CN108893477B - Babesia microti 2D41 antigen protein and application thereof - Google Patents

Abstract

The invention discloses a babesia microti 2D41 antigen protein and application thereof, wherein the amino acid sequence of the antigen protein is shown as SEQ ID NO: 8, and the coding gene sequence is shown as SEQ ID NO: shown at 7. The babesia microti 2D41 antigen protein has continuous high immune reaction with mouse serum of 14 dpi-270 dpi in specific IgG antibody reaction, shows obvious effect of reducing the blood worm disease in immune protective test, and is reduced by about 35% compared with the mean value of the blood worm disease of an adjuvant group and a natural immune group. The 2D41 antigen protein can be used for detecting or diagnosing babesiosis of a field rat, and can also be used for preparing medicines for reducing treatment and/or prevention of vermiemia.

Description

Babesia microti 2D41 antigen protein and application thereof

Technical Field

The invention relates to the field of molecular, cellular, proteomic and immunological research, in particular to preparation of babesia microti immunity protein, acquisition of immunoreaction spectrum and application in screening of candidate diagnosis antigen and immune protection vaccine.

Background

Babesiosis is a zoonosis caused by the parasitism of babesia protozoa in erythrocytes, transmitted by tick bites and blood transfusions. There are over one hundred species of babesia isolated from wild animals and livestock, among which cases in which humans are infected have been reported mainly including: microti and related pathogens, b.divergens and related pathogens, b.duncanni and related pathogens, b.venatorum. In 1 month 2011 babesiosis was listed as a national regulated infectious disease. In the united states, most cases of babesia were caused by babesia microti, and it was reported that 159 cases of 162 cases of transfusion-associated babesia were identified as babesia microti between 1979 and 2009, which is a major pathogen of human babesia. The expression difference of people infected with babesia is large, and most of healthy people are recessive infected; in patients with immunodeficiency, such as splenectomy, HIV patients, the elderly and children may suffer serious illness or even death.

At present, the diagnosis method of babesiosis comprises the following steps: blood slides were examined microscopically, antibody and PCR detected and test animals were transferred, however, each detection method has relative limitations. Oil-scopy of Giese stained peripheral blood smears is the gold standard for babesiosis diagnosis, but generally the percentage of peripheral red blood cell infection is low and the morphology is similar to that of Plasmodium; the PCR technology has high sensitivity and specificity, but is not suitable for detecting low-density zoonosis and a window-stage sample; the detection of antibodies is highly sensitive and is also suitable for the detection of low density infected samples, but it is equally not effective for the detection of windowed samples and the antibodies will still persist for a considerable period of time to show false positives after the haematemia has cleared. Due to the self-limitation of babesiosis of the field mouse and the limitation of the current detection means, misdiagnosis or missed diagnosis is easy to happen in disease screening, so that blood products are polluted to form disease transfusion transmission. The existing diagnostic antigens have the defects that samples in the early stage (window stage) of infection cannot be detected, and the disease process of the infection from the acute stage to the chronic stage cannot be reflected, such as antigens BmSA1, BmP94, BmIRA, BMN1-8, BM1542, Bm186 and the like with diagnostic effects in current literature reports.

Studies have shown that the detection of antibody levels is of great importance for screening cases of occult infection, where IgM antibodies are first produced and respond to parasites during the acute infection phase, while specific IgG is associated with a reduction in parasite numbers. However, in the course of babesiosis infection, samples in the early stage of infection (window phase) cannot be detected effectively, and the antigenic/immune response spectrum is not clear at present and the disease course cannot be monitored during the course of infection from the acute phase to the chronic phase. The disease process of babesiosis is known through an immunoreaction spectrum, and a marker antigen capable of reflecting the disease process is screened, so that the method is very important for developing a high-flux, quick and sensitive babesia microti diagnostic reagent for the field babesia to meet the requirements of disease detection and screening.

Disclosure of Invention

The technical problem to be solved by the invention is to provide one or more marker antigens of babesia microti aiming at the limitation of the existing babesia microti disease detection, which shows that the high-level response is maintained in the whole course of the disease, and the antigen has the effect of controlling the zoonosema, can be used for detecting or diagnosing the babesia microti infection, and can also be used as a candidate vaccine for preparing medicines for treating and/or preventing the zoonosema.

The invention provides an isolated gene which can encode Babesia microti 2D41 antigen protein and has a sequence shown as SEQ ID NO: shown at 7.

The invention provides a babesia microti 2D41 antigen protein, which is encoded by the separated gene, and the amino acid sequence of the babesia microti is shown as SEQ ID NO: shown in fig. 8.

The invention provides an application of another separated gene in preparing a product for treating, diagnosing or preventing babesiosis of a paddy field, wherein the separated gene can code 2D41 antigen protein of babesia microti, and the sequence of the separated gene is shown as SEQ ID NO: shown at 7. The product comprises medicines for treatment and prevention, and reagent strips, kits and the like for detection and diagnosis.

The invention also provides application of the babesia microti 2D41 antigen protein in preparation of products for treating, diagnosing or preventing babesia microti diseases. In particular to the detection or diagnosis of the babesiosis disease of the field rat and the preparation of the medicine for reducing the treatment and/or prevention of the vermiemia. The babesia microti 2D41 antigen protein has continuous high immune reaction with mouse serum of 14 dpi-270 dpi in specific IgG antibody reaction, shows obvious effect of reducing the zoonosemia in immune protective test, and is reduced by about 35% compared with the mean value of the zoonosemia of an adjuvant group and a natural immune group. The Babesia microti 2D41 antigen protein is coded by the separated gene, and the amino acid sequence of the antigen protein is shown as SEQ ID NO: shown in fig. 8.

The invention also provides application of the antibody in preparation of a preparation for treating, diagnosing or preventing babesiosis of a paddy field, wherein the antibody is specifically combined with the babesia microti 2D41 antigen protein. Further, the antibody is a monoclonal antibody.

The invention also provides a kit containing the isolated gene, the babesia microti 2D41 antigen protein or the antibody.

The invention also provides an immunochromatographic test strip which contains the Babesia microti 2D41 antigen protein.

The invention also provides a vaccine comprising a polypeptide as set forth in SEQ ID NO: 8, and 2D41 antigen protein of Babesia microti. The vaccine can effectively reduce the blood disease of the worms by about 35 percent.

The invention also provides a combined antigen protein of the babesia microti, which comprises the following components: a) the Babesia microti 2D41 antigen protein has an amino acid sequence shown as SEQ ID NO: shown in fig. 8.

The invention divides 87 babesia microti proteins obtained by two-dimensional immunoblotting and mass spectrometry into 128 orofs gene sequences, clones and expresses target genes by using a fusion cloning technology and a wheat germ cell-free protein expression system, and finally successfully obtains 87 soluble proteins. Combining with the recombinant protein Bm7 screened from the Babesia microti cDNA library, the total 88 antigens react with the serum of a normal mouse and the serum of the infected mouse in different periods through a protein chip technology to obtain the immunoreaction spectrums of the antigens, thereby knowing the distribution of the antigens in the disease process.

