3 0.24

ref|XP_001776976.1|predicted protein[Physcomitrella patens... 38.9 0.30

ref|YP_896770.1|exosporium protein[Bacillus thuringiensis s... 38.9 0.32

gb|ABU49875.1|mucin[Schistosoma mansoni] 38.1 0.43

gb|ACE88754.1|polymcrphic mucin truncated splice variant IC6... 38.1 0.52

gb|ACE88790.1|polymorphic mucin variant C7/2/25r2.1[Schisto... 38.1 0.55

gb|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|polymorphic mucin variant C7/2/25r2.4[Schisto... 37.7 0.58

gb|ACE88794.1|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|ACE88831.1|polymorphic mucin variant IC6/2/25r2.3[Schist... 37.4 0.77

ref|NP_570367.1|collagen alpha 1(I)chain precursor[Synecho... 37.4 0.84

sp|P10163.3|PRB4_HUMAN RecName：Full＝Basic salivary proline-r... 37.4 0.94

gb|ACE88795.1|polymorphic mucin variant C7/2/30r2[Schistoso... 37.0 0.98

gb|ACE88808.1|polymorphic mucin variant IC10/2/30r2.2[Schis... 37.0 1.0

gb|ACE88767.1|polymorphic mucin truncated splice variant IC9... 37.0 1.0

gb|ACE88806.1|polymorphic mucin variant IC10/2/25r2[Schisto... 37.0 1.1

gb|ABU49878.1|mucin[Schistosoma mansoni] 37.0 1.1

gb|ACE88807.1|polymorphic mucin variant IC10/2/30r2.1[Schis... 37.0 1.2

gb|ACE88813.1|polymorphic mucin variant IC11/2/30r2.2[Schis... 36.6 1.3

gb|ACE88821.1|polymorphic mucinvariant IC3/2/30r2[Schistos... 36.6 1.3

gb|ACE88758.1|polymorphic mucin truncated splice variant IC7... 36.6 1.4

gb|ACE88733.1|polymorphic mucin truncated splice variant IC1... 36.2 1.9

gb|ACE88822.1|polymorphic mucin variant IC3/2/40r2[Schistos... 35.8 2.5

gb|ACE88724.1|polymorphic mucin truncated splice variant C3/... 35.8 2.5

gb|ACE88787.1|polymorphic mucin variant C6/1/40r2.2[Schisto... 35.4 3.3

gb|ACE88728.1|polymorphic mucin truncated splice variant C4/... 35.4 3.3

gb|ACE88773.1|polymorphic mucin variant C10/1/40r2.1[Schist... 35.4 3.4

gb|ACE88788.1|polymorphic mucin variant C6/1/40r2.3[Schisto... 35.4 3.5

gb|ACE88786.1|polymorphic mucin variant C6/1/40r2.1[Schisto... 35.4 3.6

gb|ACE88774.1|polymorphic mucin variant C10/1/40r2.2[Schist... 35.0 3.8

ref|NP_664542.1|collagen-like protein SclB[Streptococcus py... 35.0 3.9

gb|ACE88753.1|polymorphic mucin truncated splice variant IC6... 35.0 4.2

gb|ACE88747.1|polymorphic mucin truncated splice variant IC4... 35.0 4.6

gb|ACE88737.1|polymorphic mucin truncated splice variant IC1... 34.7 4.7

gb|ACE88738.1|polymorphic mucin truncated splice variant IC1... 34.7 4.9

gb|ACE88746.1|polymorphic mucin truncated splice variant IC4... 34.7 5.0

gb|ACE88739.1|polymorphic mucin truncated splice variant IC1... 34.7 5.3

gb|ACE88789.1|polymorphic mucin variant C6/2/80r2[Schistoso... 34.3 6.3

gb|ACE88759.1|polymorphic mucin truncated splice variant IC7... 34.3 6.4

ref|YP_002743725.1|cell surface-anchored protein SclH[Strep... 33.9 8.4

With first example: conserved Plasmodium protein, the amino acid consistence of unkNOwn function [Plasmodium falciparum 3D7] is merely 37%.As follows:

emb|CAX64066.1|conserved Plasmodium protein，unkNOwn function[Plasmodium falciparum3D7]

Length＝1066

Score＝54.3bits(129)，Expect＝7e-06，Method：Composition-based stats.

