CN108893476A - Babesiamicrofti 2D97 antigen protein and its application - Google Patents

Abstract

The present invention discloses a kind of Babesiamicrofti 2D97 antigen protein and its application, amino acid sequence such as SEQ ID NO：Shown in 2, coding gene sequence such as SEQ ID NO：Shown in 1.Present invention obtains the immune response of Babesiamicrofti spectrums, it was found that in specific IgM antibodies reaction, the antigen protein 2D97 that higher immune response is presented with the mouse serum of the 3dpi and 7dpi of early stage is uniquely screened, can be used as the candidate for early diagnosing antigen.Escherichia coli prokaryotic expression has been carried out using the nucleotide sequence of antigen protein 2D97, its recombinant protein is successfully obtained, it shows 3 with early stage in the protein chip analysis of specific IgM antibodies, 7dpi mouse serum has higher immune response, can be used in the detection or diagnosis of 3~7 days early stages of infection by Babesia microti and/or window phase.

Description

Babesiamicrofti 2D97 antigen protein and its application

Technical field

The present invention relates to molecule, cell, proteomics and immunological investigation fields, and in particular to Babesiamicrofti is immune The preparation of albumen and the acquisition of immune response spectrum, and the application in candidate diagnosis antigen and the screening of immunoprotection vaccine.

Background technique

Babesiasis is the parasitic disease of the infecting both domestic animals and human caused by Babesia protozoon colonizes in red blood cell, passes through tick It bites and blood transfusion is propagated.The babesia separated from wild animal and domestic animal has more than 100 kinds, wherein there is the case of infection people Report mainly include：B.microti and related cause of disease, B.divergens and related cause of disease, B.duncani and related cause of disease, B.venatorum.In January, 2011, babesiasis is put into national Notifiable disease.In the U.S., most of babesia cases All it is to be had been reported that between 1979 to 2009 caused by Babesiamicrofti, has in the relevant babesia case of 162 blood transfusions It is Babesiamicrofti that 159 parts of pathogen is identified, and Babesiamicrofti is a main pathogens of mankind's babesiasis.People's sense Performance after dye babesia differs greatly, and in Healthy People, subclinical infection is presented mostly；And in immune deficiency patient, such as spleen Excision, HIV patient, old man and child etc. can then be in a bad way even dead.

Currently, the diagnostic method of babesiasis includes：Microscopy Blood piece, antibody test and PCR detection and switching experimental animal, But each detection method has opposite limitation.The oil mirror inspection of Giemsa stain peripheral blood film is the gold of babesiasis diagnosis Standard, but Peripheral Erythrocytes infection percentage is relatively low under normal conditions, and form is similar to Plasmodium；Round pcr has Very high sensibility and specificity, but it is not suitable for the detection of low-density worm mass formed by blood stasis and window phase sample；The detection of antibody has Very high sensibility is also applied for the detection of the infection sample of low-density, but it cannot equally carry out the sample of window phase Effectively detection, and after the removing of worm mass formed by blood stasis, antibody still can continue the quite a while and false positive is presented.Due to vole The self limiting of babesiasis and the limitation of current detection means are very easy to by mistaken diagnosis or fail to pinpoint a disease in diagnosis in disorder in screening, so that It causes the pollution of blood product and forms disease blood transfusion and spread through sex intercourse.The defect of existing diagnostic antigen is, can not detect infection early stage The sample of (window phase) can not also react the disease process that infection is transferred to chronic phase from acute stage, such as in current document report Antigen BmSA1, BmP94, BmIRA, BMN1-8, BM1542, Bm186 with diagnosis effect etc..

Studies have shown that the detection of antibody level is of great significance for the screening of subclinical infection case, in acute infection Stage, IgM antibody are generated at first and are responded to helminth, and specific IgG is then related with the reduction of helminth quantity.So And in the progression of infection of babesiasis, the sample of infection early stage (window phase) cannot be detected effectively, and infection is transferred to from acute stage During chronic phase, spectrotype/immune response spectrum it is not immediately clear that disease process cannot monitor.Pass through immune response Spectrum understands the disease process of babesiasis, and screening can react the significant antigen of disease process, and to developing, a kind of flux is high, fastly The needs of speed, sensitive Babesiamicrofti diagnostic reagent are to meet disease detection and screening is extremely important.

Summary of the invention

Technical problem to be solved by the present invention lies in provide one for the limitation of current Babesiamicrofti disease detection The significant antigen and its joint antigen of kind or a variety of Babesiamicroftis, are able to detect the sample of infection early stage and/or window phase, The process that disease is transferred to chronic phase from acute stage can be reacted, can serve as Babesiamicrofti diagnostic reagent and has flux high, fast Fast, accurate feature can satisfy the demand of disease detection and screening.

The present invention provides a kind of isolated gene, codified Babesiamicrofti 2D97 antigen protein, sequence such as SEQ ID NO：Shown in 1.

The present invention provides a kind of Babesiamicrofti 2D97 antigen protein, is encoded by above-mentioned isolated gene, amino acid Sequence such as SEQ ID NO：Shown in 2.

The present invention provides a kind of isolated genes in the preparation of preparation treatment, diagnosis or prevention Babesiamicrofti disease Application, a kind of above-mentioned isolated gene codified Babesiamicrofti 2D97 antigen protein, sequence such as SEQ ID NO：1 institute Show.The preparation includes the drug for treating, preventing, reagent strip, kit for detecting, diagnosing etc..

The present invention also provides a kind of Babesiamicrofti 2D97 antigen proteins in preparation treatment, diagnosis or prevention vole bar Application in the preparation of shellfish parasitosis.In particular for detecting or diagnosing Babesiamicrofti disease infection early stage and/or window phase.It is described Babesiamicrofti 2D97 antigen protein, encoded by above-mentioned isolated gene, amino acid sequence such as SEQ ID NO：2 institutes Show.

The present invention also provides a kind of antibody answering in the preparation of preparation treatment, diagnosis or prevention Babesiamicrofti disease With a kind of above-mentioned Babesiamicrofti 2D97 antigen protein of the combination of the antibody specificity.Further, the antibody For monoclonal antibody.

The present invention also provides a kind of kits, contain a kind of above-mentioned isolated gene, above-mentioned Babesiamicrofti 2D97 antigen protein or above-mentioned antibody.

The present invention also provides a kind of immuno-chromatographic test paper strips, contain above-mentioned Babesiamicrofti 2D97 antigen protein.

87 Babesiamicrofti albumen that the present invention passes through two-dimentional immunoblotting and mass spectral analysis obtains, are divided into 128 Babesiamicrofti ORFs gene order carries out target gene with fusion cloning technology and wheat germ cell-free protein expression system Clone and expression, finally have successfully been obtained 87 soluble proteins.In conjunction with the recombination egg screened from Babesiamicrofti cDNA library White Bm7 amounts to 88 antigens by protein chip technology, and mice serum is anti-after the infection with Normal Mouse Serum and different times It answers, obtains the immune response spectrum of these antigens, and then understand its distribution in disease process.

It was found that in specific IgG antibodies reaction, the Babesiamicrofti albumen of all tests and 0,3,7dpi mouse Very weak immune response is all presented in serum, does not detect or diagnosis effect；It is unique to screen in specific IgM antibodies reaction The antigen protein 2D97 that higher immune response is presented to the mouse serum of one and the 3dpi and 7dpi of early stage can be used as early stage The candidate of diagnostic antigen.Using the nucleotide sequence of antigen protein 2D97, escherichia coli prokaryotic expression is carried out, has successfully been obtained Recombinant protein Bm2D97, evaluates the recombinant protein with the mouse serum of infection by Babesia microti, in specific IgM antibodies Protein chip analysis in, recombinant protein Bm2D97, which is showed, has higher immune response, energy with 3, the 7dpi mouse serum of early stage It is enough in the detection or diagnosis of 3~7 days early stages of infection by Babesia microti and/or window phase.

It was also found that specific IgG antibodies reaction in, filtered out two it is anti-with mouse sero-immunity after acute phase The albumen 2D33 and 2D36 that should decline, can be used for reacting the disease process that Babesiamicrofti disease is transferred to chronic phase from acute stage. Using the nucleotide sequence of antigen protein 2D33 and 2D36, escherichia coli prokaryotic expression has been carried out, has successfully obtained recombination egg White Bm2D33 and Bm2D36 evaluates the recombinant antigen with the mouse serum of infection by Babesia microti, in specific IgG antibodies Protein chip analysis in, recombinant protein Bm2D33 and Bm2D36 show bright with the immune response of mouse serum after acute phase The effect of aobvious decline can be used in detecting or diagnosing the metabasis process that Babesiamicrofti disease is transferred to chronic phase from acute stage. Recombinant protein Bm2D33 and Bm2D36 shows very high specificity in the cross reaction test with pernicious malaria patients serum, Specificity 100%.Recombinant protein Bm2D33 is compared to reported BmSA1 (sensibility 0%) and shows higher sensitivity Property, sensibility 62.5%.

It was also found that having filtered out and having been held with the mouse serum of 14dpi~270dpi in specific IgG antibodies reaction First three candidate antigens has been carried out Escherichia coli protokaryon table by 4 albumen 2D5,2D29,2D41 and Bm7 of continuous high immune response It reaches, successfully obtains 1 recombinant protein Bm2D41.It is whole that recombinant protein Bm2D41 shows as disease in specific IgG antibodies Maintain high-level response.We have also carried out the evaluation as candidate vaccine to Bm2D41 and Bm7, find through recombinant protein Bm2D41 immune mouse, the peak value for being compared to control group worm mass formed by blood stasis reduce nearly 35%, and significant difference effectively inhibits tight The generation of weight worm mass formed by blood stasis.Recombinant protein Bm2D41 be compared to reported BmSA1 (sensibility 0%) show it is higher quick Perception, sensibility 50%.

The present invention also provides the joint antigen proteins of one group of Babesiamicrofti, including：

A) Babesiamicrofti 2D97 antigen protein is used as infection by Babesia microti and early diagnoses antigen；

B) at least one of both Babesiamicrofti 2D33 antigen protein, 2D36 antigen protein are used as reaction vole Babe insect infection is transferred to the diagnostic antigen of the process of chronic phase from acute stage.

