CN103897051B - A kind of clonorchis sinensis specificity PPMP type antigen - Google Patents

A kind of clonorchis sinensis specificity PPMP type antigen Download PDF

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CN103897051B
CN103897051B CN201410068195.3A CN201410068195A CN103897051B CN 103897051 B CN103897051 B CN 103897051B CN 201410068195 A CN201410068195 A CN 201410068195A CN 103897051 B CN103897051 B CN 103897051B
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clonorchis sinensis
sinensis
ppmp
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许学年
周岩
董玉婷
包意芳
徐斌
冯正
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National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
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Abstract

The specificity PPMP type antigen of a kind of clonorchis sinensis of the present invention, is the nucleotide sequence of antigen gene as SEQ? ID? shown in NO:3, additionally provide the antibody of a species specific specific antigens in conjunction with above-mentioned clonorchis sinensis.Additionally provide a kind of specificity PPMP type antigen protein of clonorchis sinensis of separation, coded by the nucleotide sequence of the specific antigens gene of above-mentioned clonorchis sinensis.Additionally provide the carrier of this specific antigens gene containing clonorchis sinensis.Present invention also offers a kind of test kit.Present invention also offers the specific antigen protein of above-mentioned clonorchis sinensis, antibody, carrier and test kit to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.Clonorchis sinensis PPMP type antigen of the present invention has higher immunogenicity, and easily many/the monoclonal antibody of preparation, can be applicable to the aspects such as Detection of antigen, and experiment proves that laboratory animal (mouse) is stronger to this antigen immune response.

Description

A kind of clonorchis sinensis specificity PPMP type antigen
The divisional application that the application is application number is " 2010101786846 ", the applying date is " on 05 19th, 2010 ", denomination of invention is " clonorchis sinensis specificity PPMP type antigen and application thereof ".
Technical field:
The invention belongs to bioengineering field, particularly relate to a kind of clonorchis sinensis, specifically the specific antigens PPMP type antigen of three kinds of clonorchis sinensises and application thereof.
Background technology:
Clonorchiasis sinensis (Clonorchiasissinensis) is the Amphixenosis caused by clonorchis sinensis ［ Clonorchissinensis, Cs ］ infects.This disease is mainly distributed in Asia, as countries such as China, Japan, Korea S's (for Korea S main human parasite), Korea, Vietnam, South East Asia.Estimate that the whole world has 3,500 ten thousand people to infect.In China, except Xinjiang, the Inner Mongol, Gansu, Qinghai, Tibet, Ningxia etc. economize, autonomous region has no except report, all the other 25 provinces, municipalities and autonomous regions and Taiwan Province and the Hong Kong Special Administrative Region all have popular report or the case report of this disease.Due to the flowing of immigrant, and the tourism of global range and economic activity day by day frequently, also get more and more in the case report of some non-Endemic Areas and developed country's (comprising North America and West Europe) this disease.
Show in the Survey on current status of important human parasitic diseases result that June ~ 2004 end of the year calendar year 2001 carries out in the whole nation (except Taiwan, Hong Kong, Macao except) according to the Ministry of Health, the result of clonorchis sinensis infection rate parasitosis Morbidity investigation more national than nineteen ninety rises 75%, and wherein Guangdong, Guangxi, Jilin 3 province (district) rise 182%, 164% and 630% respectively.Estimate that current national clonorchis sinensis the infected reaches more than 1,200 ten thousand people.Clonorchiasis sinensis is China's minority one of several parasitosis presenting ascendant trend individually, and the preventing and controlling of this disease are extremely urgent.
At south China (as Guangdong, Guangxi) and part northern area (the Korean nationality residential district as 3 provinces of Northeast China), local crowd has the custom of eating freshwater fish that is raw or that do not boil, and the bladder worm lived in the flesh of fish is ingested human body, causes the infection of people.Though these Area Inhabitants 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 not eating or seldom ate " sashimi (raw fish) " also starts to eat " sashimi (raw fish) " in the past, the local flavor eating method of present sashimi is also very prevailing in non-Endemic Area, particularly big and medium-sized cities.Simultaneously due to market access, Endemic Area transports various places to containing the fish of encysted metacercaria of clonorchis sinensis, and the number that therefore town dweller infects clonorchis sinensis has the trend of increase.
Clonorchis sinensis adult parasitizes in the hepatic duct of people or other final host, the mechanical stimulus of the secretion/meta-bolites of polypide and polypide itself, bile duct can be caused, particularly cause the inflammatory reaction of secondary bile duct, grade and moderate infection bile duct can occur that limitation is expanded, proliferation of bile duct epithelium, tube wall thickening, luminal stenosis, as merged bacteriological infection, can cause cholangitis and cholangiohepatitis, surrounding annulus hamartoplasia, can there is liver cirrhosis in late period.Evidence suggests, clonorchis sinensis infects can increase the risk that patient suffers from cholangiocarcinoma (CLG), and it is the important public health problem in one, clonorchiasis sinensis Endemic Area that clonorchis sinensis infects dependency Hepatic bile duct neoplasm.
In order to quick diagnosis, in time treatment with effectively control clonorchiasis sinensis, need the infection of clonorchis sinensis in special, sensitive, easy method detection crowd.
For the diagnosis of clonorchis sinensis patient, traditional stool examination is still the main method making a definite diagnosis clonorchiasis sinensis at present.But because patient compliance is poor, and clonorchis sinensis worm's ovum is less is easy to undetected, and the method has certain defect.
Immune diagnostic method (mainly ELISA method) is applied more widely in current preventing and controlling at the scene.But still there are some problems: have false positive in various degree to Healthy People, have certain false negative to clonorchiasis sinensis people, infecting other parasite (schistosomicide, paragonimus) also has certain cross reaction.YongTS application ELISA method detects 48 routine clonorchiasis sinensis Serum Antibodies, only has the positive reaction of 75%, but in 28 routine normal controls and 16 routine paragonimus cases, has occurred the false positive of 7.1% and 37.5% respectively.