Identities＝32/85(37％)，Positives＝40/85(47％)，Gaps＝9/85(10％)

Query 75 DPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVK 134

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

Sbjct 745 ETKEDGQKGDDKPNGDDKPNGDDKPNG---DDKPNGDDKPNGDDKPNG---DDKPNGDDK 798

Query 135 PQGDPESDGAVADDAPPSQVKPQGD 159

P GD++G DD P KP D

Sbjct 799 PNGDDKPNG---DDKPNGDDKPNSD 820

Score＝420bits(97)，Expect＝0.035，Method：Composition-based stats

Identities＝29/85(34％)，Positives＝36/85(42％)，Gaps＝9/85(10％)

Query 79 DGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGD 138

D DD P KPGD++G DD P KPGD++G DD P KP D

Sbjct 767 DKPNGDDKPNGDDKPNGDDKPNG---DDKPNGDDKPNGDDKPNG---DDKPNGDDKPNSD 820

Query 139 PESDGAVADDAPPSQVKPQGDPESD 163

D +DD S K D++

Sbjct 821 ---DKQSSDDKRNSDDKQNSDDKQN 842

Score＝41.2bits(95)，Expect＝0.053，Method：Composition-based stats.

Identities＝34/106(32％)，Positives＝42/106(39％)，Gaps＝15/106(14％)

Query 58 NADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGD 117

N DD P G+ P DD P KP GD++G DD P KP GD

Sbjct 758 NGDDKPNGDDKP-NGDDKPN-----GDDKPNGDDKPNGDDKPNG---DDKPNGDDKPNGD 808

Query 118 PESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESD 163

++G DD P S K D D +DD S K D++

Sbjct 809 DKPNG---DDKPNSDDKQSSD---DKRNSDDKQNSDDKQNSDDKQN 848

Score＝35.4bits(80)，Expect＝3.0，Method：Composition-based stats.

Identities＝34/112(30％)，Positives＝42/112(37％)，Gaps＝11/112(9％)

Query 58 NADDADGAQSSPVKGECDPESDGAV-ADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQG 116

N DD P G+ P D DD P KP GD++G DD P S K

Sbjct 770 NGDDKPNGDDKP-NGDDKPNGDDKPNGDDKPNGDDKPNGDDKPNG---DDKPNSDDKQSS 825

Query 117 DPE---SDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGA 165

D+ D +DD S K D D +DD S K D +G+

Sbjct 826 DDKRNSDDKQNSDDKQNSDDKQNSD---DKQNSDDKQNSDDKQNSDDNNGGS 874

Score＝339bits(76)，Expect＝8.4，Method：Composition-based stats.

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

Query 117 DPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVK 176

++DG DD P KPGD D P KPGD++G DP K

Sbjct 745 ETKEDGQKGDDKPNGDDKPNGD---------DKPNGDDKPNGDDKPNG---DDKPNGDDK 792

Query 177 PQGE 180

P G+

Sbjct 793 PNGD 796

Use TBlastn that nr among the GenBank and nt storehouse are carried out homology relatively, the result is following:

Search database Nucleotide collection(nr/nt)using Tblastn(search translated nucleotidedatabases 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 1e-04

gb|AC010176.12|Homo sapiens 12 BAC RP11-711K1(RoswellPark... 52.0 1e-04

ref|NM_006249.4|Homo sapiens proline-rich protein BstNI subf... 50.8 3e-04

gb|CP000724.1|Alkaliphilus metalliredigens QYMF，complete ge... 50.4 3e-04

emb|X07881.1|Human gene PRB3L for proline-rich protein G1 49.3 8e-04

ref|NG_013305.1|Homo sapiens proline-rich protein BstNI subf... 48.5 0.002

emb|X07637.1|Human PRB3 gene(PRB3S)for G1 protein，exon 3 48.1 0.002

ref|XM_001137791.1|PREDICTED：Pan troglodytes hypothetical L... 45.8 0.009

gb|AC193211.4|Pan troglodytes BAC clone CH251-646O21 from ch... 43.5 0.043

gb|AC193209.3|Pan troglodytes BAC clone CH251-562J12 from ch... 43.5 0.043

gb|AC192834.3|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|AK098523.1|Homo sapiens cDNA FLJ25657 fis，clone TST00324 40.0 0.50

ref|NM_120511.1|Arabidopsis thaliana KTF1(KOW DOMAIN-CONTAI... 39.7 0.66

gb|AC213901.5|MACACA MULATTA BAC clone CH250-175D19 from chr... 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|AF247176.1|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