One group of above-mentioned Babesiamicrofti antigen protein can be used as joint antigen, be used for detection and diagnosis Babesiamicrofti Disease can effectively detect infection early stage (window phase) 3d-7d, and can react the process that infection is transferred to chronic phase from acute stage, tool Have the advantages that flux is high, quick, sensitive, overcomes the defect of existing diagnostic antigen.Wherein, Babesiamicrofti 2D33 antigen protein It shows to react with mouse sero-immunity after acute phase in specific IgG antibodies reaction with Babesiamicrofti 2D36 antigen protein The characteristics of decline, has reacted the process that chronic phase is transferred to from acute stage.

Further, the joint antigen protein of one group of above-mentioned Babesiamicrofti also includes：C) have and continue high be immunized instead The Babesiamicrofti antigen protein answered.Further, the above-mentioned Babesiamicrofti antigen protein with lasting high immune response Selected from least one of Babesiamicrofti antigen protein 2D5,2D29,2D41 and Bm7.Further, it is above-mentioned have hold The Babesiamicrofti antigen protein of continuous high immune response is selected from least one of Babesiamicrofti antigen protein 2D41 and Bm7. By babe insect infection is made a definite diagnosis patient serum detect discovery, compared with reported BmSA1, Bm2D33 (or Bm2D36) and The recall rate of the joint antigen of Bm2D41, Bm7 composition is 62.5%, has more diagnostic value.

The present invention also provides the joint antigen proteins of one group of above-mentioned Babesiamicrofti or its recombinant protein to control in preparation Application in the preparation for the treatment of, diagnosis or prevention Babesiamicrofti disease especially can satisfy detection or diagnosis Babesiamicrofti Disease infection early stage, and detection or diagnosis are transferred to the process of chronic phase from acute stage.

One group the present invention also provides the joint antigen protein of one group of above-mentioned Babesiamicrofti of the combination of specificity is anti- Application of the body in the preparation of preparation treatment, diagnosis or prevention Babesiamicrofti disease.

The present invention also provides a kind of kit, the joint antigen protein containing one group of above-mentioned Babesiamicrofti or One group of above-mentioned antibody.

The present invention starts with from the disease process of infection by Babesia microti, establishes infection by Babesia microti mouse experiment animal Model, by two dimensional electrophoresis protein immunoblot identify worm mass formed by blood stasis peak period, antibody peak period reacting antigen, using wheat germ without Cell system expresses candidate antigens, prepares high-throughput protein chip adaptive immune response spectrum, finally anti-to the diagnosis of screening again Former and candidate vaccine is evaluated.Before the present invention, there are no go out to now refer to be immunoreacted spectrum in Babesiamicrofti disease process Acquisition and application open report.

The present invention also provides a kind of immune responses of Babesiamicrofti antigen to compose, and is using fusion cloning technology and wheat germ without thin Born of the same parents' protein expression system is cloned and has been expressed to target gene 2D1~128, and 87 soluble proteins are obtained, in conjunction with from field The recombinant protein Bm7 of Babesia muris cDNA library screening amounts to 88 antigens by protein chip technology, with Normal Mouse Serum It is reacted with mice serum after the infection of different times, the immune response spectrum of acquisition

Detailed description of the invention

Fig. 1 is the two-dimentional western blot hybridization figure of Babesiamicrofti thick leach protein.

Fig. 2 is the design of primers of In-Fusion clone PCR amplification.

Fig. 3 is that purpose gene PCR expands electrophoretogram.

Fig. 4 is that the bacterium colony PCR of part recombinant plasmid identifies electrophoretogram.

Fig. 5 is membrane-transferring device scheme of installation.

Fig. 6 is the Western-blot analysis chart of cell-free expression albumen, wherein M indicates albumen Marker.

Fig. 7 is the protein chip figure that Babesiamicrofti albumen is immunoreacted in IgG antibody, wherein A represents vole babe Worm albumen 2D3~2D54, B represent Babesiamicrofti albumen 2D55~2D128；A~j is respectively indicated：0,3,7,14,21,30, The mouse serum of 60,120,150,270dpi；1 indicates positive control, and 2 indicate that negative control, other frames represent the vole bar of screening Shellfish worm candidate antigens.

Fig. 8 is the thermal map analysis chart that Babesiamicrofti albumen is immunoreacted in specific IgG antibodies, wherein with chip The median of the fluorescence signal intensity of immune response clusters, and extent of fluorescence is 0~2500.

Fig. 9 is the protein chip figure that Babesiamicrofti albumen is immunoreacted in IgM antibody, wherein A represents vole babe Worm albumen 2D3~2D54 and Bm7, B represent Babesiamicrofti albumen 2D55~2D128 and Bm7；A~j is respectively indicated：0,3,7, The mouse serum of 14,21,30,60,120,150,270dpi；1 indicates positive control, and 2 indicate that negative control, other frames represent screening Babesiamicrofti candidate antigens.

Figure 10 is the thermal map analysis chart that Babesiamicrofti albumen is immunoreacted in specific IgM antibodies, wherein with chip The median of the fluorescence signal intensity of immune response clusters, and extent of fluorescence is 0~2500.

Figure 11 is the electrophoresis result figure of PCR amplification target gene fragment, wherein M：DNA marker, 1：2D97,2：2D33, 3：2D36,4：2D41.

Figure 12 is the expression of recombinant protein Bm2D33, Bm2D36, Bm2D41, Bm2D97 and the electrophoresis result of soluble analysis Figure, wherein M：Protein markers, 1：Recombinant clone does not induce full bacterium, and 2：Recombinant clone does not induce supernatant, and 3：Recombinant clone is not Induced precipitation, 4：The full bacterium of recombinant clone induction, 5：Recombinant clone induction supernatant, 6：Recombinant clone induced precipitation.

Figure 13 is the electrophoresis result figure of recombinant protein Bm2D33, Bm2D36, Bm2D41, Bm2D97 after purification, wherein M： Protein markers, 1：Recombinant clone does not induce full bacterium, and 2 recombinant clones induce full bacterium, and 3：Recombinant protein after purification, 4：Excision Recombinant protein after fusion tag.

Figure 14 is the mouse serum of infection by Babesia microti to recombinant antigen Bm2D33, Bm2D36, Bm2D41, Bm2D97, Bm7 ELISA evaluation result.

Figure 15 is that Babesiamicrofti patients serum evaluates the ELISA of each recombinant antigen and combined antigen, wherein A generation Each recombinant antigen of table is reacted with the ELISA of babesiasis human serum, and B represents the ELISA of combined antigen Yu babesiasis human serum Reaction, n=8.

Figure 16 is the cross reaction result of each recombinant antigen and malaria patients serum, wherein A indicates recombinant antigen and every other day The ELISA of malaria patients serum reacts, and B indicates that recombinant antigen is reacted with the ELISA of pernicious malaria patients serum, n=10.

Figure 17 is the ELISA assessment of 200 parts of malaria epidemic area live fever patient samples.

Specific embodiment

Clear, complete description will be carried out to technical solution of the present invention below, it is clear that described embodiment is this hair Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.

1 animal blood serum source of embodiment

1, experimental animal and worm strain

Experimental animal uses the BALB/c mouse of 6~8 week old, is purchased from Shanghai Laboratory Animal Research Institute of the Chinese Academy of Sciences. Babesiamicrofti worm strain is Babesia microtiPRA-99T, by experimental zoology research institute of the Chinese Academy of Medical Sciences It provides, this experiment inoculation passage Liquid nitrogen storage.

2, serum collection

The worm blood for the worm strain of standard containing Babesiamicrofti taken out from liquid nitrogen is balanced to room temperature, and raw with sterile 0.9% It manages salt water and presses 1:1 dilution proportion is simultaneously uniformly mixed.The 200 μ l of worm blood after dilution is drawn with 1ml asepsis injector, is injected into BALB/c mouse is intraperitoneal, and Babesiamicrofti is made to recover in Mice Body.The pathogen of Babesiamicrofti is observed simultaneously by microscopy It counts, (does not occur haemolysis, about 40%) infection rate of red blood cell, plucks eyeball acquisition whole blood before worm mass formed by blood stasis peaks Into anticoagulant tube, the anticoagulated whole blood of dye worm is collected.

The anticoagulated whole blood of normal BALB/c mouse is acquired, and simultaneously with 1:1 ratio is mixed with the infection anticoagulated whole blood of collection, Worm blood density is set to reach 20%, after physiological saline proportional diluted, the abdominal cavity of μ l to the 10 normal BALB/c mouse of each inoculation 200 It is interior.0 day (0days post-infection, 0dpi), i.e. normal mouse are denoted as before mouse inoculation, next day is denoted as after infection 1 day (1dpi), and start to observe and record.Since to 30dpi, daily by tail point blood sampling, and each of hereinafter being mentioned 1dpi The blood sampling of tail vein blood time point, makes thin Blood piece, observes the infection of the cause of disease volume morphing and record red blood cell of Babesiamicrofti Situation.3 days, 7 days, 14 days, 21 days, 30 days, 60 days, 120 after mouse inoculation the last week (normal mouse whole blood) and inoculation It, 150 days, the heparin sodium and blood 1 of 270 days difference tail veins acquisition about 200 μ l, 1g/L concentration of whole blood：10 mixing are anticoagulant.It adopts The tail vein whole blood of collection is centrifuged at room temperature, and 3500rpm, 10min collect serum respectively, and -20 DEG C save backup.

It the western blot hybridization of 2 Babesiamicrofti crude protein of embodiment and analyzes and identifies

Babesiamicrofti worm strain is Babesia microtiPRA-99T, by Chinese Academy of Medical Sciences experimental animal Research institute is learned to provide.Babesiamicrofti is inoculated in BALB/c mouse, anticoagulant to take whole blood, separating red corpuscle is received after cracking, centrifugation Collection precipitating, is determined as babesia polypide by microscopy.By immunohistochemistry technology, as shown in Figure 1, by the thick egg of Babesiamicrofti It is white to do western blot hybridization with the mice serum of 7dpi and 30dpi respectively, and by mass spectral analysis, identify 87 vole babe Worm albumen, the albumen identified including 8 mice serums by 7dpi.