We find that 4 proteins 2D5, 2D29, 2D41 and Bm7 which have continuous high immune response with mouse serum of 14 dpi-270 dpi are screened in a specific IgG antibody reaction, and escherichia coli prokaryotic expression is carried out on the first three candidate antigens, so that 1 recombinant protein Bm2D41 is successfully obtained. The recombinant protein Bm2D41 showed that high level of response was maintained throughout the disease in specific IgG antibodies. We also evaluated Bm2D41 and Bm7 as candidate vaccines, and found that the peak value of the recombinant protein Bm2D41 of mice immunized is reduced by nearly 35% compared with that of mice controlled by the control group, the difference is obvious, and the occurrence of severe insect blood disease is effectively inhibited. The recombinant protein Bm2D41 showed a higher sensitivity compared to the reported BmSA1 (sensitivity of 0%), sensitivity of 50%.

Drawings

FIG. 1 is a two-dimensional immunoblot hybridization diagram of a babesia microti crude extract protein.

FIG. 2 shows the primer design for In-Fusion clone PCR amplification.

FIG. 3 is the PCR amplification electrophoresis chart of the target gene.

FIG. 4 is a colony PCR identification electrophoresis of a portion of the recombinant plasmid.

Fig. 5 is a schematic view of the installation of the film transfer device.

FIG. 6 is a Western-blot analysis chart of cell-free expressed proteins, wherein M represents a protein Marker.

FIG. 7 is a protein chip diagram of the immunoreaction of Babesia microti protein in IgG antibody, wherein A represents Babesia microti protein 2D 3-2D 54, and B represents Babesia microti protein 2D 55-2D 128; a to j respectively represent: 0,3,7,14,21,30,60,120,150,270dpi of murine serum; 1 represents a positive control, 2 represents a negative control, and the other boxes represent the candidate antigens of the Babesia microti.

FIG. 8 is a chart of a thermographic analysis of the immunoreaction of Babesia microti protein in specific IgG antibodies, wherein the fluorescence signal intensity of the chip immunoreaction is clustered at the median value, and the fluorescence range is 0-2500.

FIG. 9 is a protein chip diagram of the immunoreaction of Babesia microti protein in IgM antibody, wherein A represents Babesia microti proteins 2D 3-2D 54 and Bm7, and B represents Babesia microti proteins 2D 55-2D 128 and Bm 7; a to j respectively represent: 0,3,7,14,21,30,60,120,150,270dpi of murine serum; 1 represents a positive control, 2 represents a negative control, and the other boxes represent the candidate antigens of the Babesia microti.

FIG. 10 is a chart of heat map analysis of the immunoreaction of Babesia microti protein in specific IgM antibody, wherein the median of the fluorescence signal intensity of the chip immunoreaction is clustered, and the fluorescence range is 0-2500.

FIG. 11 is a diagram showing the result of electrophoresis of a target gene fragment amplified by PCR, wherein M: DNA marker, 1: 2D97, 2: 2D33, 3: 2D36, 4: 2D 41.

Fig. 12 is a graph showing the results of electrophoresis of expression and solubility analysis of recombinant proteins Bm2D33, Bm2D36, Bm2D41, Bm2D97, wherein M: protein marker, 1: recombinant clone non-induced whole bacteria, 2: recombinant clones did not induce supernatant, 3: recombinant clones did not induce precipitation, 4: recombinant clone-induced whole bacteria, 5: recombinant clone induction supernatant, 6: precipitation was induced by recombinant cloning.

Fig. 13 is a graph showing electrophoresis results of purified recombinant proteins Bm2D33, Bm2D36, Bm2D41, and Bm2D97, wherein M: protein marker, 1: recombinant clone non-induced whole bacteria, 2 recombinant clone induced whole bacteria, 3: purified recombinant protein, 4: and (3) cutting the recombinant protein after the fusion tag is cut.

FIG. 14 shows the results of ELISA evaluation of sera of babesia microti-infected mice on recombinant antigens Bm2D33, Bm2D36, Bm2D41, Bm2D97, and Bm 7.

Fig. 15 shows the ELISA evaluation of babesia microti patient sera against each recombinant antigen and the combined antigen, where a represents the ELISA reaction of each recombinant antigen with babesia human sera, B represents the ELISA reaction of the combined antigen with babesia human sera, and n ═ 8.

FIG. 16 is a graph showing the results of cross-reactivity of each recombinant antigen with the serum of a malaria patient, wherein A represents the ELISA reaction of the recombinant antigen with the serum of a. vivax malaria patient, B represents the ELISA reaction of the recombinant antigen with the serum of a falciparum malaria patient, and n ═ 10.

FIG. 17 is an ELISA assessment of 200 spot fever patient samples from malaria endemic areas.

FIG. 18 shows the evaluation of immune effect after the third immunization of protein immunization groups, wherein A represents the evaluation of Bm2D41 immunization group IgG antibody level, and B represents the evaluation of Bm7 immunization group IgG antibody level.

FIG. 19 is a graph showing the evaluation of the immunoprotective effect of recombinant proteins, in which A represents Bm2D 41-immunized group, B represents Bm 7-immunized group, and the solid line and dotted line represent the zoonosis and antibody level after inoculation of Babesia microti, respectively.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1 animal serum sources

1. Laboratory animal and insect strain

The experimental animals are BALB/c mice of 6-8 weeks old and purchased from Shanghai research center of Chinese academy of sciences. The Babesia strain is Babesia microti

PRA-99T, provided by institute of Experimental zoology of Chinese academy of medical sciences, was inoculated with liquid nitrogen for subculture.

2. Serum collection

The blood containing the babesia microti standard insect strain taken out from the liquid nitrogen is balanced to room temperature, diluted by sterile 0.9% physiological saline according to the proportion of 1:1 and mixed evenly. And (3) sucking 200 mu l of diluted larva blood by using a 1ml sterile syringe, and injecting the larva blood into the abdominal cavity of a BALB/c mouse to recover the babesia microti in the mouse. And (3) observing pathogens of babesia microti and counting the pathogens by microscopic examination, picking eyeballs and collecting the whole blood into an anticoagulation tube before the blood disease of the babesia microti reaches a peak (hemolysis does not occur, and the infection rate of red blood cells is about 40%), and collecting the anticoagulation whole blood stained with the insects.

Simultaneously collecting anticoagulated whole blood of normal BALB/c mice, mixing the anticoagulated whole blood with the collected infected anticoagulated whole blood in a ratio of 1:1 to ensure that the density of the blood of the insects reaches 20%, diluting the blood with normal saline in equal ratio, and then inoculating 200 mu l to 10 abdominal cavities of the normal BALB/c mice respectively. Mice were scored as 0day before inoculation (0days post-infection, 0dpi), i.e., normal mice, the next day as 1 day after infection (1dpi), and observations were started. Blood was collected from the tip of the tail each day from 1dpi to 30dpi, and from each tail vein blood collection time point mentioned hereinafter, thin blood sheets were prepared, the pathogen morphology of babesia microti was observed and the infection of erythrocytes was recorded. About 200. mu.l of whole blood was collected in tail veins one week before inoculation of mice (normal mouse whole blood) and 3 days, 7 days, 14 days, 21 days, 30 days, 60 days, 120 days, 150 days, 270 days after inoculation, respectively, and heparin sodium at a concentration of 1g/L was mixed with blood 1: 10 mixing and anticoagulating. The collected tail vein whole blood is centrifuged at room temperature at 3500rpm for 10min, and serum is collected and stored at-20 ℃ for later use.