With first example: conserved Plasmodium protein, the amino acid consistence of unkNOwn function [Plasmodium falciparum 3D7] is merely 33%.As follows:

gb|BC096210.3|Homo sapiens proline-rich protein BstNI subfamily 3，mRNA(cDNA cloneMGC：116863 IMAGE：40004741)，complete cds

Length＝1058

GENE ID：5544 PRB3|proline-rich protein BstNI subfamily 3 [Homo sapiens](Over 10 PubMed links)

Score＝52.4 bits(124)，Expect＝9e-05，Method：Compositional matrix adjust.

Identities＝35/104(33％)，Positives＝37/104(35％)，Gaps＝0/104(0％)

Frame＝-2

Query 76 PESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKP 135

P G D PP PGP G D PP PGP G D PP P

Sbjct 454 PGRGGGPWDWFPPCGGGPSGFPGRGGGPWDWFPPCGGCPSGFPGRGGGPWDWFPPCGGGP 275

Query 136 QGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQG 179

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

Sbjct 274 SGFPGRGGGPWDWLPPCGGRPSGFPGGGGVRWGWFPPCGRRPSG 143

Score＝45.1bits(105)，Expect＝0014，Method：Compositional matrix adjust.

Identities＝34/109(31％)，Positives＝36/109(33％)，Gaps＝5/109(4％)

Frame＝-2

Query 83 ADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESD 142

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

Sbjct 496 WDWFPPCGGGPSGFPGRGGGPWDWFPPCGGGPSGFPGRGGGPWDWFPPCGGCPSGFPGRG 317

Query 143 GAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQ-----GEIRMRKF 186

G D PP P G P G PP +P G+R F

Sbjct 316 GGPWDWFPPCGGGPSGFPGRGGGPWDWLPPCGGRPSGFPGGGGVRWGWF 170

Score＝447bits(104)，Expect＝0.020，Method：Compositional matrix adjust

Identities＝41/136(30％)，Positives＝54/136(39％)，Gaps＝0/136(0％)

Frame＝+3

Query 59 ADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDP 118

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

Sbjct 231 GNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGQPPQGGNQSQGPPPRPGKPEGPP 410

Query 119 ESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQ 178

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

Sbjct 411 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPHPGKPEGPPPQGGNQSQGPPPRPGKPE 590

Query 179 GEIRMRKFRCQFIILR 194

G +Q R

Sbjct 591 GPPPQGGNQSQGPPPR 638

Score＝44.7bits(104)，Expect＝0.020，Method：Compositional matrix adjust.

Identities＝41/136(30％)，Positives＝54/136(39％)，Gaps＝0/136(0％)

Frame＝+3

Query 59 ADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDP 118

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

Sbjct 294 GNQSQGPPPRPGKPEGQPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPP 473

Query 119 ESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQ 178

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

Sbjct 474 PQGGNQSQGPPPHPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPE 653

Query 179 GEIRMRKFRCQFIILR 194

G +Q R

Sbjct 654 GPPPQGGNQSQGPPPR 701

Score＝43.5bits(101)，Expect＝0.049，Method：Compositionalmatrix adjust.

Identities＝39/121(32％)，Positives＝51/121(42％)，Gaps＝0/121(0％)

Frame＝+3

Query 59 ADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDP 118

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

Sbjct 357 GNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPHPGKPEGPP 536

Query 119 ESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQ 178

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

Sbjct 537 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPE 716

Query 179 G 179

G

Sbjct 717 G 719

Score＝431bits(100)，Expect＝0.055，Method：Compositional matrix adjust.

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

Frame＝-2

Query 76 PESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKP 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＝43.1bits(100)，Expect＝0.062，Method：Compositionalmatrix adjust.