The acquisition of 3 Babesiamicrofti antigen of embodiment immune response spectrum

1, experimental material and method

1.1 carriers and bacterial strain

Escherichia coli (E.coli) DH5 α is purchased from Beijing Tiangeng biochemical technology Co., Ltd.Linearized vector pEU-E01- His-TEV-MCS-N2 (restriction enzyme digestion sites Xho I, BamH I) and Babesiamicrofti genomic DNA are by this experiment It saves and provides.

1.2 laboratory apparatus and reagent

(1) key instrument：

PCR instrument；Constant-temperature metal bath；Vertical laminar flow clean bench；Full temperature concussion and cultivate case；Electro-heating standing-temperature cultivator；Platform Formula high speed refrigerated centrifuge；Compact centrifuge；NanoDrop2000 (ultramicron ultraviolet/visible light spectrophotometer)；High steam Autoclave；Decolorization swinging table；Constant-temperature mixer；85-2 type constant temperature blender with magnetic force；Nucleic acid electrophoresis apparatus；Protein electrophoresis system：BIO- RAD；Gel imager：BIO-RAD；Biochip point sample instrument：PersonalArrayerTM16；Biochip scanner.

(2) main agents and material

PCR product purifying：AxyPrep DNA clean-up Kit (Axygen, the U.S.)；PCR product cuts glue purification： AxyPrep DNA gel QIAquick Gel Extraction Kit (Axygen, the U.S.)；In-Fusion clone's connection：In-FusionTM Advantage PCR Cloning Kit (Clontech, the U.S.)；Escherichia coli culture：Liquid, solid-state LB culture medium are (certainly With)；Ampicillin powder (storing liquid 50mg/ml, Sigma, the U.S.)；Bacterium colony PCR：(the north 2 × Taq PCR MasterMix Capital Tiangeng biotech company)；Plasmid extraction：AxyPrep Plasmid DNA small volume of reagent box (Axygen, the U.S.)；DNase/ RNase-Free Water (Beijing Suo Laibao Science and Technology Ltd)；Wheat germ cell-free protein expression：Wheat Germ WEPRO7240H Expression Kit (CellFree Sciences, the U.S.)；SDS-PAGE electrophoresis：PAGE gel examination Agent box (BIO-RAD, the U.S.)；Albumen pre-dyed Marker (Thermo Scientific, Lithuania)；5 × albumen sample-loading buffer (the green skies) SDS-PAGE electrophoretic buffer (the green skies)；R250 Coomassie brilliant blue dye liquor (autogamy)；Destainer is (according to a conventional method Autogamy)；Western blot：Nitrocellulose membrane (NC film, PALL, the U.S.)；Transferring film liquid (the green skies)；BSA (Sigma, the U.S.)； Penta-His Antibody (BSA free, Qiagen, Germany)；HRP goatanti-mouse IgG (Sigma, the U.S.)； 30% hydrogen peroxide (Shanghai Ling Feng chemical reagent Co., Ltd)；DAB (Sigma, the U.S.)；Protein chip：Protein chip point sample Sample solution (the rich brilliant allusion quotation biology Co., Ltd difficult to understand in Beijing)；Brilliant core encloses the materials such as hybridization wet box (the limited public affairs of the rich brilliant allusion quotation biology difficult to understand in Beijing Department)；Brilliant core microarray optical grade epoxy substrate (the rich brilliant allusion quotation biology Co., Ltd difficult to understand in Beijing)；BSA (Sigma, the U.S.)；Alexa 546 goat anti-mouse IgM (μ-chain) (Invitrogen, the U.S.) of Fluor；Alexa Fluor 546 goat Anti-human IgG (H+L) (Invitrogen, the U.S.).

1.3 experimental method

1.3.1 design primer

Babesiamicrofti immune-related protein is analyzed with SMART, the ORF for being included according to sequence identifies embodiment 2 87 albumen be divided into 128 genetic fragments (2D1~128).Seamless clone is carried out using In-Fusion clone technology, is drawn Object design is as shown in Fig. 2, in the end of forward primer 5 ' of gene specific plus pEU-F：5 '-GGGCGGATATCTCGAG-3 ' sequence, 5 ' ends of the reverse primer of gene specific plus pEU-R：5 '-GCGGTACCCGGGATCC-3 ' sequence.Babesiamicrofti gene specific Design of primers use software PrimerPremier5.0.N-Fusion clones target gene information and primer sequence such as 1 institute of table Show

1 128 genetic fragment (2D1~128) gene informations of table and primer sequence

1.3.2 the amplification of aim sequence

The reaction system and reaction condition of PCR amplification such as table 2, shown in table 3：

The reaction system of 2 target fragment PCR amplification of table

The reaction condition of 3 target fragment PCR amplification of table

Note：* indicate that annealing temperature can be optimized according to the primer TM value difference of different genes segment

Clip size identification is carried out by 1% agarose gel electrophoresis to PCR product achieved above, there will be correct mesh The product of band send to Hua Da gene and be sequenced.If need to be tapped rubber after purification containing miscellaneous band, then send sequencing.PCR expands The 128 Babesiamicrofti genes increased, finally have successfully been obtained the DNA product of 113 target gene, including 9 quilts The genetic fragment of 7dpi mouse serological diagnosis, amplification rate are 88.3%, and electrophoresis result is as shown in Figure 3.

1.3.3In-Fusion clone

The screening of construction of recombinant plasmid and positive colony, steps are as follows：(1) 2 μ l are added in 5 μ l PCR products It in Clonging Enhancer solution, is put into PCR instrument after mixing, 37 DEG C of reaction 15min, then 80 DEG C of reaction 15min；(2) The coupled reaction system of In-Fusion clone is as shown in table 4, is put into PCR instrument after mixing and is attached reaction, and condition is 50 DEG C Connect 15min；(3) connection product is placed on ice, product is simultaneously all transferred in competent cell DH5 α, 37 by heat-shock transformed method 16~18h is cultivated on DEG C ammonia benzyl resistance LB solid medium；(4) disperse 4 single colonies of picking on each plate, be dissolved in respectively In the LB liquid medium of 10 μ l non-resistants, template of the 2 μ l bacterium solutions as bacterium colony PCR is taken, the reaction system of bacterium colony PCR and anti- Answer condition as shown in table 5, table 6；(5) PCR product is identified with 1% agarose gel electrophoresis, stripe size is correct Bacterium solution corresponding to bacterium colony (each gene selects more than two correct clones as far as possible) is transferred into the ammonia benzyl resistance LB culture medium of 3ml In, 37 DEG C of 16~18h of shake culture；(6) culture solution of each bacterium colony respectively draws 1ml, send Hua Da gene sequencing；Remaining bacterium solution takes 500 μ l add 500 μ l, 50% glycerol conservation, and -80 DEG C save backup；(7) to correct recombinant plasmid is sequenced, pass through the bacterium of preservation Kind inoculation, expands culture and extracts plasmid.Since the wheat germ cell-free protein expression system of next step requires recombinant plasmid Concentration need to reach 150ng/ μ l or more, so this, which studies each recombinant plasmid, does two parallel bacterium solution cultures.Plasmid extraction mistake Journey is detailed in Axygen Plasmid DNA Mini Kit operation manual.(8) using NanoDrop2000 to the plasmid concentration of extraction Be measured, make a record, and be stored in -80 DEG C it is spare.

4 In-fusion of table clones coupled reaction system

5 bacterium colony PCR reaction system of table

The reaction condition of 6 colony PCR amplification of table

By In-Fusion clone technology, 109 correct recombinant plasmids of sequence, including 9 are finally successfully constructed (cloning efficiency 100%) reaches 96.5% by the genetic fragment of 7dpi mouse serological diagnosis, total cloning efficiency.The bacterium colony of part recombinant plasmid PCR qualification result is as shown in Figure 4.

1.3.4 cell-free expression

Referring to the operation instruction of Wheat Germ WEPRO7240H Expression Kit, to the successful purpose base of building It is as follows because carrying out in-vitro transcription and accurate translation, operating procedure：(1) it is transcribed in vitro, the system of responsive transcription such as 7 institute of table Show, reaction in PCR instrument after mixing, condition is 37 DEG C of transcription 6h；(2) preparation of accurate translation system, system are as shown in table 8；(3) 206 1 × SUB-Amix of μ l × SGC is added in 96 orifice plates, then the translation reaction system prepared is slowly added into plate hole bottom Portion keeps stratification state, is placed in constant-temperature metal bath, 15 DEG C of reaction 20h；(4) PCR pipe is transferred to after mixing translation product In, -80 DEG C save backup.

Reaction system is transcribed in vitro in table 7

8 In Vitro Translation reaction system of table

By using the in-vitro transcription and translation of wheat germ cell-free protein expression system, 111 recombinant plasmids (including two The sequence of full genome synthesis) it is total to 87, successful expression albumen, including 7 genetic fragment institute tables by 7dpi mouse serological diagnosis The albumen reached.