Existing research data shows that the ESA of some parasitic excretory-secretory antigens (ESA) or restructuring can be applicable to the serodiagnosis of parasitosis.KimSI ESA carrys out the dynamic response of detected activity Cell of Patients With Clonorchiasis Sinensis serum corresponding antibodies, find that 30kDa and 7kDa zone and patients serum are strong positive reaction, and react more weak with the patients serum of praziquantel treatment after 6 months, wherein 7kDa zone has no the serum after fully recovering with paragonimiasis westermani, metagonimiasis yokogawai, Patients With Clonorchiasis Sinensis and plays cross reaction, specificity is stronger than 30kDa zone, thinks that 7kDa antigen can be used as the significant diagnostic antigen of reactivity clonorchiasis sinensis accordingly.The excretory-secretory antigen (ESA) of Min-HoCHOI to clonorchis sinensis detects the diagnostic value that the ELISA method of antibody and adult worm antigen (CA) detect the ELISA method of antibody and compares and assess.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%) detecting antibody with CA, shows that ESA is better than crude antigen for detecting clonorchis sinensis patients serum antibody.Therefore, compared with the CA of clonorchis sinensis, ESA has higher specificity and susceptibility as the diagnostic antigen of clonorchiasis sinensis.But traditional worm source property antigen preparation is complicated, quality controllability is lower, and preparation amount is limited, is difficult to the demand adapting to look into disease on a large scale.
In addition, because this worm parasitizes this specific position of hepatic duct, determine antigen in Host Serum and antibody horizontal lower, be difficult to detect (reason being also the good diagnostic reagent of this disease shortage at present), but in excrement sample, have the existence of ESA.YongTS etc. identify the clonorchis sinensis antigen reacted with IgE antibody, and find that the antigen of 28kDa and IgE react the strongest, this albumen is also present in excrement.The people such as SirisinhaS use monoclonal antibody ELISA(MonoclonalantibodyELISA, McAb-ELISA) detect antigen in opisthorchis viverrini patient excrement sample, the antigen minimum quantity that can detect is 0.05ng-0.1ng.Research shows, detects the specific antigen in patient excrement sample, can provide early diagnosis, existing disease patient makes a definite diagnosis and the foundation of efficacy assessment.
And diagnose the characteristic of antibody or antigen to decide the diagnosis effect of the immunological method such as antigen or Serum Antibody Detection in excrement sample and serum.Simultaneously owing to being difficult to obtain highly purified specific antigen or a large amount of natural antigens, therefore use Protocols in Molecular Biology, the technological line building specificity recombinant antigen is the only way finding suitable antigen.
Summary of the invention:
The object of the present invention is to provide specific antigens and the application thereof of three kinds of clonorchis sinensises, the specific antigens of these described three kinds of clonorchis sinensises and application thereof will solve the method specificity of prior art diagnosis clonorchiasis sinensis and the not high technical problem of susceptibility.
The invention provides a kind of clonorchis sinensis specificity PPMP type antigen (Cs34), the nucleotide sequence of described antigen gene as shown in SEQIDNO:1 or as described in the nucleotide sequence of antigen gene and SEQIDNO:1 shown in nucleotide sequence homology reach more than 90% or its part after replacing, lack or adding as shown in SEQIDNO:1 nucleotide sequence derive there is antigenic nucleotide sequence.
Present invention also offers a kind of clonorchis sinensis specificity PPMP type antigen (Cs34) protein of separation, by the nucleotide sequence of the specific antigens gene (Cs34) of above-mentioned a kind of clonorchis sinensis coded or with the nucleotide sequence homology of the specific antigens gene (Cs34) of above-mentioned a kind of clonorchis sinensis reach more than 90% nucleotide sequence coded or coded by the nucleotide sequence of the specific antigens gene (Cs34) of the above-mentioned a kind of clonorchis sinensis of part after replacing, lack or adding.
Further, the aminoacid sequence of clonorchis sinensis specificity PPMP type antigen (Cs34) albumen of above-mentioned separation as shown in SEQIDNO:4 or to the aminoacid sequence shown in SEQIDNO:4 through replacement, lack or after adding one or several amino acid by the aminoacid sequence shown in SEQIDNO:4 the aminoacid sequence that derives.
Further, the aminoacid sequence of clonorchis sinensis specificity PPMP type antigen (Cs34) albumen of above-mentioned separation as shown in SEQIDNO:7 or to the aminoacid sequence shown in SEQIDNO:7 through replacement, lack or after adding one or several amino acid by the aminoacid sequence shown in SEQIDNO:7 the aminoacid sequence that derives.
Further, the aminoacid sequence of clonorchis sinensis specificity PPMP type antigen (Cs34) albumen of above-mentioned separation as shown in SEQIDNO:8 or to the aminoacid sequence shown in SEQIDNO:8 through replacement, lack or after adding one or several amino acid by the aminoacid sequence shown in SEQIDNO:8 the aminoacid sequence that derives.
Present invention also offers a kind of antibody, specific specificity PPMP type antigen (Cs34) albumen in conjunction with above-mentioned a kind of clonorchis sinensis.
Further, described antibody is monoclonal antibody.
Present invention also offers a kind of carrier, the specific antigens gene (Cs34) containing above-mentioned a kind of clonorchis sinensis.
Present invention also offers a kind of host cell, containing above-mentioned carrier, or transform or transfection with the specificity PPMP type antigen gene (Cs34) of a kind of clonorchis sinensis described in above-mentioned.
Present invention also offers a kind of vaccine, specificity PPMP type antigen (Cs34) albumen containing above-mentioned a kind of clonorchis sinensis or above-mentioned antibody or above-mentioned carrier.
Specificity PPMP type antigen (Cs34) albumen that present invention also offers above-mentioned a kind of clonorchis sinensis is being prepared treatment, diagnosis or is being prevented the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned antibody to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned vaccine to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned carrier to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers a kind of test kit, specificity PPMP type antigen (Cs34) albumen containing above-mentioned clonorchis sinensis or above-mentioned antibody.
Present invention also offers the second clonorchis sinensis specificity PPMP type antigen (Cs2), the nucleotide sequence of described antigen gene as shown in SEQIDNO:2 or or as described in the nucleotide sequence of antigen gene and SEQIDNO:2 shown in nucleotide sequence homology reach more than 90% nucleotide sequence or its part after replacing, lack or adding as shown in SEQIDNO:2 nucleotide sequence derive there is antigenic nucleotide sequence.
Present invention also offers clonorchis sinensis specificity PPMP type antigen (Cs2) albumen that the second is separated, by the nucleotide sequence of the specific antigens gene (Cs2) of above-mentioned the second clonorchis sinensis coded or with the nucleotide sequence homology of the specific antigens gene (Cs2) of above-mentioned the second clonorchis sinensis reach more than 90% nucleotide sequence coded or coded by the nucleotide sequence of the specific antigens gene (Cs2) of the above-mentioned the second clonorchis sinensis of part after replacing, lack or adding.