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

Frame＝+3

Query 69 PVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDA 128

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

Sbjct 198 PGKPEGRPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGQPPQGGNQSQGP 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 R

Sbjct 558 QGPPPR 575

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

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

Frame＝+3

Query 59 ADDADGAQSSPVKGECDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDP 118

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

Sbjct 420 GNQSQGPPPRPGKPEGPPPQGGNQSQGPPPHPGKPEGPPPQGGNQSQGPPPRPGKPEGPP 599

Query 119 ESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDG 164

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

Sbjct 600 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGSPSQGG 737

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

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

Frame＝+3

Query 80 GAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDPESDGAVADDAPPSQVKPQGDP 139

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

Sbjct 168 GNQPQRTPPPPGKPEGRPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGQP 347

Query 140 ESDGAVADDAPPSQVKPQGDPESDGAVAGDAPPSQVKPQG 179

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

Sbjct 348 PQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEG 467

Through homology relatively, explain that clonorchis sinensis Cs1 antigen gene is the clonorchis sinensis specific antigens of a novelty.

Sequence table

< 110>Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C

< 120>specific antigens of clonorchis sinensis and purposes

<160>6

<170>PatentIn version 3.3

<210>1

<211>733

<212>DNA

< 213>clonorchis sinensis Cs1 antigen

<400>1

atttggtatg tttcaggcaa tcatggggat gaaaccgcaa cttgtatact tcatcttcat 60

tcaactcgtc actgctgagt gtttggcaga aaacatgtct gaggacattt tagacgcgga 120

agccaaattt atagaaagtc taaaagcact ccgggacata ccaaatactt caggctcgag 180

cacatctaat ttaaacgccg acgatgcgga tggtgcgcaa tcaagtccag tgaaaggaga 240

atgtgaccct gaatcagatg gagctgttgc ggatgatgcg ccaccaagtc aagtgaaacc 300

gcaaggtgac cctgaatcag atggagctgt tgcggatgat gcgccaccaa gtcaagtgaa 360

accgcaaggt gaccctgaat cagatggagc tgttgcggat gatgcgccac caagtcaagt 420

gaaaccgcaa ggtgaccctg aatcagatgg agctgttgcg gatgatgcgc caccaagtca 480

agtgaaaccg caaggtgacc ctgaatcaga tggagctgtt gcgggtgatg cgccaccaag 540

tcaagtgaaa ccgcaaggtg agattcgtat gcgtaaattc cgatgtcaat ttataattct 600

acgaagaatc atatcatagt taatcccgga ttgatgttct agaaaactag tgatgagagt 660

tgacaataaa ttgttaaatt ttctggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaa 733

<210>2

<211>198

<212>PRT

< 213>clonorchis sinensis Cs1 antigen

<400>2

Met Gly Met Leu Pro Gly Leu Val Thr Pro Ile Pro Ile Gly Leu Val

1 5 10 15

Thr Ala Gly Cys Leu Ala Gly Ala Met Ser Gly Ala Ile Leu Ala Ala

20 25 30

Gly Ala Leu Pro Ile Gly Ser Leu Leu Ala Leu Ala Ala Ile Pro Ala

35 40 45

Thr Ser Gly Ser Ser Thr Ser Ala Leu Ala Ala Ala Ala Ala Ala Gly

50 55 60

Ala Gly Ser Ser Pro Val Leu Gly Gly Cys Ala Pro Gly Ser Ala Gly

65 70 75 80

Ala Val Ala Ala Ala Ala Pro Pro Ser Gly Val Leu Pro Gly Gly Ala

85 90 95

Pro Gly Ser Ala Gly Ala Val Ala Ala Ala Ala Pro Pro Ser Gly Val

100 105 110

Leu Pro Gly Gly Ala Pro Gly Ser Ala Gly Ala Val Ala Ala Ala Ala

115 120 125

Pro Pro Ser Gly Val Leu Pro Gly Gly Ala Pro Gly Ser Ala Gly Ala

130 135 140 145

Val Ala Ala Ala Ala Pro Pro Ser Gly Val Leu Pro Gly Gly Ala Pro

150 155 160

Gly Ser Ala Gly Ala Val Ala Gly Ala Ala Pro Pro Ser Gly Val Leu

165 170 175

Pro Gly Gly Gly Ile Ala Met Ala Leu Pro Ala Cys Gly Pro Ile Ile

180 185 190

Leu Ala Ala Ile Ile Ser

195

<210>3

<211>234

<212>PRT

< 213>pET28b-CS1 recombinant protein

<400>3

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Asp Pro Asn Ser Ala Arg Gly Lys Pro Gln Leu Val Tyr Phe Ile Phe