1.3.5 Western blot analysis

To the cell-free expression of target gene, this experiment using protein immunoblot (Western-blot) technology into Row analysis, operates as follows：(1) preparation of PAGE gel：The PAGE gel that this experiment uses is to use BIO-RAD public The polyacrylamide gel of department's production premixes reagent, prepares 12% separation gel and 5% concentration glue, is put in 4 after gel sets It DEG C saves backup；(2) loading sample preparation：The protein product of the 12 cell-free expression of μ l is taken, 3 μ 5 × albumen of l are added and buffer loading Liquid mixes well, and boiling water bath boils 5~10min；(3) protein electrophoresis：The sample and albumen Marker prepared respectively takes on 10 μ l Sample, 80V electrophoresis 30min, then 120V electrophoresis 60min；(4) transferring film operates as follows：1) suitable NC film will be reduced to be placed in methanol 30~60s is activated, is then transferred in transferring film liquid and impregnates together with filter paper；2) gel, NC film, filter paper are discharged as shown in Figure 5 (black side is cathode, and white side is anode), steps up fixed access electrophoresis tank, fills it up with transferring film liquid and be followed by the swimming that is powered, in ice Under the conditions of bath, 220mA transferring film 90min；3) it closes：NC film is taken out, incubation box is put into, after clear water rinses, with 3%BSA (PBS Dilution) 4 DEG C of closings are overnight；4) it washs：Confining liquid is outwelled, NC film is rinsed 3 times with PBST, each 5min；5) primary antibody is incubated for：It will Penta-His antibody presses 1: 2000 dilution proportion with PBS, is placed on shaking table and is incubated at room temperature 1h；6) it washs：Outwell primary antibody incubation Liquid, NC film are rinsed 3 times with PBST, each 5min；7) secondary antibody is incubated for：By the sheep anti-mouse antibody of HRP label with PBS by 1: 4000 Dilution proportion is placed on shaking table and is incubated at room temperature 1h；8) it washs：Outwell secondary antibody Incubating Solution, NC film PBST rinses 3 times, every time 5min；9) it develops the color：By DAB: PBS: 30%H₂O₂The ratio of=2mg: 3ml: 0.9 μ l prepares developing solution, the NC film leaching after being incubated for It steeps in developing solution and (pays attention to being protected from light when colour developing), rinsed when purpose band to appear immediately to terminate reaction, and with filter paper by NC Film blots；10) it photographs to record, and the filter paper of the NC film after display is wrapped up into 4 DEG C of preservations.

The results of immunoblot analysis of albumen is as shown in fig. 6, hair when wherein having several protein electrophoresis by the system expression Migration is given birth to.The high-flux clone and expression of results of Babesiamicrofti genetic fragment are summarized as shown in table 9：

The high-flux clone of 9 Babesiamicrofti genetic fragment of table and expression

1.3.6 the preparation of protein chip and immuning hybridization obtain immune response spectrum

This experiment expresses albumen using optical grade epoxy substrate as carrier, by the wheat germ cell-free that the above experiment obtains middle acquisition The Bm7 (amino acid sequence is as shown in SEQ ID NO.9) screened from cDNA library before (2D3~2D128) and this laboratory, The point sample of protein sample is carried out by using chip point sample instrument.Experiment is compareed using BmSA1 as positive protein, with the wheat of plasmid-free As negative control group, the concrete operations of chip preparation are as follows by embryo cell-free translation system and PBST：(1) sample preparation：Take egg White chip point sample sample solution and each 6 μ l of expression protein liquid are mixed, and are drawn 10 μ l and are added in 384 orifice plates, centrifugation is placed on standby on ice With；(2) instrument prepares：Ultrasonic pond and the work top of point sample instrument are cleaned, and adds ddH in ultrasonic pond₂O is to graduation mark；Humidification Device ddH₂O will add to graduation mark；The waste liquid in waste liquid bottle is outwelled, the ddH in washer bottle is filled₂O；Start instrument and corresponding Operating software completes pattern match and self-test；(3) fence is pasted：2 × 6 holes (9cm × 9cm) fence is selected in experiment, uses fence Stickup and compaction tool, stick in epoxy group on piece for fence, are placed in chip Yang Yi point sample area, prepare point sample；(4) parameter is set It sets：After demarcating the initial position of 384 orifice plates, the parameters such as punch block, slide, pre- point sample slide, dot matrix, sample, cleaning are set gradually； The albumen matrix of this experimental setup 10 × 10, each albumen sample repeat point sample 2 times, 10 matrixes of each albumen point sample；(5) point Sample and preservation：Confirm whether work in every is ready, clicks " beginning " and carry out point sample；The slide that point sample is completed can carry out next It walks hybrid experiment or is sealed in wet box and save backup for 4 DEG C；Pay attention to：Slide after point sample should stand half an hour or more, make albumen Sufficiently in conjunction with slide.

The immuning hybridization of protein chip determines optimal reactant firstly the need of the dilution of optimization serum and fluorescence secondary antibody System, detailed process is as follows for hybrid experiment:(1) it closes：50 μ are added in the fence of each matrix of protein chip made above L 3%BSA (PBS dilution) is placed in hybridization wet box, and 4 DEG C of closings are overnight；Before cleaning, hybridization wet box is put into 37 DEG C of constant temperature The re-closed 1h of case；(2) it cleans：The confining liquid on chip is dried, is placed in deionized water and rinses once, then is shaken with PBST shaking table Washing 3 times, each 5min, 1000g are centrifuged 3min drying；(3) it is incubated for primary antibody：By 10 of different times after infection Babesiamicrofti Only mixing mice serum (0,3,7,14,21,30,60,120,150,270dpi) is diluted simultaneously in 1: 100 ratio with PBST It mixes, 50 μ l is taken to be added sequentially in each albumen matrix respectively, be placed in hybridization wet box, 37 DEG C of reaction 1h；(4) it cleans：Clearly Wash the same step (2) of operation；(5) it is incubated for secondary antibody：By Alexa Fluor 546goatanti-mouse IgG or Alexa Fluor 546goat anti-mouse IgM antibody is diluted mixing with PBST by 1: 200, and 50 μ l is taken to be added in each albumen matrix, It is placed in hybridization wet box, 37 DEG C of reaction 1h (after secondary antibody is added, caution of operation is protected from light)；(6) it cleans：The same step of cleaning operation (2)； (7) it scans：Protein chip after hybridization is placed in chip scanner, ScanArray Express software is set The suitable parameter of version 4.0 (PerkinElmer) selects the wavelength of λ=532nm to carry out chip scanning.Save picture simultaneously Read data；(8) data are analyzed：With negative sample after the mean fluorescence intensity of sample to be detected after background correction and background correction Mean fluorescence intensity ratio >=2 as positive samples refer to.Use Multi-array experiment viewer (MeV) thermal map that software carries out protein chip is drawn and is analyzed；Each albumen of the acquisition hybridized by protein chip is immunized Response spectrum carries out hierarchical clustering by R language (www.r-project.org/), and Europe is respectively adopted in distance algorithm and clustering method Family name's distance and ward.D2 method.

In specific IgG antibodies reaction, most of Babesiamicrofti albumen and 0,3,7dpi mouse serum all presents non- Often weak immune response (Fig. 7:A~c), and very high immune response (Fig. 7 is then presented with the mouse serum of 14~150dpi:D~ I), it is immunoreacted degree in 270dpi and is declined (Fig. 7:j).The specific IgG antibodies immune response spectrum of each albumen is such as It shown in Fig. 8, is clustered by R language analysis and hclust function, 88 albumen are divided into 4 groups altogether, respectively Group1 (11 eggs It is white), Group2 (47 albumen), Group3 (29 albumen) and Group4 (the only recombinant protein of the prokaryotic expression of 1 purifying), Respectively represent high, medium, lower and high immune response group.Protein-bonded immune response spectrum and grouping situation, respectively from four 4 albumen 2D5,2D29,2D41 (amino for having lasting high immune response with the mouse serum of 14dpi~270dpi are filtered out in group Acid sequence such as SEQ ID NO.8) and Bm7.In addition, also anti-with mouse sero-immunity after acute phase from filtering out two in Group3 The albumen 2D33 (amino acid sequence such as SEQ ID NO.4) and 2D36 (amino acid sequence such as SEQ ID NO.6) that should be remarkably decreased.

In specific IgM antibodies reaction, in addition to 14,21,30dpi mouse serum is in most of Babesiamicrofti albumen Existing outer (Fig. 9 of stronger immune response:D~f), and very low immune response is then all presented with the mouse serum in other periods.Often The specific IgM immune response spectrum of a albumen as shown in Figure 10, is clustered by R language analysis and hclust function, 88 albumen It is divided into 4 groups, respectively Group1 (19 albumen), Group2 (50 albumen) altogether, Group3 (18 albumen) and Group4 (1 recombinant protein).Composed according to immune response, do not show difference between first three groups, only one screened from The mouse serum of the albumen 2D97 (amino acid sequence such as SEQ ID NO.2) of Group3 and the 3dpi and 7dpi of early stage present higher Immune response, the albumen can be used as the candidate of early diagnosis antigen.

Prokaryotic expression, purifying and the evaluation of embodiment 4 Babesiamicrofti biomarker candidate 2D33,2D36,2D41,2D97

1 experimental material and method

1.1 plasmids, bacterial strain

Escherichia coli (E.coli) DH5 α, BL21 (ED3) are purchased from Beijing Tiangeng biochemical technology Co., Ltd.Plasmid PET21a, pET28a, pET42a are saved by this laboratory and are provided, and plasmid pSmart1 is limited purchased from Changzhou world people and biology Company.

1.2 sample collections and ethics statement

8 parts of Babesiamicrofti patients serum samples are provided by preclinical medicine teacher Yuan Chengxunjia seminar of Fudan University.Between Day malaria and each 10 parts of malignant malaria patients serum's sample are by Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C Chen Jun tiger Teacher provides.Experiment passes through Giemsa staining microscopy sample also from 200 parts of fever patient samples of malaria epidemic area Tengchong on-site collection This pathogen, is feminine gender；By detection of nucleic acids, tertian fever positive sample 14 are found, malignant malaria is 4 positive, wherein wrapping Coinfection containing an example.Normal human serum sample from Suzhou Medical College is as negative control.The acquisition of blood sample is by China Country of disease prevention and control center prevention of parasitic diseases control institute the Research papers committee ratify.It is all to participate in providing sample Individual all endorsed informed consent form, and give explanation to purposes, potential risk and the interests of collecting sample.

1.3 laboratory apparatus and reagent

(1) key instrument

PCR instrument；Vertical laminar flow clean bench；Full temperature concussion and cultivate case；Electro-heating standing-temperature cultivator；Desk type high speed freeze from The heart；Compact centrifuge；NanoDrop2000 (ultramicron ultraviolet/visible light spectrophotometer)；High-pressure steam sterilizing pan；Nov nucleic acid Swimming instrument；Protein electrophoresis system；Gel imager；Ultrasonic Cell Disruptor；Protein chromatographic purification system；Microplate reader.