Further, the aminoacid sequence of clonorchis sinensis specificity PPMP type antigen (Cs2) albumen of the separation of above-mentioned separation as shown in SEQIDNO:5 or to the aminoacid sequence shown in SEQIDNO:5 through replacement, lack or after adding one or several amino acid by the aminoacid sequence shown in SEQIDNO:5 the aminoacid sequence that derives.
Present invention also offers the second antibody, specific specificity PPMP type antigen (Cs2) albumen in conjunction with above-mentioned the second clonorchis sinensis.
Further, described antibody is monoclonal antibody.
Present invention also offers the second carrier, the specific antigens gene (Cs2) containing above-mentioned the second clonorchis sinensis.
Further, the aminoacid sequence of above-mentioned carrier is as shown in SEQIDNO:9.
Present invention also offers the second host cell, containing above-mentioned the second carrier, or transform or transfection with the specificity PPMP type antigen gene (Cs2) of the second clonorchis sinensis described in above-mentioned.
Present invention also offers the second vaccine, specificity PPMP type antigen (Cs2) albumen of clonorchis sinensis be separated containing above-mentioned the second or the antibody described in the second or above-mentioned the second carrier.
Specificity PPMP type antigen (Cs2) albumen that present invention also offers above-mentioned the second clonorchis sinensis is being prepared treatment, diagnosis or is being prevented the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned the second antibody to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned the second vaccine to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers above-mentioned the second carrier to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers the second test kit, specificity PPMP type antigen (Cs2) albumen containing above-mentioned the second clonorchis sinensis or above-mentioned the second antibody.
Present invention also offers the third clonorchis sinensis specificity PPMP type antigen (Cs3), the nucleotide sequence of described antigen gene as shown in SEQIDNO:3 or or as described in the nucleotide sequence of antigen gene and SEQIDNO:3 shown in nucleotide sequence homology reach more than 90% nucleotide sequence or its part after replacing, lack or adding as shown in SEQIDNO:3 nucleotide sequence derive there is antigenic nucleotide sequence.
Present invention also offers clonorchis sinensis specificity PPMP type antigen (Cs3) albumen that the third is separated, by the nucleotide sequence of the specific antigens gene (Cs3) of above-mentioned a kind of clonorchis sinensis coded or with the nucleotide sequence homology of the specific antigens gene (Cs3) of above-mentioned a kind of clonorchis sinensis reach more than 90% nucleotide sequence coded or coded by the nucleotide sequence of the specific antigens gene (Cs3) of the above-mentioned a kind of clonorchis sinensis of part after replacing, lack or adding.
Further, the above-mentioned aminoacid sequence of clonorchis sinensis specificity PPMP type antigen (Cs3) albumen that the third is separated as shown in SEQIDNO:6 or to the aminoacid sequence shown in SEQIDNO:6 after replacing, lack or adding one or several amino acid by the aminoacid sequence shown in SEQIDNO:6 the aminoacid sequence that derives.
Present invention also offers the third antibody, specific specificity PPMP type antigen (Cs3) albumen in conjunction with the third above-mentioned clonorchis sinensis.
Further, described antibody is monoclonal antibody.
Present invention also offers the third carrier, the specific antigens gene (Cs3) containing the third above-mentioned clonorchis sinensis.
Further, the aminoacid sequence of the third above-mentioned carrier is as shown in SEQIDNO:10.
Present invention also offers the third host cell, containing the third above-mentioned carrier, or transform or transfection with the specificity PPMP type antigen gene (Cs3) of the third above-mentioned clonorchis sinensis.
Present invention also offers the third vaccine, specificity PPMP type antigen (Cs3) albumen containing the third above-mentioned clonorchis sinensis or the third above-mentioned antibody or the third carrier.
Specificity PPMP type antigen (Cs3) albumen that present invention also offers the third clonorchis sinensis is being prepared treatment, diagnosis or is being prevented the application in the medicine of clonorchiasis sinensis.
Present invention also offers the third above-mentioned antibody to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers the third above-mentioned vaccine to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers the third above-mentioned carrier to treat in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
Present invention also offers the third a kind of test kit, specificity PPMP type antigen (Cs3) albumen containing the third above-mentioned clonorchis sinensis or the third above-mentioned antibody.
Term " expression vector " herein, refers to bacterial plasmid, yeast plasmid and other various virus vector conventional in this area.The carrier be suitable in the present invention includes but not limited to: the carrier (prokaryotic expression carrier) of expressing in bacterium, the carrier (as pichia vector) of expressing in yeast, the carrier (retroviral vector, adenovirus carrier etc.) of expressing in mammalian cell.In a preferred embodiment, described expression vector is coli expression carrier.Those skilled in the art can utilize a series of technology such as DNA recombinant technology, build containing encoding fusion protein of the present invention DNA sequence dna, suitable transcribe the expression vector with the particular element such as translational control sequence, promotor and selected marker.Above-mentioned carrier can be used to the host cell transformed, transfection is suitable, so that the fusion rotein required for obtaining.
Host cell of the present invention can be prokaryotic cell prokaryocyte, also can be eukaryotic cell, e.g., and bacterial cell, mammalian cell etc.Host cell, after conversion or transfection contain the gene order of encoding fusion protein of the present invention, namely forms through engineering approaches cell or cell strain, can be used for producing required fusion rotein.Those skilled in the art can select suitable carrier, host cell rightly, and know and how carrier high-efficiency is transformed or is transfected in 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 the eukaryotic cells such as mammalian cell.
Host cell of the present invention can be cultivated by ordinary method, induce and express required fusion rotein, comprises fermenting process and purifying process.The albumen of above-mentioned expression or can be secreted into periplasmic, extracellular in cell, on cytolemma.As required, can utilize the physics of fusion rotein, chemistry and other biological characteristics, carry out separation and purification.Method includes but not limited to: split bacterium, centrifugal, saltouts, molecular sieve chromatography, ion-exchange chromatography, adsorption chromatography, reverse chromatograms, and the sex change of routine, renaturation process etc., and these methods are all well-known to those skilled in the art.