Example 2 immunoblot hybridization and analytical identification of Babesia microti crude protein

The Babesia strain is Babesia microti

PRA-99T, provided by the institute of Experimental zoology, academy of Chinese medical sciences. The babesia microti is inoculated to a BALB/c mouse, whole blood is obtained through anticoagulation, red blood cells are separated, precipitate is collected after cracking and centrifugation, and the babesia microti body is determined through microscopic examination. Through an immunoomics technology, as shown in figure 1, the crude protein of babesia microti is respectively subjected to immunoblotting hybridization with mouse serum of 7dpi and 30dpi, and through mass spectrometry, 87 babesia microti proteins are identified, wherein the 87 babesia microti proteins comprise 8 proteins identified by the mouse serum of 7 dpi.

Example 3 acquisition of antigen immunoreaction spectra of Babesia microti

1. Experimental materials and methods

1.1 vectors and strains

Escherichia coli (e.coli) DH5 α was purchased from kyoto biochemical technologies, ltd. The linearized vector pEU-E01-His-TEV-MCS-N2 (restriction endonuclease cleavage site Xho I, BamH I) and Babesia microti genomic DNA were provided by the present experimental storage.

1.2 laboratory instruments and reagents

(1) The main apparatus is as follows:

a PCR instrument; constant temperature metal bath; a vertical laminar flow clean bench; a full-temperature shaking incubator; an electric heating constant temperature incubator; performing bench high-speed freezing centrifugation; a small centrifuge; NanoDrop2000 (ultramicro uv/vis spectrophotometer); a high-pressure steam sterilization pot; a decoloring shaking table; a constant-temperature blending device; 85-2 type constant temperature magnetic stirrer; a nucleic acid electrophoresis apparatus; protein electrophoresis system: BIO-RAD; gel imager: BIO-RAD; biochip spotting instrument: personal Arrayer 16; a biochip scanner.

(2) Principal reagents and materials

And (3) PCR product purification: AxyPrep DNA clean-up Kit (Axygen, USA); and (3) cutting and purifying PCR products: AxyPrep DNA gel recovery kit (Axygen, usa); In-Fusion cloning ligation: in-fusion (TM) Advantage PCR Cloning Kit (Clontech, USA); and E, culturing escherichia coli: liquid and solid LB culture medium (self-prepared); ampicillin powder (stock 50mg/ml, Sigma, USA); colony PCR: 2 × Taq PCR MasterMix (Beijing Tiangen Biotech); plasmid extraction: AxyPrep plasmid DNA minikits (Axygen, usa); DNase/RNase-Free Water (Beijing Sorboard technologies, Inc.); wheat germ cell-free protein expression: the Wheat Germ WEPRO7240H Expression Kit (CellFree Sciences, USA); SDS-PAGE electrophoresis: SDS-PAGE gel kit (BIO-RAD, USA); protein pre-staining Marker (Thermo Scientific, Litao wang); 5 × protein loading buffer (Biyuntian); SDS-PAGE running buffer (Biyun day); r250 coomassie brilliant blue dye liquor (self-prepared); decolorizing solution (prepared by a conventional method); western blot: nitrocellulose membrane (NC membrane, PALL, usa); membrane transferring liquid (Biyuntian); BSA (Sigma, usa); Penta-His Antibody (BSA free, Qiagen, Germany); HRP Goatanti-mouse IgG (Sigma, USA); 30% hydrogen peroxide (shanghai Lingfeng Chemicals, ltd); DAB (Sigma, usa); protein chip: protein chip spotting and loading liquid (Beijing Boao Crystal dictionary, Bio Inc.); materials such as a crystal core surrounding hybridization wet box (Beijing Boao Crystal classic biology, Ltd.); a chip microarray optical-grade epoxy substrate (Beijing bo ao crystal dictionary, Bio/Co., Ltd.); BSA (Sigma, usa); alexa Fluor 546goat anti-mouse IgM (μ -chain) (Invitrogen, USA); alexa Fluor 546goat anti-human IgG (H + L) (Invitrogen, USA).

1.3 Experimental methods

1.3.1 design of primers

The babesia microti immune-related protein was analyzed by SMART, and the 87 proteins identified in example 2 were divided into 128 gene fragments (2D 1-128) based on the ORF contained in the sequence. Seamless cloning was performed by In-Fusion cloning, primer design as shown In FIG. 2, pEU-F added to the 5' end of the gene specific forward primer: 5'-GGGCGGATATCTCGAG-3' sequence, pEU-R added to the 5 ' end of the gene specific reverse primer: 5'-GCGGTACCCGGGATCC-3' sequence. Primers specific for babesia microti gene were designed using software primerpremier 5.0. The information of the target gene and the primer sequence of the n-Fusion clone are shown in Table 1

1128 gene segments (2D 1-128) in table, gene information and primer sequences

1.3.2 amplification of sequences of interest

The reaction system and reaction conditions for the PCR amplification are shown in table 2 and table 3:

TABLE 2 reaction System for PCR amplification of target fragments

TABLE 3 reaction conditions for PCR amplification of fragments of interest

Note: indicates that the annealing temperature can be optimized according to different primer TM values of different gene segments

The PCR products obtained above were subjected to 1% agarose gel electrophoresis for fragment size identification, and the products with the correct target band were sent to Huada gene for sequencing. If the hybrid band is contained, the sequence is sequenced after tapping purification.

The 128 babesia microti genes amplified by PCR finally successfully obtain the DNA products of 113 target genes, wherein the DNA products comprise 9 gene fragments identified by 7dpi mouse serum, the amplification rate is 88.3 percent, and the electrophoresis result is shown in figure 3.

1.3.3In-Fusion cloning

Constructing recombinant plasmids and screening positive clones, comprising the following steps: (1) adding 5 mul of PCR product into 2 mul of cloning Enhancer solution, mixing uniformly, putting into a PCR instrument, reacting for 15min at 37 ℃, and reacting for 15min at 80 ℃; (2) the ligation reaction system of In-Fusion clone is shown In Table 4, and the ligation reaction system is put into a PCR instrument for ligation reaction after being uniformly mixed, and the ligation reaction is carried out for 15min under the condition of 50 ℃; (3) placing the ligation products on ice, transferring all the products into a competent cell DH5 alpha by a heat shock transformation method, and culturing for 16-18 h on an ampicillin-resistant LB solid culture medium at 37 ℃; (4) 4 single colonies were scattered and picked on each plate, and dissolved in 10. mu.l of non-resistant LB liquid medium, 2. mu.l of the bacterial liquid was taken as a template for colony PCR, and the reaction system and reaction conditions of colony PCR are shown in tables 5 and 6; (5) identifying the PCR product by using 1% agarose gel electrophoresis, transferring bacterial liquid corresponding to a colony with a correct band size (more than two correct clones are selected as much as possible for each gene) into 3ml of ampicillin-resistant LB culture medium, and performing shake culture at 37 ℃ for 16-18 h; (6) absorbing 1ml of culture solution of each colony, and sending the culture solution to Huahua Dagen for sequencing; collecting the residual bacterial liquid 500 μ l, adding 500 μ l 50% glycerol, and preserving at-80 deg.C; (7) and inoculating the recombinant plasmid with correct sequencing through the preserved strain, performing amplification culture and extracting the plasmid. As the next wheat germ cell-free protein expression system requires that the concentration of the recombinant plasmid needs to reach more than 150 ng/mul, each recombinant plasmid is cultured by two parallel bacterial liquids in the research. The plasmid extraction process is detailed in an Axygen plasmid DNA miniprep kit operating manual. (8) The concentration of the extracted plasmid was determined using a NanoDrop2000, recorded and stored at-80 ℃ until use.