(2) main agents and material

PCR product purifying：AxyPrep DNA clean-up Kit (Axygen, the U.S.)；In BamH1, Xho1 are restricted Enzyme cutting and Buffer3, NEB, the U.S.；Seamless clone：In-Fusion HD Cloning Kits (Clontech, the U.S.)；Large intestine Bacillus culture：Liquid, solid-state LB culture medium Nacl 10g, Tryptone 10g, Yeast Extract 5g, (Agar 15g) adds Deionized water is settled to 1L.Ampicillin powder (storing liquid 50mg/ml, Sigma, the U.S.)；Kanamycins antibiotic powder (storing liquid 10mg/ml, Sigma, the U.S.)；Bacterium colony PCR：(Beijing Tiangeng biotechnology is public by 2 × Taq PCR MasterMix Department)；Plasmid extraction：AxyPrep Plasmid DNA small volume of reagent box (Axygen, the U.S.)；IPTG powder (storing liquid 1mol/L, Sigma, the U.S.)；PMSF powder (storing liquid 1mol/L, Sigma, the U.S.)；Inclusion body protein purifying：PAGE glue albumen micro time It receives kit (Sangon Biotech (Shanghai) Co., Ltd.)；GST purification system：1ml GSTrap HP purification column, GE are public Department, Sweden；Buffer A:PBS pH7.4；Buffer B:(eluent):1mMDTT,10mM Glutathione pH7.4；Ni Column purification system：5ml HisTrap FF purification column, GE company, Sweden；Buffer A:20mM Tris,50mM NaCl,pH 8.0；Buffer B (eluent):20mM Tris,50mM NaCl,500mM imidazole,pH 8.0；Protein tag excision： SUMO protease reagent (Shanghai Suo Laibao Biotechnology Co., Ltd)；His protein purification medium filler, GE company, Sweden；Egg White concentration mensuration：Branford kit (Beijing Tiangeng biology Co., Ltd)；ELISA experiment：HRP goat anti-mouse IgG (Sigma, the U.S.)；HRP goat anti-mouse IgM (Sigma, the U.S.)；HRP goat anti-human IgG (Sigma, the U.S.)；HRP goat anti-human IgM (Sigma, the U.S.)；Immune protective test：Freund's Complete adjuvant (Sigma Aldrich, the U.S.)；Freund's incomplete adjuvant(Sigma Aldrich, the U.S.).

1.4 experimental method

1.4.1 design of primers and synthesis

According to the nucleic acid sequence of the biomarker protein 2D97,2D33,2D36,2D41 of screening, (nucleotide sequence is respectively SEQ ID NO.1,3,5,7), with 5.0 software of PREMIER be respectively albumen prokaryotic expression design specific primer.Load used Body is respectively pET42a, pET21a, pSmart1, pET28a, and restriction enzyme restriction enzyme site selects BamH1 and Xho1.Experiment Using seamless clone technology, carrier sequence is introduced respectively at 5 ' ends of specific primer：F：5 '-GGGATATCGGGGATCC and R： 5 '-GGTGGTGGTGCTCGAG, primer sequence are as shown in table 10.Primer is synthesized by Huada gene company.

The primer sequence of 10 Babesiamicrofti recombinant clone of table

Note：Underscore italicized item is restriction enzyme site sequence

1.4.2 the building of prokaryotic expression carrier

The amplification of target gene using the pEU-E01 recombinant plasmid containing target gene for diluting 100 times as template, carries out respectively PCR amplification, reaction system and reaction condition such as table 2, shown in table 3.Use restriction enzyme Xho1 and BamH1 by carrier matter simultaneously Grain carries out double digestion processing, and double digestion system is as shown in table 11, and reaction condition is 37 DEG C and stays overnight.With 1% Ago-Gel to PCR Product and digestion products carry out electroresis appraisal.If product band is single, directly product can be carried out with PCR cleaning agents box pure Change；If product contains miscellaneous band, need that target stripe is carried out to cut glue purification.

The endonuclease reaction system of 11 double digestion of table

Coupled reaction system is shown in Table 12.After reactant is mixed, 37 DEG C of reaction 15min, then 50 DEG C of reaction 15min, it completes The connection of carrier and target gene.Connection product is all transferred in competent cell E.coliDH5 α, in the LB of corresponding resistant 16~18h is cultivated on solid medium.Bacterium colony PCR identification uses universal primer T7 promoter primer:5'- TAATACGACTCACTATAGGG-3 ' and T7 Termina tor Primer:5'-GCTAGTTATTGCTCAGCGG-3'.Screening Positive colony after the LB liquid medium culture of corresponding resistant, extract plasmid simultaneously send to Hua Da gene sequencing.Bacterium colony PCR's Reaction system and reaction condition is as shown in table 3-4,3-5：

12 coupled reaction system of table

Using the pEU-E01 recombinant plasmid containing 2D97,2D33,2D36,2D41 genetic fragment as template, PCR reaction, electricity are carried out Swimming testing result shows that the target fragment size of amplification is consistent with theory, respectively may be about 984,432,519 and 687bp, such as Figure 11 It is shown, wherein M indicates DNA marker, and 1 indicates 2D97, and 2 indicate 2D33, and 3 indicate 2D36, and 4 indicate 2D41.The mesh of Successful amplification Segment it is purified after, carrier pET42a, pET21a, pSmart1, pET28a after being connected respectively to digestion, recombinant plasmid warp Positive colony screening and sequencing identification are consistent with objective gene sequence, successfully construct recombinant plasmid.

1.4.3 recombinant protein inducing expression and soluble analysis

Recombinant plasmid Bm2D97, Bm2D33, Bm2D36, Bm2D41 that success constructs are transformed into E.coliBL21 respectively (DE3) in competent cell, picking monoclonal colonies are cultivated in corresponding resistant LB culture medium, and 220rpm shakes under the conditions of 37 DEG C Overnight incubation.The culture bacterium solution of 500 μ l is taken to be added in the sterile glycerol of 500 μ l 50%, mixing, which is placed in -80 DEG C, saves bacterium Kind.The bacterium solution of overnight incubation 3~4h of shake culture in the culture medium of 2 pipe corresponding resistants is transferred to by 5% inoculum concentration again (to inhale Luminosity A600 value reaches 0.6~0.8), wherein an effective final concentration of 0.1mmol/L (Bm2D33) and 1mmol/L are taken respectively The IPTG of (Bm2D36, Bm2D41, Bm2D97) is induced, and another pipe does not induce as a control group, cultivates 4h under the conditions of 37 DEG C. After inducing expression, 1ml bacterium solution 12000rpm is respectively taken to be centrifuged 1min, abandon supernatant, collects thallus.Thallus is resuspended with PBS, 200w, 3s are opened, under conditions of 5s is closed, with Ultrasound Instrument ultrasound 20 times.Full liquid after 40 μ l ultrasounds, remaining full liquid are left and taken respectively 12000rpm is centrifuged 1min, separation supernatant precipitating, and precipitating is resuspended again with the PBS of 960 μ l.Full liquid, supernatant are taken respectively It is added in 10 μ l 5 × albumen sample-loading buffers with each 40 μ l is precipitated, sample 10min is boiled in boiling water bath, prepares albumen loading Liquid.The expression of albumen and its solubility are analyzed by 12% SDS-PAGE.Deposition condition is as previously mentioned, electrophoresis terminates Afterwards, 1h is dyed with Coomassie Brillant Blue solution, then is decolourized with destainer to visible clearly protein band, taken pictures and save electrophoretogram.

After 4 recombinant plasmids are transformed into the expression of E.coli BL21 (DE3) competent cell respectively, through final concentration 1mmol/L IPTG induction, 2D97,2D36 gene obtain inclusion body expression, and 2D41 gene obtains solubility expression, and molecular weight of albumen is respectively 38KDa (apparent molecular weight 55KDa), 19KDa and 39KDa (13KDa containing SUMO-tag, apparent molecular weight 20KDa)；Through Final concentration 0.1mmol/L IPTG induction, 2D33 (GST-tag 26kDa) gene obtain solubility expression, and molecular weight of albumen is 42KDa；4 recombinant proteins are named as Bm2D97, Bm2D36, Bm2D41 and Bm2D33 respectively, their amino acid sequence point It is not consistent with 2D97,2D36,2D41 and 2D33.SDS-PAGE electrophoretic analysis result is as shown in the figure (such as Figure 12).In Figure 12, M table Show that protein markers, 1 expression recombinant clone do not induce full bacterium, 2 expression recombinant clones do not induce supernatant, and 3 indicate recombinant clone Non- induced precipitation, 4 indicate that recombinant clone induces full bacterium, and 5 indicate that recombinant clone induces supernatant, and 6 indicate recombinant clone induced precipitation.

1.4.4 the purifying of recombinant protein

(1) great expression of recombinant protein：1) bacterial strain is recovered：The preservation strain for determining inducible expression is inoculated into accordingly In the 3ml LB culture medium of resistance, 220rpm, 37 DEG C of shake cultures are stayed overnight；2) expand culture：The bacterium solution of overnight incubation is pressed 5% Inoculum concentration be transferred in the culture medium of 50ml corresponding resistant, 220rpm, (600 value of absorbance A reaches 37 DEG C of shake culture 3h 0.6~0.8), then by the bacterium solution according to 5% inoculum concentration expand culture into the LB culture medium of 1L, according to the item expressed in a small amount Part carries out inducing expression；3) bacterium is received：Under the conditions of 4 DEG C, 4000rpm is centrifuged 15min, collects thallus, repeats this step to 1L bacterium solution Thallus all collected；4) thallus is handled：After the thallus of collection is resuspended with the bufferA of 40~45ml, under the conditions of 4 DEG C 4000rpm is centrifuged 15min and cleans thallus, abandons supernatant, thallus is resuspended with bufferA again, and the albumen of final concentration of 1mM is added Enzyme inhibitor PMSF, -80 DEG C save backup.