By the clonorchis sinensis adult cDNA expression library built the present inventor, with the clonorchis sinensis patient pooled serum picking up from Guangxi Zhuang Autonomous Region, use immunoscreening screening clonorchis sinensis adult cDNA expression library.Obtain 51 positive colonies altogether, have 44 clones to complete order-checking.Find wherein to repeat tandem sequence containing specificity KPPMPGDRDA or QPPMPGGRDA in 3 routine protein amino acid sequence, because of all containing PPMP sequence signature, therefore called after clonorchis sinensis PPMP type antigen.And such antigen repeating tandem sequence containing KPPMPGDRDA is called PPMPI type antigen, that repeats tandem sequence containing QPPMPGGRDA is then called PPMPII type antigen.Through tetraploid rice, clonorchis sinensis PPMP type antigen of the present invention is the specific antigens also not having disclosed clonorchis sinensis novelty up to now.But with the GRA(glycin-rich protein of clonorchis sinensis), Glycinerichantigen2(glycin-rich protein 2), the rich proline protein of PRA() and PRA2(richness proline protein 2) etc. have higher homology.By the expression and purification of clonorchis sinensis antigen PPMPI type antigens c s2 antigen and PPMPII type antigens c s3 antigen, further established the clonorchiasis sinensis diagnosis elemental method of high specific and susceptibility; And the easy many/monoclonal antibody of preparation, the different aspects such as the scientific research of clonorchiasis sinensis and control can be applied.Test kit of the present invention can also be used to detect clonorchiasis sinensis.
Accompanying drawing illustrates:
Fig. 1, Fig. 2 are clonorchis sinensis PPMP type antigen gene Alignment analytical resultss.In figure, 1-3 sequence is the aminoacid sequence of clonorchis sinensis PPMP type antigen gene Cs34, Cs2 and Cs3 that the present inventor obtains, GRA is Glycinerichantigen sequence, GRA2 is Glycinerichantigen2 sequence, PRA is Prolinerichantigen sequence (note: the present inventor revises coding incorrect in former sequence), and PRA2 is Prolinerichantigen2 sequence.
Cs34 antigen full length amino acid sequence is sent to SignalP3.0Server(network address by Fig. 3: http://www.cbs.dtu.dk/services/SignalP/), uses neural network (NN) to carry out online signal peptide shearing site analysis chart.
Cs34 antigen full length amino acid sequence is sent to SignalP3.0Server(network address by Fig. 4: http://www.cbs.dtu.dk/services/SignalP/), uses hidden markov model (HMM) to carry out online signal peptide shearing site analysis chart.
Fig. 5 is the agarose gel electrophoresis figure of pBluescriptSK_Cs2 phagemid EcoRI/XhoI double digestion 1%, M:DNAMarker; 1:pBluescriptSK_Cs2 plasmid EcoRI/XhoI double digestion result (755bp).
Fig. 6 is the agarose gel electrophoresis figure of 1% of pET28b plasmid EcoRI/XhoI double digestion, M:DNAMarker; 1:pET28b plasmid NcoI/XhoI double digestion result.
Fig. 7 is the agarose gel electrophoresis figure of 1% of pBluescriptSK_Cs3 phagemid and pET28b plasmid EcoRI/XhoI double digestion, M:DNAMarker; 1:pET28b plasmid NcoI/XhoI double digestion result.2:pBluescriptSK_Cs3 plasmid EcoRI/XhoI double digestion result (1157bp).
Fig. 8 is the polyacrylamid gel electrophorogram of pET28b-Cs2 [BLR (DE3) Host Strains] expression of recombinant proteins qualification 12%, M:ProteinMarker(FERMENTASMBI); NI:pET28b-Cs2 does not induce; I:pET28b-Cs2 induces; S: induction supernatant; P: induced precipitation.
Fig. 9 is the polyacrylamid gel electrophorogram of pET28b-Cs3 [BLR (DE3) pLysS Host Strains] expression of recombinant proteins qualification 12%, M:ProteinMarker(FERMENTASMBI); NI:pET28b-Cs3 does not induce; I:pET28b-Cs3 induces; S: induction supernatant; P: induced precipitation.
Figure 10 is pET28b-Cs2 [BLR (DE3) Host Strains] soluble proteins purifying 12% polyacrylamide gel electrophoresis figure, M:Proteinmarker; 1:Ni-NTA purified stream fluid (containing 10mM imidazoles); 2-3: containing 20mM imidazole wash liquid pipe 4,6; 4-9: manage containing 50mM imidazole elution 1-6; 9-11: manage containing 100mM imidazole elution 1-3; 12-14: manage containing 250mM imidazole elution 1-3, wherein, pET28b-Cs2 soluble proteins concentrates in the elutriant containing 50mM-100mM imidazoles.
Figure 11 is pET28b-Cs3 [BLR (DE3) pLysS Host Strains] soluble proteins purifying 12% polyacrylamide gel electrophoresis figure, M:Proteinmarker; 1:Ni-NTA purified stream fluid (containing 10mM imidazoles); 2-3: containing 20mM imidazole wash liquid pipe 4,6; 4-9: manage containing 50mM imidazole elution 1-6; 10-12: manage containing 100mM imidazole elution 1-3; 13-14: manage containing 250mM imidazole elution 1-2, wherein, pET28b-Cs3 soluble proteins concentrates in the elutriant containing 50mM-100mM imidazoles.
Figure 12 is that pET28b-CS2 soluble purified protein ELISA method detects all kinds of serum antibody scatter diagram.
Figure 13 is that pET28b-CS3 soluble purified protein ELISA method detects all kinds of serum antibody scatter diagram.
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It is important to note that these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many changes of the present invention, to change concerning one of ordinary skill in the art be all obviously, and these equivalent modifications, limited range of the present invention all should be belonged to.
Embodiment:
The following stated experimental technique, does not illustrate, all according to " Molecular Cloning: A Laboratory guide ", Science Press in 2002) described method carries out.
The structure of embodiment 1 clonorchis sinensis Schistosoma japonicum
With the encysted metacercaria of clonorchis sinensis picking up from Guangxi Zhuang Autonomous Region, infect cat 5 by administration by gavage.After 40 days, check faecal egg, then cut open and kill, collect clonorchis sinensis adult, clean with the normal saline flushing of sterilizing, Liquid nitrogen storage.
Utilize the total serum IgE of TRIzol (GIBCO/BRL company) test kit extracting clonorchis sinensis adult (1 gram of clonorchis sinensis adult hematocrit, Liquid nitrogen storage).
Adopt mRNA purification kit (mRNAPurificationKit, Amersham company), purified mRNA from the total serum IgE extracted.Concrete steps are shown in product description.