TABLE 4 In-fusion cloning ligation reaction System

TABLE 5 colony PCR reaction System

TABLE 6 reaction conditions for PCR amplification of colonies

Through In-Fusion cloning technology, 109 recombinant plasmids with correct sequences are finally and successfully constructed, 9 gene fragments (the cloning rate is 100%) identified by 7dpi mouse serum are included, and the total cloning rate reaches 96.5%. The colony PCR identification of the partially recombinant plasmid is shown in FIG. 4.

1.3.4 cell-free expression

Referring to the application instruction of the Wheat Germ WEPRO7240H Expression Kit, the successfully constructed target gene is subjected to in vitro transcription and translation Expression, and the operation steps are as follows: (1) performing in vitro transcription, wherein the system of transcription reaction is shown in Table 7, uniformly mixing, and performing reaction in a PCR instrument under the condition of transcription at 37 ℃ for 6 h; (2) preparing a translation expression system, wherein the system is shown in a table 8; (3) adding 206 mu l of 1 XSUB-Amix XSGC into a 96-well plate, slowly adding the prepared translation reaction system to the bottom of the plate hole, keeping the layering state, placing in a constant-temperature metal bath, and reacting for 20h at 15 ℃; (4) the translation products are mixed evenly and transferred to a PCR tube to be stored at-80 ℃ for standby.

TABLE 7 in vitro transcription reaction System

TABLE 8 in vitro translation reaction System

By in vitro transcription and translation using a wheat germ cell-free protein expression system, 87 proteins were successfully expressed from a total of 111 recombinant plasmids (including two sequences synthesized from the entire gene), including 7 proteins expressed from gene fragments identified in 7dpi mouse serum.

1.3.5 Western blot analysis

For the cell-free expression condition of the target gene, the experiment adopts a protein immunoblotting (Western-blot) technology for analysis, and the operation is as follows: (1) preparation of SDS-PAGE gels: the SDS-PAGE gel used in the experiment is prepared by using a polyacrylamide gel premixing reagent produced by BIO-RAD company to prepare 12% of separation gel and 5% of concentrated gel, and the gel is stored at 4 ℃ for later use after being solidified; (2) sample preparation: adding 3 mul of 5 Xprotein buffer sample loading liquid into 12 mul of cell-free expressed protein product, fully mixing, and boiling in a boiling water bath for 5-10 min; (3) protein electrophoresis: respectively taking 10 mul of prepared sample and protein Marker, carrying out 80V electrophoresis for 30min, and carrying out 120V electrophoresis for 60 min; (4) the operation of film transfer is as follows: 1) placing the NC membrane which is properly cut into methanol for activating for 30-60 s, and then transferring the NC membrane and the filter paper into a membrane transferring solution for soaking; 2) the gel, NC membrane, and filter paper were discharged as shown in fig. 5 (black side is the negative electrode, white side is the positive electrode),clamping and fixedly connecting the gel membrane into an electrophoresis tank, adding the membrane transfer liquid fully, then switching on electrophoresis, and carrying out membrane transfer for 90min at 220mA under the ice bath condition; 3) and (3) sealing: taking out the NC membrane, putting the NC membrane into an incubation box, rinsing the NC membrane by clear water, and blocking the NC membrane overnight at 4 ℃ by using 3% BSA (PBS) for dilution; 4) washing: pouring off the blocking solution, and rinsing the NC membrane with PBST for 3 times, 5min each time; 5) primary antibody incubation: diluting the Penta-His antibody with PBS according to the proportion of 1: 2000, and placing the diluted antibody on a shaking table to incubate for 1h at room temperature; 6) washing: pouring off the primary anti-incubation liquid, and rinsing the NC membrane with PBST for 3 times, 5min each time; 7) and (3) secondary antibody incubation: diluting a goat anti-mouse antibody marked by HRP (horse radish peroxidase) with PBS (phosphate buffer solution) according to the proportion of 1: 4000, and placing the diluted goat anti-mouse antibody on a shaking table for incubation for 1h at room temperature; 8) washing: pouring out the secondary antibody incubation solution, and rinsing the NC membrane with PBST for 3 times, 5min each time; 9) color development: according to the proportion of DAB, PBS and 30% H₂O₂Preparing a developing solution according to the ratio of 2mg to 3ml to 0.9 mul, soaking the incubated NC membrane in the developing solution (taking care to avoid light during developing), immediately rinsing to terminate the reaction when a target strip appears, and sucking the NC membrane by using filter paper; 10) and recording by photographing, and wrapping the displayed NC membrane by using filter paper for storage at 4 ℃.

The results of immunoblot analysis of the proteins are shown in FIG. 6, where several proteins expressed by this system migrated upon electrophoresis. The results of high throughput cloning and expression of babesia microti gene fragments are summarized in table 9:

TABLE 9 high throughput cloning and expression of Babesia muricatum gene fragments

1.3.6 preparation of protein chips and immunohybridization to obtain an immunoreaction profile

In the experiment, an optical-grade epoxy substrate is used as a carrier, wheat germ cell-free expression protein (2D 3-2D 128) obtained in the experiment and Bm7 (an amino acid sequence is shown as SEQ ID NO. 9) screened from a cDNA library in the laboratory before are subjected to spotting of a protein sample by using a chip spotting instrument. The experiment uses BmSA1 as a positive protein control, a plasmid-free wheat germ cell-free translation system and PBST as a negative control group, and the specific operation of chip preparation is as follows: (1) sample preparation: egg taking deviceUniformly mixing the white chip sample application sample solution and the expression protein solution by 6 mul respectively, sucking 10 mul, adding into a 384-pore plate, centrifuging and placing on ice for later use; (2) preparing an instrument: scrubbing the ultrasonic pool and the working table of the sample applicator, and adding ddH in the ultrasonic pool₂O to the scale mark; ddH for humidifier₂O will be added to the scale line; pouring the waste liquid in the waste liquid bottle, and filling the ddH in the cleaning bottle₂O; starting an instrument and corresponding operating software to complete pattern matching and self-checking; (3) fence pasting: 2 multiplied by 6 hole (9cm multiplied by 9cm) fences are selected for the experiment, a fence pasting and pressing tool is used for pasting the fences on an epoxy substrate, the epoxy substrate is placed in a sample application area of a chip sample instrument, and sample application is prepared; (4) setting parameters: after the initial position of the 384-hole plate is calibrated, parameters such as a needle frame, a slide, a pre-spotting slide, a dot matrix, a sample, cleaning and the like are sequentially set; in the experiment, 10 x 10 protein matrixes are set, each protein sample is spotted repeatedly for 2 times, and each protein sample is spotted with 10 matrixes; (5) spotting and storing: confirming whether each work is ready, clicking 'start' to sample; the spotted slide can be used for next hybridization experiment or sealed in a wet box for storage at 4 ℃ for later use; note that: the spotted slide should be kept still for more than half an hour to ensure that the protein is fully combined with the slide.