(2) insoluble albumen rubber tapping purifying：To the protein B m2D36 and Bm2D97 of inclusion body expression, using raw work biology work The micro QIAquick Gel Extraction Kit of PAGE glue albumen of journey Co., Ltd carries out purification and recovery to destination protein, and purification step is as follows：1) will The thallus of the inclusion body protein of great expression dissolves at room temperature, opens in 400w, 5s, under conditions of 10s is closed, with Ultrasound Instrument ultrasound 99 After secondary, 4 DEG C, 15000g is centrifuged 30min (raising speed 6, reduction of speed 2), abandons supernatant, and precipitating is resuspended with suitable bufferA, every 800 μ l Portion is dispensed into the centrifuge tube of 1.5ml, and -80 DEG C save backup；2) the precipitating suspended matter dispensed in 1) is taken, 200 μ l 5 are added × albumen sample-loading buffer, boils 10min in boiling water bath after mixing, and albumen sample solution is made；3) SDS-PAGE points of configuration 12% From glue and 5% concentration glue, comb is not added when preparing concentration glue, the albumen sample solution made is layered on concentration glue and carries out electrophoresis； 4) purpose band is completely scaled off from entire block glue, is put into the centrifuge tube of 50ml after dyeing and decoloration by protein adhesive, is used ddH₂O is cleaned three times；5) adhesive tape is cut into segment to be placed in centrifuge tube, with tissue grinder instrument by colloidal grinding at tiny broken Piece, after solution A 4ml is added, room temperature shakes 16~18h on decolorization swinging table；6) under room temperature, 12000rpm is centrifuged 15min, inhales It takes supernatant into 50ml centrifuge tube, adds the B solution of 20ml pre-cooling, 4 DEG C of standing 30min after mixing；7) under room temperature, 12000rpm is centrifuged 15min, abandons supernatant, retains the white precipitate of tube bottom, and centrifuge tube is placed in draught cupboard, make residual liquid Volatilization is clean；8) with after suitable PBS solution dissolution precipitating, 20 μ l are taken to prepare albumen sample solution, with SDS-PAGE electrophoresis point mirror Determine protein sample.Protein solution is saved backup in -80 DEG C.

(3) affinity purification of soluble protein：Soluble protein Bm2D33 uses GST affinity purification system, and Bm2D41 uses Ni Column purification system, concrete operations are as follows：1) thallus of great expression soluble protein is dissolved at room temperature, is opened in 400w, 5s, 10s Under conditions of pass, after Ultrasound Instrument ultrasound 99 times, 4 DEG C, 15000g is centrifuged 30min (raising speed 6, reduction of speed 2), and supernatant is transferred to newly 50ml centrifuge tube in, it is primary to repeat centrifugation, collects supernatant；2) it is rinsed with water purification column, stablizes (about 5 to UV value A column volume), then purification column is balanced with about 5 column volume buffer A, UV value is reset；3) albumen supernatant is passed through into pressure pump It after purification column after being loaded to balance, is rinsed with buffer A and removes non-specific binding albumen, be down to baseline to UV value hereinafter, dividing (100% buffer is not used when GST is purified directly with the buffer B of 10%, 30%, 50%, 80%, 100% gradient flushing B is rinsed), destination protein is collected depending on appearance situation；4) albumen of the different component of collection is subjected to SDS-PAGE electrophoresis, identifies mesh Albumen and its purity；5) 5 column volumes are rinsed again with 100% buffer B after collection albumen, sufficiently removing affinity media Upper binding protein is sufficiently rinsed and is saved with water and 20% ethyl alcohol respectively again later.

(4) excision of protein fusion label：Bm2D41 albumen contains SUMO fusion tag, needs to cut off next to carry out Functional verification experiment.Digestion is tested using digestion on column by the way of, and configuration digestion system is as shown in table 13, by digestion system and 1ml His protein purification medium filler is sufficiently mixed, and 4 DEG C of shaking tables shake digestion 16h.4 DEG C, 500g is centrifuged 5min, collects supernatant Liquid is the destination protein liquid after digestion, after SDS-PAGE electroresis appraisal, packing be stored in -80 DEG C it is spare.The processing of filler With preserving type with the processing of the above purification column.

13 SUMO protease digestion system of table

The measurement of protein concentration samples Branford method, and concrete operations are referring to kit specification.Inclusion body protein The Bm2D97 and Bm2D36 micro QIAquick Gel Extraction Kit of PAGE glue albumen is purified, and through SDS-PAGE electrophoretic analysis, list has successfully been obtained The recombinant protein of one band (shown in Figure 13).Soluble protein Bm2D41 and Bm2D33 are purified with affinity media, through SDS-PAGE Electrophoretic analysis obtains the relatively single fusion protein of band.Bm2D41 albumen after purification, through SUMO protease digestion system It cuts off protein fusion label, obtains the relatively single recombinant protein of purpose band (shown in Figure 13).In Figure 13, M indicates protein Marker, 1 expression recombinant clone do not induce full bacterium, and 2 indicate that recombinant clones induce full bacterium, and 3 indicate recombinant protein after purification, and 4 Recombinant protein after indicating excision fusion tag.

1.4.5 the evaluation of recombinant protein

This experiment evaluates recombinant protein by ELISA experimental technique, except Bm2D97 uses IgM antibody (including HRP Goat anti-mouse IgM and HRP goat anti-human IgM) it detects outside, remaining recombinant antigen uses IgG antibody (including HRP goat anti-mouse IgG and HRP goat anti-human IgG) is detected.

Pass through optimum experimental albumen peridium concentration, serum and secondary antibody dilution.Best peridium concentration recombination after optimization Protein B m2D97, Bm2D33, Bm2D36, Bm2D41, Bm7 are respectively 2 μ g/ml, 2 μ g/ml, 2 μ g/ml, 1 μ g/ml, 5 μ g/ml, As reference, best peridium concentration is respectively 1 μ g/ml and 5 μ g/ml by the crude protein of Babesiamicrofti and reported BmSA1.

(1) evaluation of the mouse serum of infection by Babesia microti to recombinant antigen

Recombinant protein Bm2D97, Bm2D33, Bm2D36, Bm2D41, Bm7 are coated with by the above peridium concentration, respectively with infection 3 days after Babesiamicrofti, 7 days, 14 days, 21 days, 30 days, 60 days, 120 days, 150 days, 270 days mice serums and normal mouse Each 10 part (1 of serum：100 dilutions) it is incubated for, secondary antibody HRP goat anti-mouse IgM and HRP goat anti-mouse IgG presses 1：It is incubated for after 5000 dilutions, the immune response trend of recombinant antigen is evaluated with this.

In the protein chip analysis of specific IgM antibodies, the cell-free expression albumen 2D97 of screening, through prokaryotic expression and Recombinant antigen B m2D97 is obtained after purification, is evaluated with the mice serum of different infective stages, is showed the 3dpi mouse blood with early stage There is higher immune response clearly.The immune response trend of recombinant antigen is as shown in figure 14.From the protein chip of specific IgG antibodies It analyzes in result, cell-free expression the albumen 2D41 and Bm7 of screening, through prokaryotic expression and after purification, from different infective stages Mice serum reaction, Recombinant antigen B m2D41 and Bm7 present persistently high immune response；The cell-free expression egg of screening After white 2D33 and 2D36 expression and purification, evaluated with the mice serum of different infective stages, Recombinant antigen B m2D33 and Bm2D36 table Reveal the immune response trend that decline is reacted after acute phase.The immune response trend of recombinant antigen is as shown in figure 14.

(2) evaluation of the Babesiamicrofti patients serum to recombinant antigen

Recombinant protein Bm2D97, Bm2D33, Bm2D41, Bm7, BmSA1 press the above peridium concentration, respectively with 8 parts of babesias Patients serum and 10 portions of normal human serums (1：100 dilutions) it is incubated for, secondary antibody HRP goat anti-human IgM and HRP goat Anti-human IgG presses 1：It is incubated for after 10000 dilutions, recombinant protein is evaluated as the sensibility of candidate diagnosis antigen using this.

Using normal person and babesiasis human serum to recombinant antigen Bm2D97, Bm2D33, Bm2D41, Bm7 and BmSA1 into Row elisa assay, as a result as shown in figure 15, wherein Bm2D33 and Bm2D41 is compared to reported BmSA1 (sensibility is 0%) higher sensibility is shown, respectively 62.5% and 50%.In the elisa assay to combined antigen, more antigen groups Conjunction Bm2D33+Bm2D41+Bm7 is compared to Bm2D33+Bm2D41 and shows higher sensibility, the sensitivity of two combined antigens Property is respectively 62.5% and 25%, as a result as shown in figure 15.

(3) cross reaction of recombinant antigen and malaria patients serum

It is incubated for the recombinant antigen coating in (2) with secondary antibody, primary antibody uses 10 parts of tertian fevers and malignant malaria patients serum respectively With 10 portions of normal human serums (1：100 dilutions) it is incubated for, the cross reaction of recombinant antigen Yu malaria patients serum is evaluated with this.

Elisa assay is carried out to each recombinant antigen using normal person and malaria patients serum, as a result as shown in figure 16, Bm7, BmSA1, Bm2D33, Bm2D41 and Bm2D97 for the specificity of vivax malaria sample detection be respectively 90%, 80%, 70%, 60% and 30%；Specificity for malignant malaria pattern detection is respectively 100%, 80%, 100%, 80% and 60%. In the cross reaction with malaria patients serum, Bm7 and Bm2D33 show very high specificity, in particular for malignant malaria disease The specificity of human serum is 100%.