Clonorchis sinensis Schistosoma japonicum adopts directed cloning method, utilizes ZAP-cDNASynthesisKit(Stratagene company) build.Reference reagent box specification sheets operates, and main process comprises:
The synthesis of 1.cDNA first chain, the poly T primer of application containing Xho I restriction enzyme site, in order to make the destruction of cDNA first chain unrestricted restriction endonuclease in building-up process, the dCTP in dNTP is substituted with 5-methyl dCTP, make the DNA hemimethylation of synthesis, thus when Xho I digested cdna, the non-methylation sites being only positioned at poly T primer can be cleaved;
The synthesis of 2.cDNA second chain, digests the RNA in the first chain synthetic product mRNA-DNA heterozygote by RNaseH, and the cDNA fragment of generation synthesizes the second chain as primer under archaeal dna polymerase I acts on;
3., with the end-filling of PfuDNA polysaccharase by the double-strand cDNA of synthesis, add the joint (adaptor) containing EcoR I restriction enzyme site at 5 ' end of the double-stranded DNA filled afterwards, and make joint phosphorylation;
4. digest with restriction enzyme Xho I, postdigestive double-stranded DNA, by SepharoseCL-2B gel filtration chromatography, carries out fractional separation, removes free joint;
5. the DNA fragmentation after separation is merged concentrated, be connected on Uni-ZAPXR carrier;
6. finally pack phage protein shell with packing extract (Gigapack III GoldPackingExtract, Stratagene).
After clonorchis sinensis Schistosoma japonicum has built, further library capacity and Insert Fragment mean length etc. are detected.
Detection computations result, the capacity of clonorchis sinensis Schistosoma japonicum is 1.43 × 106pfu/ml.
From library, random picking 16 recombinant clones carry out pcr amplification, and 1% agarose gel electrophoresis is identified PCR primer.Recombinant clone expanding fragment length scope is at 0.6kb ~ 2kb.Average Insert Fragment length is 1.1kb.
Restructuring insertion rate is 99.6%.
The immunoscreening in embodiment 2cDNA library
Phage-infect:
Getting XL1-blueMRF ' bacterium liquid 200 μ l mixes (predefined according to library titre with appropriate cDNA library phagocytosis body fluid, about 3000 plaques/every plate), 37 DEG C of incubations, after 15 minutes, add top-layer agar (topagar) mixing of 3ml48 DEG C, are laid on immediately on NZY culture medium flat plate.Set at room temperature 10 minutes, hatches in 42 DEG C.
The abduction delivering of fusion rotein:
Observe plaque to occur rear (about 3.5 hours), cover dull and stereotyped with the nitrocellulose filter (Hybond-Cextra, Amersham, NC) soaked through IPTG (15mM/L) 30 minutes, cultivate 3.5 hours again in 37 DEG C; Take out dull and stereotyped 4 DEG C of coolings 15 minutes, then mark on film and plate with pin.Film is taken off and is placed in TBST and washes 3 times, each 10 minutes, be then dipped in NC film and be immersed in confining liquid, room temperature, slight oscillatory, close and spend the night.
NC film is taken out from confining liquid, washes film 2 times with TBST, each 5 minutes.Add clonorchis sinensis patient pooled serum (picking up from Guangxi Zhuang Autonomous Region) 5ml/ film, slight oscillatory 3 hours under room temperature.Film is washed 3 times again, each 10 minutes with TBST.React 1 hour under adding two anti-(5ml/ film) i.e. goat-anti people alkaline phosphatase conjugate (AP-GAH, BIO-RAD, 1:3000 dilute) room temperatures, wash film 3 times with TBST, each 10 minutes, wash film 2 times with TBS, each 5 minutes, wash away residual Tween20.
NC film is taken out from TBS, blots unnecessary solution with the filter paper of Whatman3MM, put into APbuffer and soak NC film 3 minutes.
Diluting NBT to final concentration 0.3mg/ml, BCIP final concentration with ColorDevelopmentSolution is that 0.15mg/ml(BCIP should dropwise add in the NBT diluted, and prevents precipitation from being formed.), be mixed with NBT-BCIP nitrite ion.NC film is immersed in NBT-BCIP nitrite ion, in the dark carries out color reaction until positive spots is high-visible.Color development stopping is reacted.
According to the corresponding position of the positive spot of NC film on former culture plate, picking positive plaque, then make positive bacteriophage mono-clonal through multiple sieve, three sieves.
It is pBluescriptSK_ phagemid and extraction phagemid that embodiment 3 phage deletes cyclisation
E.coliXL1-blueMRF ' is overnight incubation in LB substratum (containing 10mMMgSO4,0.2% maltose, 15 μ g/mlTet).Next day, get nutrient solution 100 μ l and be transferred in new LB substratum, 37 DEG C, 200rpm shaking culture about 2 hours.Nutrient solution through 2000g, centrifugal 10 minutes, precipitum, resuspended to OD with 10mMMgSO4 600=1.0.In microbial culture pipe, add 200 μ lE.coliXL1-blueMRF ' re-suspension liquid, SMbuffer that 50 μ l contain positive bacteriophage and 1 μ l helper phage.Microbial culture pipe to be positioned in 37 DEG C of water-baths 15 minutes.Then 3mlLB is added, in 37 DEG C of shaking culture 5 hours.Take out microbial culture pipe, 65 DEG C of heating 20 minutes, then 3000g, centrifugal 15 minutes, proceeded to supernatant in a new centrifuge tube, is placed in 4 DEG C of preservations.
SOLR bacterium is shaking culture, then 2000g in LB substratum (containing 10mMMgSO4,50 μ g/mlKan), centrifugal 10 minutes, resuspended to OD with 10mMMgSO4 600=1.0, in 1.5mlEp centrifuge tube, add the supernatant conserving liquid 1 μ l of 200 μ lSOLR and above-mentioned preparation, 37 DEG C of incubations 15 minutes, then take out 25 μ l and be spread evenly across LB(containing 100 μ g/mlAmp) on agar plate, be inverted overnight incubation for 37 DEG C.
The bacterium colony that substratum grows, is the SOLR bacterium colony of the pBluescriptSK_ phagemid containing clonorchis sinensis cDNA Insert Fragment.
AxyPrep plasmid DNA small volume of reagent box [love pursue progress biotechnology (Hangzhou) company limited] is used to extract pBluescriptSK_ phagemid.
The separation of embodiment 4 clonorchis sinensis PPMP type antigen gene
The positive colony that screening obtains is pBluescriptSK_ plasmid through deleting cyclisation.Checked order by Shanghai Invitrogen company, obtain part mRNA (cDNA) sequence of 3 clonorchis sinensis PPMP type antigen genes altogether.Be clonorchis sinensis Cs34, Cs2 and Cs3 gene respectively, Insert Fragment length is respectively 1044,734 and 1136 bases.
MRNA (cDNA) sequence of clonorchis sinensis Cs34 gene is as shown in SEQIDNO:1.