35 40 45

Ile Gln Leu Val Thr Ala Glu Cys Leu Ala Glu Asn Met Ser Glu Asp

50 55 60

Ile Leu Asp Ala Glu Ala Lys Phe Ile Glu Ser Leu Lys Ala Leu Arg

65 70 75 80

Asp Ile Pro Asn Thr Ser Gly Ser Ser Thr Ser Asn Leu Asn Ala Asp

85 90 95

Asp Ala Asp Gly Ala Gln Ser Ser Pro Val Lys Gly Glu Cys Asp Pro

100 105 110

Glu Ser Asp Gly Ala Val Ala Asp Asp Ala Pro Pro Ser Gln Val Lys

115 120 125

Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala Val Ala Asp Asp Ala Pro

130 135 140

Pro Ser Gln Val Lys Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala Val

145 150 155 160

Ala Asp Asp Ala Pro Pro Ser Gln Val Lys Pro Gln Gly Asp Pro Glu

165 170 175

Ser Asp Gly Ala Val Ala Asp Asp Ala Pro Pro Ser Gln Val Lys Pro

180 185 190

Gln Gly Asp Pro Glu Ser Asp Gly Ala Val Ala Gly Asp Ala Pro Pro

195 200 205

Ser Gln Val Lys Pro Gln Gly Glu Ile Arg Met Arg Lys Phe Arg Cys

210 215 220

Gln Phe Ile Ile Leu Arg Arg Ile Ile Ser

225 230

<210>4

<211>211

<212>PRT

< 213>pET28b-Cs1-NcoI recombinant protein

<400>4

Met Gly Met Lys Pro Gln Leu Val Tyr Phe Ile Phe Ile Gln Leu Val

1 5 10 15

Thr Ala Glu Cys Leu Ala Glu Asn Met Ser Glu Asp Ile Leu Asp Ala

20 25 30

Glu Ala Lys Phe Ile Glu Ser Leu Lys Ala Leu Arg Asp Ile Pro Asn

35 40 45

Thr Ser Gly Ser Ser Thr Ser Asn Leu Asn Ala Asp Asp Ala Asp Gly

50 55 60

Ala Gln Ser Ser Pro Val Lys Gly Glu Cys Asp Pro Glu Ser Asp Gly

65 70 75 80

Ala Val Ala Asp Asp Ala Pro Pro Ser Gln Val Lys Pro Gln Gly Asp

85 90 95

Pro Glu Ser Asp Gly Ala Val Ala Asp Asp Ala Pro Pro Ser Gln Val

100 105 110

Lys Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala Val Ala Asp Asp Ala

115 120 125

Pro Pro Ser Gln Val Lys Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala

130 135 140

Val Ala Asp Asp Ala Pro Pro Ser Gln Val Lys Pro Gln Gly Asp Pro

145 150 155 160

Glu Ser Asp Gly Ala Val Ala Gly Asp Ala Pro Pro Ser Gln Val Lys

165 170 175

Pro Gln Gly Glu Ile Arg Met Arg Lys Phe Arg Cys Gln Phe Ile Ile

180 185 190

Leu Arg Arg Ile Ile Ser Lys Leu Ala Ala Ala Leu Glu His His His

195 200 205

His His His

210

<210>5

<211>187

<212>PRT

< 213>pET28a-CS1DS-NcoI recombinant protein

<400>5

Met Ser Glu Asp Ile Leu Asp Ala Glu Ala Lys Phe Ile Glu Ser Leu

1 5 10 15

Lys Ala Leu Arg Asp Ile Pro Asn Thr Ser Gly Ser Ser Thr Ser Asn

20 25 30

Leu Asn Ala Asp Asp Ala Asp Gly Ala Gln Ser Ser Pro Val Lys Gly

35 40 45

Glu Cys Asp Pro Glu Ser Asp Gly Ala Val Ala Asp Asp Ala Pro Pro

50 55 60

Ser Gln Val Lys Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala Val Ala

65 70 75 80

Asp Asp Ala Pro Pro Ser Gln Val Lys Pro Gln Gly Asp Pro Glu Ser

85 90 95

Asp Gly Ala Val Ala Asp Asp Ala Pro Pro Ser Gln Val Lys Pro Gln

100 105 110

Gly Asp Pro Glu Ser Asp Gly Ala Val Ala Asp Asp Ala Pro Pro Ser

115 120 125

Gln Val Lys Pro Gln Gly Asp Pro Glu Ser Asp Gly Ala Val Ala Gly

130 135 140

Asp Ala Pro Pro Ser Gln Val Lys Pro Gln Gly Glu Ile Arg Met Arg

145 150 155 160

Lys Phe Arg Cys Gln Phe Ile Ile Leu Arg Arg Ile Ile Ser Lys Leu

165 170 175

Ala Ala Ala Leu Glu His His His His His His

180 185

<210>6

<211>703

<212>mRNA