The immune hybridization of the protein chip firstly needs to optimize the dilution of serum and fluorescent secondary antibody and determine the optimal reaction system, and the specific process of the hybridization experiment is as follows (1) closing: add 50. mu.l of 3% BSA (PBS) diluted in the pens of each matrix of the protein chip prepared above, place in the hybridization wet box, block overnight at 4 ℃; before cleaning, putting the wet hybridization box into a 37 ℃ constant temperature box and sealing for 1 h; (2) cleaning: spin-drying the sealing liquid on the chip, placing the sealing liquid in deionized water for washing once, then using a PBST shaking table to shake and wash for 3 times, 5min for each time, and centrifuging for 3min at 1000g for spin-drying; (3) incubating the primary antibody: diluting 10 mixed mouse sera (0, 3,7,14,21,30,60,120,150 and 270dpi) infected with babesia microti at different periods with PBST according to the proportion of 1: 100, mixing uniformly, respectively taking 50 mul, sequentially adding into each protein matrix, placing in a hybridization wet box, and reacting for 1h at 37 ℃; (4) cleaning: the cleaning operation is the same as the step (2); (5) incubation of secondary antibody: diluting and uniformly mixing Alexa Fluor 546goat anti-mouse IgG or Alexa Fluor 546goat anti-mouse IgM antibody with PBST according to the ratio of 1: 200, adding 50 ul of the mixture into each protein matrix, placing the protein matrix into a hybridization wet box, and reacting for 1h at 37 ℃ (after adding a secondary antibody, operating and keeping out of light); (6) cleaning: the cleaning operation is the same as the step (2); (7) scanning: the hybridized protein chip is placed in a chip scanner, appropriate parameters of ScanArray Express software version4.0(PerkinElmer) are set, and the chip scanning is carried out by selecting the wavelength of which lambda is 532 nm. Saving the picture and reading data; (8) and (3) data analysis: and taking the ratio of the fluorescence intensity mean value of the sample to be detected after the background is subtracted to the fluorescence intensity mean value of the negative sample after the background is subtracted to be more than or equal to 2 as the reference of the positive sample. Thermographic mapping and analysis of the protein chips using Multi-array experimental viewer (MeV) software; the immunoreaction spectrum of each protein obtained by protein chip hybridization is subjected to hierarchical clustering by an R language (www.r-project. org /), and the distance algorithm and the clustering method respectively adopt an Euclidean distance method and a ward.D2 method.

In the specific IgG antibody reaction, most of the babesia microti proteins showed very weak immune reaction with 0,3 and 7dpi mouse serum (FIG. 7: a-c), but showed very high immune reaction with 14-150 dpi mouse serum (FIG. 7: d-i), and the degree of immune reaction was reduced at 270dpi (FIG. 7: j). The specific IgG antibody immunoreactivity profile for each protein is shown in fig. 8, and 88 proteins were grouped into 4 groups, Group1(11 proteins), Group2(47 proteins), Group3(29 proteins) and Group4 (only 1 purified prokaryotically expressed recombinant protein), representing high, medium, low and very high immunoreactivity groups, respectively, by R language analysis and hclust functional clustering. The immune response spectrum and grouping condition of the binding proteins, 4 proteins 2D5, 2D29, 2D41 (amino acid sequence is shown as SEQ ID NO.8) and Bm7 which have sustained high immune response with mouse serum of 14 dpi-270 dpi are respectively screened from four groups.

In the specific IgM antibody reaction, most of Babesia microti proteins showed strong immunoreaction with mouse serum of 14,21 and 30dpi (FIG. 9: d-f), but showed very low immunoreaction with mouse serum of other stages. The specific IgM immunoreactivity profile for each protein is shown in FIG. 10, and 88 proteins were grouped into 4 groups, Group1(19 proteins), Group2(50 proteins), Group3(18 proteins) and Group4(1 recombinant protein), respectively, by R language analysis and hclust functional clustering. According to the immune response spectrum, the first three groups do not show difference, only one protein 2D97 (amino acid sequence is shown as SEQ ID NO.2) selected from Group3 shows higher immune response with early mouse serum of 3dpi and 7dpi, and the protein can be used as a candidate for early diagnosis antigen.

Example 4 prokaryotic expression, purification and evaluation of Babesia microti candidate biomarkers 2D33, 2D36, 2D41, 2D97

1 Experimental materials and methods

1.1 plasmids, strains

Escherichia coli (e.coli) DH5 α, BL21(ED3) were purchased from beijing tiangen biochemical technologies, inc. Plasmids pET21a, pET28a, pET42a were all provided for storage in the laboratory, and pSmart1 was purchased from Changzhou Tiandi and BioLimited.

1.2 sample Collection and ethical statement

8 serum samples of Babesia microti patients were provided by the task team of the trained teacher of the basic medical college of the university of Compound Dan. 10 portions of each of the human serum samples of vivax and falciparum malaria were provided by the aged tiger teacher, who was the central parasite disease prevention and control institute of Chinese disease prevention and control. 200 samples of fever patients are collected from the soaring site of a malaria epidemic area in the experiment, and pathogens of the samples are negative through giemsa staining microscopy; through nucleic acid detection, 14 positive vivax malaria samples and 4 positive falciparum malaria samples are found, wherein one case of coinfection is included. A normal human serum sample from suzhou medical school served as a negative control. The collection of blood samples was approved by the scientific ethics committee of the national institute for the prevention and control of parasitic diseases, central for the prevention and control of diseases in china. All individuals participating in providing the sample signed informed consent and provided instructions on the use, potential risks, and benefits of collecting the sample.

1.3 Experimental instruments and reagents

(1) Main instrument

A PCR instrument; a vertical laminar flow clean bench; a full-temperature shaking incubator; an electric heating constant temperature incubator; performing bench high-speed freezing centrifugation; a small centrifuge; NanoDrop2000 (ultramicro uv/vis spectrophotometer); a high-pressure steam sterilization pot; a nucleic acid electrophoresis apparatus; a protein electrophoresis system; a gel imager; an ultrasonic crusher; a protein chromatography purification system; a microplate reader.

(2) Principal reagents and materials

And (3) PCR product purification: AxyPrep DNA clean-up Kit (Axygen, USA); BamH1, Xho1 restriction enzyme and Buffer3, NEB, usa; seamless cloning: In-Fusion HD Cloning Kits (Clontech, USA); and E, culturing escherichia coli: 10g of liquid and solid LB culture medium Nacl, 10g of Tryptone, 5g of Yeast Extract (Agar 15g) and deionized water are added to make the volume of the solution constant to 1L. (ii) a Ampicillin powder (stock 50mg/ml, Sigma, USA); kanamycin antibiotic powder (stock 10mg/ml, Sigma, usa); colony PCR: 2 × Taq PCR MasterMix (Beijing Tiangen Biotech); plasmid extraction: AxyPrep plasmid DNA minikits (Axygen, usa); IPTG powder (stock 1mol/L, Sigma, USA); PMSF powder (stock solution 1mol/L, Sigma, USA); purifying the inclusion body protein: a PAGE collagen micro-recovery kit (Biotechnology engineering (Shanghai) Co., Ltd.); GST purification system: 1ml GSTrap HP purification column, GE, Sweden; buffer A is PBS pH7.4; buffer B (eluent) 1mM DTT,10mM Glutathionone pH 7.4; ni column purification system: 5ml HisTrap FF purification column, GE, Sweden; buffer A20 mM 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 solibao biotechnology limited); his protein purification media packing, GE company, sweden; protein concentration determination: branford kit (beijing tiangen bio ltd); ELISA experiment: HRP coat anti-mouse IgG (Sigma, USA); HRP coat anti-mouse IgM (Sigma, USA); HRP coat anti-human IgG (Sigma, USA); HRP coat anti-human IgM (Sigma, USA); immunoprotection assay: freund's complete adjivant (Sigma Aldrich, USA); freund's incomplete adjust adapt (Sigma Aldrich, USA).