MRNA (cDNA) sequence of clonorchis sinensis Cs2 gene is as shown in SEQIDNO:2.
MRNA (cDNA) sequence of clonorchis sinensis Cs3 gene is as shown in SEQIDNO:3.
The protein amino acid sequence that the cDNA fragment inserted in Cs34, Cs2 and Cs3 clone is derived is as shown in SEQIDNO:4-6, and Cs34, Cs2 and Cs3 albumen is respectively containing 265,157 and 284 amino acid.
The aminoacid sequence of clonorchis sinensis Cs34 gene is as shown in SEQIDNO:4.
The aminoacid sequence of clonorchis sinensis Cs2 gene is as shown in SEQIDNO:5.
The aminoacid sequence of clonorchis sinensis Cs3 gene is as shown in SEQIDNO:6.
Embodiment 5 clonorchis sinensis Cs34 Antigenic homology compares
Use Blastp to carry out tetraploid rice to nr storehouse in GenBank, result is as follows:
Be 55% with the amino acid identity of first case: antigenCs44 [Clonorchissinensis], homology is 63%.As follows:
The Alignment of embodiment 6 clonorchis sinensis PPMP type antigen gene analyzes
The 3 routine PPMP type antigen aminoacid sequences that the present inventor finds, use the sequence such as Omiga2.0 software and Glycinerichantigen together to carry out multisequencing alignment analysis, manually after adjustment, alignment analyzes as shown in Figures 1 and 2.Alignment analyzes and shows: clonorchis sinensis PPMP antigen gene is a Lei Duo homologous gene family.Cs34 with the Cs2 sequence of PPMPI type antigen is substantially identical, have identical " KPPMPGDRDA " ten amino acid repeat tandem sequence, but the C of Cs2 gene holds disappearance, and Cs34 gene contains part C end structure.And ten amino acid repetition tandem sequences of the Cs3 gene of PPMPII type are " QPPMPGGRDA ", two amino acid in repeated strings array structure, are had to there occurs change, the same disappearance of C end of this gene.Such antigen is consistent with N and the C end structure sequence height of GRA, GRA2, PRA and PRA2 albumen of clonorchis sinensis, and containing very hydrophobic district, is made up of a large amount of hydrophobic amino acids.Can push away thus: the full length amino acid sequence of Cs34 antigen as shown in SEQIDNO:7, containing 268 amino acid.
The SignalP of embodiment 7 clonorchis sinensis Cs34 antigen gene analyzes
Cs34 antigen full length amino acid sequence is sent to SignalP3.0Server(network address: http://www.cbs.dtu.dk/services/SignalP/), use neural network (NN) to carry out the analysis of online signal peptide shearing site, result is as Fig. 3 and calculation result below:
Use hidden markov model (HMM) to carry out the analysis of online signal peptide shearing site, result is as Fig. 4 and calculation result below:
According to SignalP analytical results, show clonorchis sinensis Cs34 antigen really containing signal peptide structure.Signal peptide shearing site is between the 20th and the 21st amino acid.The aminoacid sequence (containing 248 amino acid) of the ripe Cs34 antigen after signal peptide is sheared is as shown in SEQIDNO:8.Containing 248 amino acid, predicted molecular weight is 26.61kDa.
Although Cs2 and Cs3 gene disappearance N terminal sequence as can be seen here, the whole coding regions containing ripe antigen protein.
The structure of embodiment 8pET28b-Cs2 expression vector
By pBluescriptSK_Cs2 phagemid, use EcoRI/XhoI(NEB company) restriction enzyme, 37 DEG C of double digestions spend the night.Digestion products 1% agarose gel electrophoresis (see figure 5), under ultraviolet lamp, the object fragment of cutting and separating, uses E.Z.N.A.Ultra-Sep gelExtractionKit(Omiga company) purifying recovery object fragment (755bp).
PET28b expression vector uses EcoRI/XhoI(NEB company equally) restriction enzyme makes double digestion, and 37 DEG C of reactions are spent the night.Digestion products 1% agarose gel electrophoresis (see figure 6), under ultraviolet lamp, the object fragment of cutting and separating, uses E.Z.N.A.Ultra-Sep gelExtractionKit(Omiga company) purifying recovery.
Object fragment is connected by 8:1 mol ratio with carrier segments, containing T4DNA ligase enzyme (NEB company) 1ul in linked system, 10 × T4ligasebuffer2ul, deionized water, it is that 20ul 16 DEG C of connections in 1.5mlEppendorf pipe are spent the night that object fragment and carrier segments amount to volume, is built into the recombinant plasmid of the prokaryotic expression of pET28b-Cs2.The aminoacid sequence (containing 196 amino acid) of this recombinant protein is as shown in SEQIDNO:9.Predicted molecular weight is 20.98kDa.
The expression vector connected is converted into DH5a, and after extracting plasmid, checking insertion sequence is errorless.And further Transformed E .coliBLR (DE3) competent cell.
The structure of embodiment 9pET28b-Cs3 expression vector
By pBluescriptSK_Cs3 phagemid, use EcoRI/XhoI(NEB company) restriction enzyme, 37 DEG C of double digestions spend the night.Digestion products 1% agarose gel electrophoresis (see figure 7), under ultraviolet lamp, the object fragment of cutting and separating, uses E.Z.N.A.Ultra-Sep gelExtractionKit(Omiga company) purifying recovery object fragment (755bp).
PET28b expression vector uses EcoRI/XhoI(NEB company equally) restriction enzyme makes double digestion, and 37 DEG C of reactions are spent the night.Digestion products 1% agarose gel electrophoresis (see figure 7), under ultraviolet lamp, the object fragment of cutting and separating, uses E.Z.N.A.Ultra-Sep gelExtractionKit(Omiga company) purifying recovery.
Object fragment is connected by 8:1 mol ratio with carrier segments, containing T4DNA ligase enzyme (NEB company) 1ul in linked system, 10 × T4ligasebuffer2ul, deionized water, it is that 20ul 16 DEG C of connections in 1.5mlEppendorf pipe are spent the night that object fragment and carrier segments amount to volume, is built into the recombinant plasmid of the prokaryotic expression of pET28b-Cs3.The aminoacid sequence (containing 323 amino acid) of this recombinant protein is as shown in SEQIDNO:10.Predicted molecular weight is 33.05kDa.
The expression vector connected is converted into DH5a, and after extracting plasmid, checking insertion sequence is errorless.And further Transformed E .coliBLR (DE3) pLysS competent cell.