< 213>clonorchis sinensis Cs1 antigen gene

<400>6

gaaaccgcaa cttgtatact ttatcttcat tcaactggtc actgctgagt gtttggcaga 60

aaacatgtct gaggacattt tagacgcgga agccaaattt atagaaagtc taaaagcact 120

ccgggacata ccaaatactt caggctcgag cacatctaat ttaaacgccg acgatgcgga 180

tggtgcgcaa tcaagtccag tgaaaggaga atgtgaccct gaatcagatg gagctgttgc 240

ggatgatgcg ccaccaagtc aagtgaaacc gcaaggtgac cctgaatcag atggagctgt 300

tgcggatgat gcgccaccaa gtcaagtgaa accgcaaggt gaccctgaat cagatggagc 360

tgttgcggat gatgcgccac caagtcaagt gaaaccgcaa ggtgaccctg aatcagatgg 420

agctgttgcg gatgatgcgc caccaagtca agtgaaaccg caaggtgacc ctgaatcaga 480

tggagctgtt gcgggtgatg cgccaccaag tcaagtgaaa ccgcaaggtg agattcgtat 540

gcgtaaattc cgatgtcaat ttataattct acgaagaatc atatcatagt taatcccgga 600

ttgatgttct agaaaactag tgatgagagt tgacaataaa ttgttaaatt ttctggaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 703

Claims (13)

1. the specific antigens of a clonorchis sinensis, it is characterized in that: said antigenic coding gene sequence is shown in SEQ ID NO:1.

2. an antibody is characterized in that: the specific antigens of the described a kind of clonorchis sinensis of its specific combination claim 1.

3. a kind of antibody as claimed in claim 2 is characterized in that: described antibody is monoclonal antibody.

4. isolating protein is characterized in that: its aminoacid sequence be by in the sequence shown in the SEQ ID NO:1 from 23 to the coded aminoacid sequence of 617 bit bases.

5. isolating protein, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO:3, shown in SEQ ID NO:4 or shown in SEQ ID NO:5.

6. carrier, it is characterized in that: said carrier contains the encoding sox of the specific antigens of the described a kind of clonorchis sinensis of claim 1.

7. host cell, it is characterized in that: described cell contains the described carrier of claim 6, and perhaps described cell is transformed or transfection by the encoding sox of the specific antigens of the described a kind of clonorchis sinensis of claim 1.

8. vaccine; It is characterized in that: the specific antigens that comprises the described a kind of clonorchis sinensis of claim 1; The perhaps described antibody of claim 2; The perhaps described isolating protein of claim 4, the perhaps described isolating protein of claim 5, the perhaps described carrier of claim 6.

9. the application in the medicine that the specific antigens of the described a kind of clonorchis sinensis of claim 1 is treated in preparation, diagnosis perhaps prevents clonorchiasis sinensis.

10. the application in the medicine that the described antibody of claim 2 is treated in preparation, diagnosis perhaps prevents clonorchiasis sinensis.

The application in the medicine that 11. claim 4 or 5 described isolating protein are treated in preparation, diagnosis perhaps prevents clonorchiasis sinensis.

12. the described vaccine of claim 8 is treated the application in the medicine that perhaps prevents clonorchiasis sinensis in preparation.

13. a test kit is characterized in that: contain the described antigen of claim 1, perhaps the described antibody of claim 2.

Images