1.4 Experimental methods

1.4.1 primer design and Synthesis

Specific primers are designed for prokaryotic expression of the protein by using PREMIER5.0 software according to the nucleic acid sequences (the nucleotide sequences are respectively SEQ ID NO.1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7) of the screened biomarker proteins 2D97, 2D33, 2D36 and 2D 41. The vectors used were respectively pET42a, pET21a, pSmart1 and pET28a, and the restriction sites were BamH1 and Xho 1. The seamless cloning technology is utilized in the experiment, vector sequences are respectively introduced into the 5' ends of the specific primers: f: 5' -GGGATATCGGGGATCC and R: 5' -GGTGGTGGTGCTCGAG, the primer sequences are shown in Table 10. The primers were synthesized by Huada Gene Co.

TABLE 10 primer sequences for Babesia microti recombinant cloning

Note: underlined italics as sequence of restriction sites

1.4.2 construction of prokaryotic expression vectors

The target gene was amplified by PCR using 100-fold diluted pEU-E01 recombinant plasmid containing the target gene as a template, and the reaction system and reaction conditions are shown in tables 2 and 3. The vector plasmid was subjected to double digestion with restriction enzymes Xho1 and BamH1 under the conditions of 37 ℃ overnight as shown in Table 11. And carrying out electrophoretic identification on the PCR product and the enzyme digestion product by using 1% agarose gel. If the product has a single band, the PCR cleaning kit can be directly used for purifying the product; if the product contains a miscellaneous band, the target band needs to be subjected to gel cutting and purification.

TABLE 11 digestion reaction System for double digestion

The ligation reaction system is shown in Table 12. Mixing the reactants, reacting at 37 ℃ for 15min, and reacting at 50 ℃ for 15min to complete the connection of the vector and the target gene. Transferring all the connection products into competent cells E.coliDH5 alpha, and culturing for 16-18 h on an LB solid culture medium with corresponding resistance. Colony PCR identification used the universal primers T7promoter Primer: 5'-TAATACGACTCACTATAGGG-3' and T7 terminator Primer: 5'-GCTAGTTATTGCTCAGCGG-3'. After the screened positive clones are cultured by a liquid LB culture medium with corresponding resistance, plasmids are extracted and sent to Huada gene sequencing. The reaction system and reaction conditions of colony PCR are shown in tables 3-4 and 3-5:

TABLE 12 ligation reaction System

PCR reaction was carried out using pEU-E01 recombinant plasmid containing 2D97, 2D33, 2D36, 2D41 gene fragments as a template, and the results of electrophoresis showed that the size of the target fragment amplified was about 984, 432, 519 and 687bp, respectively, as shown in FIG. 11, where M represents a DNA marker, 1 represents 2D97, 2 represents 2D33, 3 represents 2D36, and 4 represents 2D 41. The successfully amplified target fragments are respectively connected to enzyme-digested vectors pET42a, pET21a, pSmart1 and pET28a after purification, and the recombinant plasmids are consistent with the target gene sequence through positive clone screening and sequencing identification, so that the recombinant plasmids are successfully constructed.

1.4.3 recombinant protein inducible expression and solubility assays

The successfully constructed recombinant plasmids Bm2D97, Bm2D33, Bm2D36 and Bm2D41 are respectively transformed into E.coliBL21(DE3) competent cells, and monoclonal colonies are picked up and cultured in a corresponding resistant LB culture medium and are subjected to shake culture at 220rpm at 37 ℃ overnight. Adding 500 μ l of the culture bacterial liquid into 500 μ l of 50% sterilized glycerol, mixing, and storing at-80 deg.C. And then inoculating the overnight-cultured bacterial liquid into 2 tubes of correspondingly resistant culture media according to the inoculum size of 5 percent for shake culture for 3-4 h (the absorbance A600 value reaches 0.6-0.8), respectively taking one tube for induction by IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mmol/L (Bm2D33) and 1mmol/L (Bm2D36, Bm2D41 and Bm2D97), taking the other tube as a control group for non-induction, and culturing for 4h at 37 ℃. After induction expression, 1ml of each of the bacterial solutions was centrifuged at 12000rpm for 1min, and the supernatant was discarded to collect the cells. The cells were resuspended in PBS and sonicated 20 times in a sonicator at 200w, 3s on, 5s off. 40. mu.l of the whole solution after sonication was left, the remaining whole solution was centrifuged at 12000rpm for 1min, the supernatant and the pellet were separated, and the pellet was resuspended in 960. mu.l of PBS. The whole solution, the supernatant and the precipitate were taken and 40. mu.l each was added to 10. mu.l of 5 Xprotein loading buffer, and the sample was boiled in a boiling water bath for 10min to prepare a protein loading solution. Protein expression and its solubility were analyzed by 12% SDS-PAGE. And (3) after the electrophoresis is finished, dyeing for 1h by using a Coomassie brilliant blue solution, decoloring to obtain a visible and clear protein band by using a decoloring solution, and photographing and storing an electrophoretogram.

After 4 recombinant plasmids are respectively transformed into E.coli BL21(DE3) competent cells for expression, the expression is induced by IPTG with the final concentration of 1mmol/L, the 2D97 and 2D36 genes obtain inclusion body expression, the 2D41 gene obtains soluble expression, the protein molecular weights are respectively 38KDa (apparent molecular weight is 55KDa), 19KDa and 39KDa (containing SUMO-tag 13KDa and the apparent molecular weight is 20 KDa); inducing by IPTG with final concentration of 0.1mmol/L, 2D33(GST-tag 26kDa) gene obtains soluble expression, and the molecular weight of protein is 42 KDa; the 4 recombinant proteins were named Bm2D97, Bm2D36, Bm2D41 and Bm2D33, respectively, and their amino acid sequences were identical to 2D97, 2D36, 2D41 and 2D33, respectively. The results of SDS-PAGE electrophoretic analysis are shown in FIG. 12. In FIG. 12, M represents a protein marker, 1 represents a recombinant clone-non-induced whole bacterium, 2 represents a recombinant clone-non-induced supernatant, 3 represents a recombinant clone-non-induced precipitate, 4 represents a recombinant clone-induced whole bacterium, 5 represents a recombinant clone-induced supernatant, and 6 represents a recombinant clone-induced precipitate.

1.4.4 purification of recombinant proteins

(1) Large scale expression of recombinant proteins: 1) recovering the strain: inoculating the preserved strain with the determined inducible expression into 3ml LB culture medium with corresponding resistance, and performing shake culture at 220rpm and 37 ℃ overnight; 2) and (3) amplification culture: transferring the overnight-cultured bacterial liquid into 50ml of a corresponding resistant culture medium according to the inoculation amount of 5%, carrying out shake culture at 220rpm and 37 ℃ for 3h (the absorbance A600 value reaches 0.6-0.8), carrying out amplification culture on the bacterial liquid into 1L of LB culture medium according to the inoculation amount of 5%, and carrying out induction expression according to the condition of small-amount expression; 3) collecting bacteria: centrifuging at 4000rpm for 15min at 4 deg.C, collecting thallus, and repeating the steps until thallus of 1L of bacterial liquid is completely collected; 4) and (3) thallus treatment: and (3) after the collected thalli are re-suspended by 40-45 ml of bufferA, centrifuging at 4000rpm for 15min at 4 ℃, washing the thalli, discarding supernatant, re-suspending the thalli by the bufferA, adding a protease inhibitor PMSF with the final concentration of 1mM, and storing at-80 ℃ for later use.