The expression identification of embodiment 10 recombinant protein and purifying
The expressive host bacterium transformed is inoculated in the LB substratum (Kan containing 50 μ g/ml) of 4ml, and in 37 DEG C, 200rpm, shaking culture is to OD 600when=0.6, the IPTG that taking-up 2ml bacterium liquid adds 2 μ l1M induces, and remaining 2ml bacterium liquid does not add IPTG in contrast, 37 DEG C, 200rpm, shaking culture 4 hours.By cultured bacterium liquid in 4 DEG C, within centrifugal 10 minutes, collect thalline, supernatant discarded through 5000rpm.The resuspended thalline of PBS of 200 μ l0.15M is added respectively in induction pipe and control tube.From induction pipe and control tube, take out the re-suspension liquid of 5 μ l respectively, add 2 × SDS-PAGE sample loading buffer 5 μ l, after mixing, boil sex change in 5 minutes in 100 DEG C.SDS-PAGE analysis (for 5%, separation gel is 12% or 16% to concentrated glue) is carried out with 8 μ l/ hole samples.Deposition condition is concentrated glue 80V, separation gel 100V.After electrophoresis terminates, dye 4 hours with staining fluid, then with destainer decolouring, until protein band is high-visible.
Result:
PET28b-Cs2, pET28b-Cs3 be 37 DEG C of inductions under the IPTG condition of 1mM, respectively 22, the visible expression of recombinant proteins band in 39Ku place.Recombinant protein only has expression in supernatant.As shown in Figure 8,9.
The purifying of recombinant protein
Bacterium liquid (500ml) after induction, through 4000rpm, 4 DEG C, centrifugal 10 minutes, abandons most supernatant.Stick with paste the ratio adding 5ml in every gram of bacterium, in thalline, add BugBuster protein Extraction agent (NOvagen company), and add 5KurLysozyme(every gram bacterium paste, NOvagen company) and 125uBenzonase nuclease (every gram of bacterium is stuck with paste, NOvagen company), after the precipitation that fully suspends, shaken at room temperature 30min.Then in 4 DEG C, centrifugal 10 minutes of 9000rpm.
The purifying of soluble proteins
Get the Ni-NTAAgarose(Qiagen company of 1ml) fill post, the PBS adding a large amount of 0.15M rinses, the ethanol that removing is residual.
Induction of bacterial supernatant liquor after centrifugal is joined in the Ni-NTAAgarose handled well, is placed on shaking table, shaken at room temperature 60-90 minute, albumen is fully combined with Ni-NTAAgarose.
After stopping oscillation, treat Ni-NTAAgarose sedimentation, open the stopper below pillar, collect the liquid flowed out.
Wash post twice with the WashingBuffer of 4 times of bed bodies, collect the liquid flowed out.
Then wash pillar with the ElutionBuffer containing different concns imidazoles of 2-4 times of bed body, collect elutriant respectively.
The liquid of collection is carried out the analysis of SDS-PAGE electrophoresis detection, detect the effect of protein purification.PET28b-Cs2 in E.coliBLR (DE3) Host Strains, the soluble recombinant protein purification result of pET28b-Cs3 in E.coliBLR (DE3) pLysS Host Strains as shown in Figure 10,11.
Embodiment 11 enzyme linked immunosorbent assay detects antibody
1. indirect ELISA method detects the antibody method in serum
With recombinant protein coated elisa plate (Nunc company, 96 orifice plates), 4 DEG C are spent the night; Close 1 hour for 1%BSA37 DEG C; Add the clonorchis sinensis patient of 1:100 dilution, normal human serum 100ul respectively, 37 DEG C are reacted 1.5 hours; The HRP adding 100 μ l marks goat anti-human igg (Sigma company, 1:10000 dilutes), and 37 DEG C are reacted 1 hour; Add substrate TMB(Tian Gen company) colour developing, microplate reader 450nm reading numerical values.
2.pET28b-Cs2 soluble purified protein detects antibody effects evaluation
To the 35 parts of clonorchis sinensis patients serums picking up from Hengxian County, pick up from 36 portions of normal human serums in the city of Guangxi of the non-Endemic Area of clonorchiasis sinensis, 15 routine Schistosoma japonicum patients serums, 15 routine paragonimus patients serums and 13 routine cysticercus cellulosae patients serums, detect the antibody in serum with above-mentioned indirect ELISA method.The bag of the soluble purified protein of recombinant antigen pET28b-Cs2 is 0.5ug/ml by concentration, every hole 100ul.
ELISA result is as follows:
PET28b-Cs2 soluble purified protein detects specific antibody result of indirect ELISA in serum
36 parts of normal human serum mean OD value=0.047, SD=0.0247.Add that 2 times of SD are for judging criterion with normal human serum average, OD value is greater than 0.096 for positive.
In the normal human serum of 36 parts of excrement inspection feminine genders, 2 examples are had to be greater than 0.096.Specificity=34/36 × the 100%=94.4% of the ELISA method of antibody is detected with pET28b-CS2 insolubility purifying protein.
35 parts of excrement inspection positive serum mean OD value=0.218, SD=0.175, wherein 26 routine serum OD values are greater than 0.096.Susceptibility=26/35 × the 100%=74.3% of the ELISA method set up.
Total coincidence rate=(34+26)/(the 36+35) × 100%=84.5% of this ELISA method.
In 15 routine Schistosoma japonicum patients serums, 15 routine paragonimus patients serums and 13 routine cysticercus cellulosae patients serums, respectively have the OD value of an example to be greater than 0.096, false positive rate is respectively 6.67%, 6.67% and 7.69%.This ELISA method specificity is higher, with schistosomicide, paragonimus, cysticercus cellulosae antibody without obvious cross reaction, and the same normal human serum of false positive rate.
The scatter diagram of the specific antibody that pET28b-Cs2 soluble purified recombinant protein ELISA method detects in all kinds of serum is shown in Figure 12.
3.pET28b-Cs3 soluble purified protein detects antibody effects evaluation
To the 35 parts of clonorchis sinensis patients serums picking up from Hengxian County, pick up from 36 portions of normal human serums in the city of Guangxi of the non-Endemic Area of clonorchiasis sinensis, 15 routine Schistosoma japonicum patients serums, 15 routine paragonimus patients serums and 13 routine cysticercus cellulosae patients serums, detect the antibody in serum with above-mentioned indirect ELISA method.The bag of the soluble purified protein of restructuring pET28b-Cs3 is 0.5ug/ml by concentration, every hole 100ul.
ELISA result is as follows:
PET28b-Cs3 soluble purified protein detects specific antibody result of indirect ELISA in serum
36 parts of normal human serum mean OD value=0.037, SD=0.0146.Add that 2 times of SD are for judging criterion with normal human serum average, OD value is greater than 0.066 for positive.