(4) assessment of malaria epidemic area scene fever patient sample

200 parts of fever patient blood samples extract DNA using DNA extraction kit, detect coding vole with round pcr The specific fragment of the 18SrRNA of babesia.In the research to 200 parts of fever patient sample nucleic acids, the tertian fever positive 14 is found A, malignant malaria is 4 positive, including the sample of 1 tertian fever and malignant malaria nucleic acid coinfection.In the field to 200 parts of samples In Babesia muris detection of nucleic acids, 10 positive samples, but the coinfection without finding babesia and plasmodium nucleic acid are found.Recombination Protein B m2D97, Bm2D33, Bm2D41, Bm7, the crude protein of Babesiamicrofti and BmSA1 are by the above optimization concentration coating, respectively With 200 parts of fever patients and 45 portions of normal human serums (1：100 dilutions) it is incubated for, secondary antibody is incubated for (2).In conjunction with detection of nucleic acids knot Fruit analyzes the potential propagation risk of Babesiamicrofti disease of malaria epidemic area.

In the research to 200 parts of malaria epidemic area patient-heated serum's samples, using Babesiamicrofti thick leach protein, BmSA1, Bm7, Bm2D33, Bm2D41 and Bm2D97 recombinant antigen carry out elisa assay, as a result as shown in figure 17, in 200 parts of blood In clear, there are 32 and 34 parts of positive serums by Recombinant antigen B m2D41 and the Bm7 test positive of lasting high reaction, middle-jiao yang, function of the spleen and stomach respectively Property serum in respectively include 1 part, positive and each 7 parts of the plasmodium molecule of 2 parts of Babesiamicrofti molecules it is positive；There are 32 parts of positives Serum Recombinant antigen B m2D33 test positive, wherein respectively includes 2 parts of Babesiamicrofti molecule positives and 9 parts in positive serum Plasmodium molecule is positive；There are 27 parts of positive serums by Recombinant antigen B m2D97 test positive, is wherein wrapped respectively in positive serum It includes 1 part of Babesiamicrofti molecule positive and 10 parts of plasmodium molecules is positive；Recombinant protein BmSA1 and Babesiamicrofti thick leach protein As control, 38 and 58 parts of positive serums are detected respectively, and 1 part, 3 parts of Babesiamicroftis point are wherein respectively included in positive serum It is sub positive and 14 parts, 9 parts of plasmodium molecule it is positive.In addition, research also found, as shown in table 14：In the positive sample detected In this, Babesiamicrofti molecule is negative and the highest of the sample proportion of antibody positive, about 72%~85%.

PCR the and ELISA positive findings analysis of 200 parts of 14 malaria epidemic area of table live fever patient samples

In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, it is not of the invention to limit Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in In protection scope of the present invention.