(2) And (3) insoluble protein tapping and purifying: the proteins Bm2D36 and Bm2D97 expressed by the inclusion body are purified and recovered by adopting a PAGE collagen trace recovery kit of biological engineering Co., Ltd, and the purification steps are as follows: 1) dissolving the thalli of the inclusion body protein expressed in large quantity at room temperature, performing ultrasonic treatment on the thalli for 99 times by using an ultrasonic instrument under the conditions of 400w, 5s on and 10s off, centrifuging the thalli at 4 ℃ and 15000g for 30min (the speed is increased by 6 and the speed is decreased by 2), discarding supernatant, re-suspending the precipitate by using a proper amount of buffer A, and subpackaging every 800 mu l of the suspension into a centrifugal tube of 1.5ml and preserving the suspension at-80 ℃ for later use; 2) adding 200 μ l of 5 × protein loading buffer solution into the precipitate suspended matter subpackaged in 1), mixing, and boiling in boiling water bath for 10min to obtain protein loading solution; 3) preparing 12% SDS-PAGE separation gel and 5% concentrated gel, spreading the prepared protein sample solution on the concentrated gel for electrophoresis without adding a comb when preparing the concentrated gel; 4) after staining and decolorizing the protein gel, the target band was completely cut from the whole gel block, placed in a 50ml centrifuge tube, and then treated with ddH₂Cleaning for three times; 5) cutting the adhesive tape into small sections, placing the small sections in a centrifuge tube, grinding the colloid into fine fragments by using a tissue grinder, adding 4ml of solution A, and then shaking on a decoloring shaking table at room temperature for 16-18 h; 6) centrifuging at 12000rpm for 15min at room temperature, collecting supernatant, adding 20ml of pre-cooled B solution, mixing, and standing at 4 deg.C for 30 min; 7) centrifuging at 12000rpm for 15min at room temperature, discarding the supernatant, retaining the white precipitate at the bottom of the tube, and placing the tube in a fume hood to completely volatilize the residual liquid; 8) after dissolving the precipitate with an appropriate amount of PBS solution, 20. mu.l of the protein loading solution was prepared and the protein samples were identified by SDS-PAGE electrophoresis. The protein solution was stored at-80 ℃ for future use.

(3) Affinity purification of soluble protein: soluble protein Bm2D33 adopts a GST affinity purification system, Bm2D41 adopts a Ni column purification system, and the specific operation is as follows: 1) dissolving thallus for expressing soluble protein in large amount at room temperature, ultrasonic treating at 400w for 5s and 10s for 99 times, centrifuging at 4 deg.C and 15000g for 30min (increasing speed 6 and decreasing speed 2), transferring the supernatant to a new 50ml centrifuge tube, centrifuging again, and collecting the supernatant; 2) washing the purification column with water, after the UV value is stable (about 5 column volumes), balancing the purification column with about 5 column volumes buffer A, and clearing the UV value; 3) loading the protein supernatant onto a purification column after equilibrium through a pressure pump, washing with buffer A to remove non-specific binding protein, respectively washing with 10%, 30%, 50%, 80% and 100% gradient buffer B (directly washing with 100% buffer B during GST purification) when the UV value is reduced to be below a baseline, and collecting target protein according to the peak condition; 4) carrying out SDS-PAGE electrophoresis on the collected proteins with different components to identify the target protein and the purity thereof; 5) after protein collection, the column was washed with 100% buffer B for 5 column volumes to remove the bound protein from the affinity medium, and then washed with water and 20% ethanol, respectively, and stored.

(4) Cleavage of the protein fusion tag: the Bm2D41 protein contains a SUMO fusion tag and requires excision for the next functional validation experiment. The enzyme digestion experiment adopts an on-column enzyme digestion mode, an enzyme digestion system is configured as shown in table 13, the enzyme digestion system is fully mixed with 1ml of His protein purification medium filler, and the mixture is subjected to shaking and enzyme digestion for 16 hours at a temperature of 4 ℃ in a shaking table. Centrifuging at 4 deg.C for 5min at 500g, collecting supernatant as enzyme-digested target protein solution, identifying by SDS-PAGE electrophoresis, packaging, and storing at-80 deg.C. The treatment and preservation of the packing are the same as the treatment of the purification column above.

TABLE 13 SUMO protease cleavage System

Measurement of protein concentration the Branford method was sampled, and the specific procedures were performed with reference to the kit instructions.

The inclusion body proteins Bm2D97 and Bm2D36 are purified by a PAGE collagen trace recovery kit, and are analyzed by SDS-PAGE electrophoresis, so that the recombinant protein with a single band is successfully obtained (shown in figure 13). Soluble proteins Bm2D41 and Bm2D33 were purified using affinity media and analyzed by SDS-PAGE electrophoresis to obtain fusion proteins with relatively single bands. The purified Bm2D41 protein is subjected to protein fusion tag excision by a SUMO protease enzyme digestion system, and a recombinant protein with a relatively single target band is obtained (shown in FIG. 13). In FIG. 13, M represents a protein marker, 1 represents a recombinant clone-uninduced whole bacterium, 2 represents a recombinant clone-induced whole bacterium, 3 represents a purified recombinant protein, and 4 represents a recombinant protein from which a fusion tag has been cleaved.

1.4.5 evaluation of recombinant proteins

Recombinant proteins were evaluated by ELISA assay techniques, except Bm2D97 was detected using IgM antibodies (including HRP coat anti-mouse IgM and HRP coat anti-human IgM), and the remaining recombinant antigens were detected using IgG antibodies (including HRP coat anti-mouse IgG and HRP coat anti-human IgG).

Protein coating concentration, serum and secondary antibody dilution were optimized by experiment. The optimized optimal coating concentration of the recombinant proteins Bm2D97, Bm2D33, Bm2D36, Bm2D41 and Bm7 are respectively 2 mu g/ml, 1 mu g/ml and 5 mu g/ml, the crude protein of the Babesia microti and the reported BmSA1 are taken as references, and the optimal coating concentration is respectively 1 mu g/ml and 5 mu g/ml. The ELISA assay was performed as in 2.2.3.6.

(1) Evaluation of recombinant antigens by mouse serum infected with Babesia microti

The recombinant proteins Bm2D97, Bm2D33, Bm2D36, Bm2D41 and Bm7 are coated according to the coating concentration, and are respectively incubated with mouse serum which is infected with Babesia microti for 3 days, 7 days, 14 days, 21 days, 30 days, 60 days, 120 days, 150 days and 270 days and normal mouse serum which are respectively 10 parts (diluted by 1: 100), and the secondary antibody HRP coat anti-mouse IgM and the HRP coat anti-mouse IgG are respectively coated according to the ratio of 1: after 5000 dilution, incubation was performed to evaluate the immunoreaction tendency of the recombinant antigen.