In the normal human serum of 36 parts of excrement inspection feminine genders, 2 examples are had to be greater than 0.066.Specificity=34/36 × the 100%=94.4% of the ELISA method of antibody is detected with pET28b-Cs3 soluble purified protein.
35 parts of excrement inspection positive serum mean OD value=0.235, SD=0.170, wherein 30 routine serum OD values are greater than 0.066.Susceptibility=30/35 × the 100%=85.7% of the ELISA method set up.
Total coincidence rate=(34+30)/(the 36+35) × 100%=90.1% of this ELISA method.
In 15 routine Schistosoma japonicum patients serums, 15 routine paragonimus patients serums and 13 routine cysticercus cellulosae patients serums, respectively have the OD value of 1 example to be greater than positive dividing value, false positive rate is respectively 6.67%, 6.67% and 7.69%.This ELISA method specificity is higher, with schistosomicide, paragonimus, cysticercus cellulosae antibody without obvious cross reaction, and the same normal human serum of false positive rate.
The scatter diagram of the specific antibody that pET28b-Cs3 soluble purified recombinant protein ELISA method detects in all kinds of serum is shown in Figure 13.
Embodiment 12 clonorchis sinensis Cs2 antigen immunogenicity detects
With the soluble purified recombinant protein of pET28b-Cs2, immunity 5 Balb/c mouse.Every mouse antigen inoculation 30ug, inoculation first uses Freund's complete adjuvant (Sigma company), subcutaneous injection; Every 3 weeks, freund 's incomplete adjuvant (Sigma company) is used to strengthen, abdominal injection, altogether reinforced immunological 4 times.Get mouse tail blood, indirect ELISA method detects mouse immune effect.With pET28b-Cs2, pET28b-Cs3 soluble purified protein (concentration is for 0.5ug/ml, 100ul) coated elisa plate respectively, two resist the HRP for Sigma company to mark sheep anti-mouse igg, and 1:5000 dilutes.
Result: with pET28b-Cs2, pET28b-Cs3 soluble purified protein coated elisa plate respectively, indirect ELISA method detects.The mean OD value of immune serum is respectively 0.500,0.517, SD and is respectively 0.019,0.019; The OD value of Normal Mouse Serum is respectively 0.075,0.072; The OD value of PBST blank is respectively 0.058,0.061.
Concrete outcome is as shown in the table:
The letter of clonorchis sinensis Cs2 recombinant protein immune effect connects ELISA detected result
Experimental result shows, pET28b-Cs2 recombinant protein, the immunogenicity that tool is very strong, and experiment mice can produce strong immunne response to recombinant antigen, easily many/the monoclonal antibody of preparation.The all aminoacid sequence feature of aminoacid sequence containing Cs34 antigen of Cs2 recombinant protein, the antigenic determinant of Cs2 has contained all antigenic determinants of Cs34.Therefore known: Cs34 antigen also has good immunogenicity.Also strong cross reaction is there is more in resist of the mouse of recombinant protein to Cs3 recombinant protein.
Embodiment 13 clonorchis sinensis Cs3 antigen immunogenicity detects
With the soluble purified recombinant protein of pET28b-Cs3, immunity 5 Balb/c mouse.Every mouse antigen inoculation 30ug, inoculation first uses Freund's complete adjuvant (Sigma company), subcutaneous injection; Every 3 weeks, freund 's incomplete adjuvant (Sigma company) is used to strengthen, abdominal injection, altogether reinforced immunological 4 times.Get mouse tail blood, indirect ELISA method detects mouse immune effect.With pET28b-Cs3 soluble purified protein (concentration is for 0.5ug/ml, 100ul) coated elisa plate, two resist the HRP for Sigma company to mark sheep anti-mouse igg, and 1:5000 dilutes.
Result: with pET28b-Cs3 soluble purified protein coated elisa plate, indirect ELISA method detects.The mean OD value of immune serum is that 0.500, SD is respectively 0.023; The OD value of Normal Mouse Serum is 0.076; The OD value of PBST blank is 0.059.
Concrete outcome is as shown in the table:
The letter of clonorchis sinensis Cs3 recombinant protein immune effect connects ELISA detected result
#1 mouse 0.474
#2 mouse 0.495
#3 mouse 0.543
#4 mouse 0.489
#5 mouse 0.501
Normal mice 0.076
PBST 0.059
Experimental result shows, pET28b-Cs3 recombinant protein, the immunogenicity that tool is very strong, and experiment mice can produce strong immunne response to recombinant antigen, easily many/the monoclonal antibody of preparation.

Claims (13)

1. a clonorchis sinensis specificity PPMP type antigen gene, is characterized in that: described antigen gene nucleotide sequence is as shown in SEQIDNO:3.
2. the clonorchis sinensis specificity PPMP type antigen protein be separated, is characterized in that: its aminoacid sequence is as shown in SEQIDNO:6.
3. an antibody, is characterized in that: the specificity PPMP type antigen protein of the specific clonorchis sinensis in conjunction with a kind of separation according to claim 2.
4. a kind of antibody as claimed in claim 3, is characterized in that: described antibody is monoclonal antibody.
5. a carrier, is characterized in that: containing a kind of clonorchis sinensis specificity PPMP type antigen gene according to claim 1.
6. carrier as claimed in claim 5, is characterized in that: the aminoacid sequence of its recombinant protein of expressing is as shown in SEQIDNO:10.
7. a host cell, is characterized in that: containing carrier according to claim 5, or described cell a kind of clonorchis sinensis specificity PPMP type antigen gene according to claim 1 transforms or transfection.
8. a vaccine, is characterized in that: the specificity PPMP type antigen protein of the clonorchis sinensis containing a kind of separation according to claim 2 or carrier according to claim 5.
9. the specificity PPMP type antigen protein of the clonorchis sinensis of a kind of separation according to claim 2 is treated in preparation, is diagnosed or prevent the application in the medicine of clonorchiasis sinensis.
10. antibody according to claim 3 is treated in preparation, is diagnosed or prevent the application in the medicine of clonorchiasis sinensis.
11. carriers according to claim 5 are treated in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
12. vaccines according to claim 8 are treated in preparation, diagnose or prevent the application in the medicine of clonorchiasis sinensis.
13. 1 kinds of test kits, is characterized in that: the specificity PPMP type antigen protein of the clonorchis sinensis containing a kind of separation according to claim 2 or antibody according to claim 3.
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