Sequence table

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

<120>Babesiamicrofti 2D97 antigen protein and its application

<130> CPC-NP-18-101030

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 984

<212> DNA

<213> 2D97

<400> 1

cacttatatt cgctaaaacg gtttcaagtg gatagtgtcg tggttgatgc cgtttaccat 60

gcggaaaata attttataac tgcatctact gaccaaacct attacttaac tagtgtaaac 120

gagactgata ttcaacagct agatcaacta ctcgaattag gtgaatggca tgaagtgccc 180

agtgaaatcg aagaacctcc tgataaacaa gagatagaaa acacatctga agaagaaaca 240

attaataaga atgaatctga tgaattattt aagtctagta taactccgtt cattatacga 300

gacggttata tttactttaa atttgtcgat atggccaacg aatcccaagg tgttgaaatt 360

gacaaatgcg tatttatcgc cgacgcttct aagaatgaat tagccattct gatcaaaggt 420

gaagtacagg aacatgaaat gttcatattt tccctaccta ctaaacaaga ctttgattat 480

tggaaaaaca atctagtgga acatgggttg aatgagcaac aaaatgatat gaataacgat 540

gatggcaaat tttaccgttc caattctggc aagattaatc aacaagcttg tgtggtgacc 600

aaaggccaaa tgcaattgtt caaagactat aatgatgacg tttcggagcc attgataaca 660

tacgtaagct catcaacaaa agtttctata aatgaggaaa aacgggaaat tattatggaa 720

tccacgtcta cgcatggaaa taggagcaga attactttgg attgcaccaa tccgaaggaa 780

ttcgtaaggt ggaaatcggc gttgattttg gctggattta tatctggtaa ctcagtcaat 840

agcgataaca taaatggact caaaaagtat gttttcccaa taaaactctt taaaacatct 900

gataacaatg atagtggaag gagggctttt gtaattaaat ccaattatgt ggcattgtat 960

atcagcagca cttctaaaga tcca 984

<210> 2

<211> 328

<212> PRT

<213> 2D97

<400> 2

His Leu Tyr Ser Leu Lys Arg Phe Gln Val Asp Ser Val Val Val Asp

1 5 10 15

Ala Val Tyr His Ala Glu Asn Asn Phe Ile Thr Ala Ser Thr Asp Gln

20 25 30

Thr Tyr Tyr Leu Thr Ser Val Asn Glu Thr Asp Ile Gln Gln Leu Asp

35 40 45

Gln Leu Leu Glu Leu Gly Glu Trp His Glu Val Pro Ser Glu Ile Glu

50 55 60

Glu Pro Pro Asp Lys Gln Glu Ile Glu Asn Thr Ser Glu Glu Glu Thr

65 70 75 80

Ile Asn Lys Asn Glu Ser Asp Glu Leu Phe Lys Ser Ser Ile Thr Pro

85 90 95

Phe Ile Ile Arg Asp Gly Tyr Ile Tyr Phe Lys Phe Val Asp Met Ala

100 105 110

Asn Glu Ser Gln Gly Val Glu Ile Asp Lys Cys Val Phe Ile Ala Asp

115 120 125

Ala Ser Lys Asn Glu Leu Ala Ile Leu Ile Lys Gly Glu Val Gln Glu

130 135 140

His Glu Met Phe Ile Phe Ser Leu Pro Thr Lys Gln Asp Phe Asp Tyr

145 150 155 160

Trp Lys Asn Asn Leu Val Glu His Gly Leu Asn Glu Gln Gln Asn Asp

165 170 175

Met Asn Asn Asp Asp Gly Lys Phe Tyr Arg Ser Asn Ser Gly Lys Ile

180 185 190

Asn Gln Gln Ala Cys Val Val Thr Lys Gly Gln Met Gln Leu Phe Lys

195 200 205

Asp Tyr Asn Asp Asp Val Ser Glu Pro Leu Ile Thr Tyr Val Ser Ser

210 215 220

Ser Thr Lys Val Ser Ile Asn Glu Glu Lys Arg Glu Ile Ile Met Glu

225 230 235 240

Ser Thr Ser Thr His Gly Asn Arg Ser Arg Ile Thr Leu Asp Cys Thr

245 250 255

Asn Pro Lys Glu Phe Val Arg Trp Lys Ser Ala Leu Ile Leu Ala Gly

260 265 270

Phe Ile Ser Gly Asn Ser Val Asn Ser Asp Asn Ile Asn Gly Leu Lys

275 280 285

Lys Tyr Val Phe Pro Ile Lys Leu Phe Lys Thr Ser Asp Asn Asn Asp

290 295 300

Ser Gly Arg Arg Ala Phe Val Ile Lys Ser Asn Tyr Val Ala Leu Tyr

305 310 315 320

Ile Ser Ser Thr Ser Lys Asp Pro

325

<210> 3

<211> 432

<212> DNA

<213> 2D33

<400> 3

ggattagaag atgctgtagc gggaacggag ttactggtag ctaaagatgg tgatgatatt 60

gaggaattga aagtggaggt aatgcgagac atatcttcaa tatttgactg tgttgatcga 120

tccggagtgg gggtttactt gatggctagt accctagggt cactagaggc tttacttcaa 180

tttataaatg ggcaaaagat accagtattt tgtgtcaaca ttggggcagt gcaaaagaag 240

gatgtcaaga aggctagcat aatgttggag aaaaggccag aatatgcggc tatacttgca 300

tttgacgtca aagttacaca agatgcagaa aaggaggccg aaacgttggg agttacaatt 360

ttttgtgctg atatcattta ccacctctct gataggttca ccaagcacat ggaggagcag 420

agggaactat ac 432

<210> 4

<211> 144

<212> PRT

<213> 2D33

<400> 4

Gly Leu Glu Asp Ala Val Ala Gly Thr Glu Leu Leu Val Ala Lys Asp

1 5 10 15

Gly Asp Asp Ile Glu Glu Leu Lys Val Glu Val Met Arg Asp Ile Ser

20 25 30

Ser Ile Phe Asp Cys Val Asp Arg Ser Gly Val Gly Val Tyr Leu Met

35 40 45

Ala Ser Thr Leu Gly Ser Leu Glu Ala Leu Leu Gln Phe Ile Asn Gly

50 55 60

Gln Lys Ile Pro Val Phe Cys Val Asn Ile Gly Ala Val Gln Lys Lys

65 70 75 80

Asp Val Lys Lys Ala Ser Ile Met Leu Glu Lys Arg Pro Glu Tyr Ala

85 90 95

Ala Ile Leu Ala Phe Asp Val Lys Val Thr Gln Asp Ala Glu Lys Glu

100 105 110

Ala Glu Thr Leu Gly Val Thr Ile Phe Cys Ala Asp Ile Ile Tyr His

115 120 125

Leu Ser Asp Arg Phe Thr Lys His Met Glu Glu Gln Arg Glu Leu Tyr

130 135 140

<210> 5

<211> 519

<212> DNA

<213> 2D36

<400> 5

atgggtggag ctgtccctct atcaaattac atctcaccga tcacaaacgc cgtatctaac 60

acaatggaga aactcattaa ttctcaattg ggctatacaa tgctatacgg aatacctgtt 120

atctttgggc ttagacagat cagccgctat aatagactcg ctaaattgag attcgatgac 180

attgggtacc agataaggcg ggcggattcg ataggcaata ggtggattgc ctgcaagtac 240

tttttcgttg gtaccgtact agcaccacta actggctgtg caatcgcata caaaatatac 300

gcttgttcac ttggcgaaaa ttaccaagtg aacaagttga tctttcacga catcaccagg 360

caatttggaa atggcatccg cattgctaat gacatacaaa ccgaatttat ccgggaaaat 420

ttgacggtgg ataacagaat atccgaatcg tttaagcgta tgtctacgga gctcaagcag 480

gacgccagca aggtgaaaag taaacttggt ttaatttaa 519

<210> 6

<211> 172

<212> PRT

<213> 2D36

<400> 6

Met Gly Gly Ala Val Pro Leu Ser Asn Tyr Ile Ser Pro Ile Thr Asn

1 5 10 15

Ala Val Ser Asn Thr Met Glu Lys Leu Ile Asn Ser Gln Leu Gly Tyr

20 25 30

Thr Met Leu Tyr Gly Ile Pro Val Ile Phe Gly Leu Arg Gln Ile Ser

35 40 45

Arg Tyr Asn Arg Leu Ala Lys Leu Arg Phe Asp Asp Ile Gly Tyr Gln

50 55 60

Ile Arg Arg Ala Asp Ser Ile Gly Asn Arg Trp Ile Ala Cys Lys Tyr

65 70 75 80

Phe Phe Val Gly Thr Val Leu Ala Pro Leu Thr Gly Cys Ala Ile Ala

85 90 95

Tyr Lys Ile Tyr Ala Cys Ser Leu Gly Glu Asn Tyr Gln Val Asn Lys

100 105 110

Leu Ile Phe His Asp Ile Thr Arg Gln Phe Gly Asn Gly Ile Arg Ile

115 120 125

Ala Asn Asp Ile Gln Thr Glu Phe Ile Arg Glu Asn Leu Thr Val Asp

130 135 140

Asn Arg Ile Ser Glu Ser Phe Lys Arg Met Ser Thr Glu Leu Lys Gln

145 150 155 160

Asp Ala Ser Lys Val Lys Ser Lys Leu Gly Leu Ile

165 170

<210> 7

<211> 687

<212> DNA

<213> 2D41

<400> 7

ggtatcgaaa ctgttggtgg cgtcatgact aagattatcc cccgtaacac tgtggttcca 60

acaaagaaat cacaaatgtt ctctacttat atggacaatc aaaccaccgt cactatccaa 120

gtttaccaag gagaacgttc aatgacacaa cacaatactc tgctcggaag atttgacctt 180

actggtattg cccctgcacc tcgtaacact cctcaaatca atgtcacatt tgaaatcgac 240

actaatggca ttgtgagtgt atcagctgag gataaaggta gtggtaagag tcagaaaatt 300

accataaccg cggaacaagg caggttatca aaggctgaga ttgaaagaat gatcgaagag 360

tcggagagat atgctcagga ggataaggaa atgatggaac gtgttgaatc caagaatacg 420

cttgatggtt acatcgctag tatgaagagg agtgtggaag ataaggataa gttggctgat 480

aagattgagg aagatgacaa gaccacaatt ttgaacgcct tgaaagatgc tgagacgtgg 540

cttttccaaa atccagaggc ggatactgat gagtataaat ccaaactcaa gtcactggaa 600

gaaatttgta atcctattat acaaaaattg taccagggca atgcagccgg ggaatccaat 660

tatgattctt acggggatga gttatga 687

<210> 8

<211> 228

<212> PRT

<213> 2D41

<400> 8

Gly Ile Glu Thr Val Gly Gly Val Met Thr Lys Ile Ile Pro Arg Asn

1 5 10 15

Thr Val Val Pro Thr Lys Lys Ser Gln Met Phe Ser Thr Tyr Met Asp

20 25 30

Asn Gln Thr Thr Val Thr Ile Gln Val Tyr Gln Gly Glu Arg Ser Met

35 40 45

Thr Gln His Asn Thr Leu Leu Gly Arg Phe Asp Leu Thr Gly Ile Ala

50 55 60

Pro Ala Pro Arg Asn Thr Pro Gln Ile Asn Val Thr Phe Glu Ile Asp

65 70 75 80

Thr Asn Gly Ile Val Ser Val Ser Ala Glu Asp Lys Gly Ser Gly Lys

85 90 95

Ser Gln Lys Ile Thr Ile Thr Ala Glu Gln Gly Arg Leu Ser Lys Ala

100 105 110

Glu Ile Glu Arg Met Ile Glu Glu Ser Glu Arg Tyr Ala Gln Glu Asp

115 120 125

Lys Glu Met Met Glu Arg Val Glu Ser Lys Asn Thr Leu Asp Gly Tyr

130 135 140

Ile Ala Ser Met Lys Arg Ser Val Glu Asp Lys Asp Lys Leu Ala Asp

145 150 155 160

Lys Ile Glu Glu Asp Asp Lys Thr Thr Ile Leu Asn Ala Leu Lys Asp

165 170 175

Ala Glu Thr Trp Leu Phe Gln Asn Pro Glu Ala Asp Thr Asp Glu Tyr

180 185 190

Lys Ser Lys Leu Lys Ser Leu Glu Glu Ile Cys Asn Pro Ile Ile Gln

195 200 205

Lys Leu Tyr Gln Gly Asn Ala Ala Gly Glu Ser Asn Tyr Asp Ser Tyr

210 215 220

Gly Asp Glu Leu

225

<210> 9

<211> 217

<212> PRT

<213> Bm7

<400> 9

Met His Ile Asn Tyr Lys Leu Ile Ile Thr Gly Leu Val Ser Ile Ala

1 5 10 15

Leu Ala Thr Ser Ile Thr Leu Ala Val Ile Tyr Leu Pro Asn Arg Ser

20 25 30

Cys Pro Gly Asn Asn Gly Val Gly Gly Gly Ser Gly Asp Asn Asn Ser

35 40 45

Gly Ile Ile Pro Asn Asp Pro His Pro Cys Cys Asn Asn Leu Arg Gln

50 55 60

Lys Pro Gln Tyr Gln Thr Lys Pro Glu Asn Glu Leu Val Asn Asp Asp

65 70 75 80

Arg Asp Leu Asn Phe Asn Lys Ile Arg Gly Gly Lys Gln Ile Ile Thr

85 90 95

Phe Thr Val Pro Ser Ile Asp Asp Leu Lys Asn Lys Arg Leu Ser Asp

100 105 110

Ser Glu Phe Ile Leu Ser Glu Lys Ala Asn Pro Leu Ile Ser Ser Gly

115 120 125

Asp Ser Lys Asn Val Ile Val Phe Glu Val Lys Asn Asp Asn Glu Lys

130 135 140

Leu Met Gly Ser Val Glu Val Gly Gln Trp Glu Val Thr Ile Thr Thr

145 150 155 160

Ser Cys Ile Arg Arg Ile Val Ile Phe Asp Ser Asn Glu Val Ser Asp

165 170 175

Asn Ile Pro Met Tyr Ile Tyr Ile Val Asp Tyr Phe Glu Gly Gly Asn

180 185 190

Ser Thr Val Ser Lys Phe Phe Phe Ala Asn Asn Arg Trp Asn Ala Asp

195 200 205

Phe Thr Asn His Thr Pro Asn Ala Ala

210 215

Claims (12)

1. a kind of isolated gene, which is characterized in that its sequence such as SEQ ID NO：Shown in 1.

2. a kind of Babesiamicrofti 2D97 antigen protein, which is characterized in that its amino acid sequence such as SEQ ID NO：Shown in 2.

3. a kind of isolated gene as described in claim 1 is in the system of preparation treatment, diagnosis or prevention Babesiamicrofti disease Application in agent.

4. a kind of Babesiamicrofti 2D97 antigen protein as claimed in claim 2 is in preparation treatment, diagnosis or prevention vole Application in the preparation of babesiasis.

5. a species specific combination as claimed in claim 2 a kind of Babesiamicrofti 2D97 antigen protein preparation treatment, Application in the preparation of diagnosis or prevention Babesiamicrofti disease.

6. such as the described in any item applications of claim 3-5, which is characterized in that for detecting or diagnosing the infection of Babesiamicrofti disease Early stage and/or window phase.

7. a kind of kit, which is characterized in that containing such as SEQ ID NO：A kind of isolated gene, such as SEQ ID shown in 1 NO：The combination such as SEQ ID NO of Babesiamicrofti 2D97 antigen protein shown in 2 or specificity：Vole babe shown in 2 The antibody of worm 2D97 antigen protein.

8. the joint antigen protein of one group of Babesiamicrofti, which is characterized in that including：

A) Babesiamicrofti 2D97 antigen protein, amino acid sequence such as SEQ ID NO：Shown in 2；

B) at least one of both Babesiamicrofti 2D33 antigen protein, 2D36 antigen protein；The 2D33 antigen protein Amino acid sequence such as SEQ ID NO：Shown in 4, the amino acid sequence of the 2D36 antigen protein such as SEQ ID NO：Shown in 6.

9. the joint antigen protein of one group of Babesiamicrofti as claimed in claim 8, which is characterized in that also include：C) have Continue the Babesiamicrofti antigen protein of high immune response；Preferably, the vole babe with lasting high immune response Worm antigen protein is selected from least one of Babesiamicrofti antigen protein 2D41 and Bm7；The amino of the 2D41 antigen protein Acid sequence such as SEQ ID NO：Shown in 8, the amino acid sequence of the Bm7 antigen protein such as SEQ ID NO：Shown in 9.

10. as the described in any item one group of Babesiamicrofti of claim 8-9 joint antigen protein preparation treatment, diagnosis or Person prevents the application in the preparation of Babesiamicrofti disease.

11. a kind of kit, the joint antigen protein containing the described in any item one group of Babesiamicrofti of such as claim 8-9.

12. a kind of antigen-reactive of Babesiamicrofti is composed, which is characterized in that a kind of Babesiamicrofti antigen immune response spectrum is Target gene 2D1~128 are cloned and expressed using fusion cloning technology and wheat germ cell-free protein expression system, are obtained 87 soluble proteins were obtained, in conjunction with the recombinant protein Bm7 screened from Babesiamicrofti cDNA library, 88 antigens is amounted to and passes through egg White chip technology is reacted with mice serum after the infection of Normal Mouse Serum and different times, the immune response spectrum of acquisition.