From the protein chip analysis result of the specific IgG antibody, the screened cell-free expression proteins 2D41 and Bm7 are subjected to prokaryotic expression and purification, and then react with mouse serum at different infection stages, and the recombinant antigens Bm2D41 and Bm7 both show sustained high immune response, and have the best effect. The immunoreaction trends of the recombinant antigens are shown in FIG. 14.

(2) Evaluation of recombinant antigen by using babesia microti patient serum

The recombinant proteins Bm2D97, Bm2D33, Bm2D41, Bm7 and BmSA1 were incubated with 8 parts of babesia patient serum and 10 parts of normal human serum (1: 100 dilution), respectively, at the above coating concentrations, and the secondary antibodies HRP coat anti-human IgM and HRP coat anti-human IgG were each expressed as 1: 10000 dilution and incubation, thereby evaluating the sensitivity of the recombinant protein as a candidate diagnostic antigen.

ELISA analysis of the recombinant antigens Bm2D97, Bm2D33, Bm2D41, Bm7 and BmSA1 using normal human and babesiasis human serum showed higher sensitivity, 62.5% and 50%, respectively, as shown in FIG. 15, in which Bm2D33 and Bm2D41 compared to the reported BmSA1 (sensitivity of 0%). In the ELISA assay for the combined antigens, the multiple antigen combination Bm2D33+ Bm2D41+ Bm7 showed higher sensitivity compared to Bm2D33+ Bm2D41, with 62.5% and 25% sensitivity for the two combined antigens, respectively, and the results are shown in fig. 15.

(3) Cross-reactivity of recombinant antigens with malaria patient serum

The cross-reactivity of the recombinant antigen with human serum of malaria was evaluated by incubating with the recombinant antigen coating and secondary antibody of (2) and primary antibody incubated with 10 parts of vivax and falciparum serum and 10 parts of normal human serum (1: 100 dilution), respectively.

ELISA analysis of each recombinant antigen using sera from normal human and malaria patients gave the results shown in FIG. 16, with specificity of detection for P.vivax samples of Bm7, BmSA1, Bm2D33, Bm2D41 and Bm2D97 of 90%, 80%, 70%, 60% and 30%, respectively; the specificity of detection for the falciparum malaria sample was 100%, 80%, 100%, 80% and 60%, respectively. In cross-reaction with the sera of malaria patients, Bm7 and Bm2D33 showed very high specificity, especially for 100% of the sera of plasmodium falciparum patients.

(4) Assessment of in situ fever patient samples in malaria endemic areas

200 parts of fever patient blood sample, extracting DNA by using a DNA extraction kit, and detecting the specific segment of 18S rRNA of the coded Babesia microti by using a PCR technology. In the study of 200 febrile patient samples, 14 positive vivax malaria and 4 positive falciparum malaria samples were found, including 1 vivax and falciparum malaria nucleic acid co-infected sample. In the nucleic acid test of babesia microti on 200 samples, 10 positive samples were found, but no co-infection of babesia and plasmodium nucleic acid was found.

The recombinant proteins Bm2D97, Bm2D33, Bm2D41, Bm7, Babesia microti crude protein and BmSA1 were coated at the optimized concentrations above, and were incubated with 200 parts of febrile patients and 45 parts of normal human serum (1: 100 dilution), respectively, and the secondary antibody incubation was performed as in (2). And (3) analyzing the potential transmission risk of the babesiosis disease of the field mouse in the malaria epidemic area by combining the nucleic acid detection result.

In the study of 200 serum samples of fever patients in malaria epidemic areas, ELISA analysis is carried out by using crude Babesia microti protein, BmSA1, Bm7, Bm2D33, Bm2D41 and Bm2D97 recombinant antigens, and the results are shown in FIG. 17, and in 200 sera, 32 and 34 positive sera are detected to be positive by continuous high-reaction recombinant antigens Bm2D41 and Bm7, wherein the positive sera respectively comprise 1, 2 Babesia microti molecules positive and 7 plasmodium molecules positive; the recombinant protein BmSA1 and the babesia microti crude extract protein are used as controls, and 38 parts of positive serum and 58 parts of positive serum are detected respectively, wherein the positive serum respectively comprises 1 part of positive molecular of babesia microti, 3 parts of positive molecular of babesia microti and 14 parts of positive molecular of plasmodium. In addition, the study also found that, as shown in table 14: among the detected positive samples, the proportion of the babesia microti molecular negative and antibody positive samples is the highest, about 72-85%.

TABLE 14 analysis of PCR and ELISA Positive results for 200 samples of field-febrile patients in malaria endemic area

1.4.6 immunoprotective assay

Using 6-week-old BALB/c female mice as test subjects, 4 groups of 4 animals were set up, i.e., Bm2D 41-immunized group, Bm 7-immunized group, adjuvant group, and natural-immunized group. The protocol is as in table 15:

TABLE 15 immunoprotection test protocol

Immunization was performed by subcutaneous multiple injections at the back, 50. mu.g of protein per immunization. One week after the third immunization, all tail veins of 4 groups of mice were collected, and sera were collected to determine the antibody level of each group of mice by ELISA detection technique. When the antibody level of the protein immunization group is obviously higher than that of the adjuvant group, 4 groups of 16 mice are all injected with 1 × 10 in the abdominal cavity⁷Red blood cells infected with babesia. The day of infection was recorded as 0dpi, starting at 3dpi, and then every other day, the tip of the tail was sampled and smeared once, up to 29 dpi. During the period, blood is collected in tail veins of 7,14,21 and 29dpi respectively, serum is separated, and the change of antibody level in the dynamics process of the hemozoonosis is detected by an ELISA technology. For significance analysis in the study, one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests were performed by GraphPad Prism version 5.0 software.

The recombinant antigen with continuous high immune response has the potential of candidate vaccine, so the recombinant proteins Bm2D41 and Bm7 are adopted to immunize mice, and after babesia microti is inoculated, the immunoprotection effect of the recombinant proteins is evaluated by counting zoonosis and detecting the change of antibody level. The experimental results show that: after the third immunization, the serum specific IgG antibody level of the mice in Bm2D41 and Bm7 immunization groups is obviously higher than that of the adjuvant group (P)<0.001) as in FIG. 18, 4 groups of 16 mice were inoculated with 1X 10 mice in total⁷Red blood cells infected with babesia microti.

Counting the blood worms of each group, except 13dpi in the Bm2D41 immune group, the blood worms of the Bm2D41 immune group are significantly lower than those of the adjuvant group or the natural immune group (P <0.05) in the period of 7dpi to 21dpi, when the blood worms of the 7dpi reach the peak value, the mean values of the blood worms of the adjuvant group and the natural immune group are 25.4 percent and 23.0 percent, while the mean value of the Bm2D41 immune group is 15.9 percent, and the blood worms are reduced by about 35 percent (A in figure 19 represents the Bm2D41 immune group); the Bm7 immune group showed no significant change in vermiemia compared to the adjuvant group or the natural immune group (B of fig. 19 indicates Bm7 immune group). In addition, experiments show that the adjuvant has an influence on the vermiemia of the host to a certain extent, and the adjuvant group is slightly lower than the natural immunity group. By the end of the observation period of the experiment, the hemorrhizia of each group was already at a low level, but babesia microti was not completely cleared. The results of the specific antibody level measurements are shown in figure 19: the antibody level remained high after three days in the protein immunization group until the end of the experiment, and no significant changes occurred before and after inoculation of babesia muricata and during the dynamics of the zoonosis.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Chinese disease prevention and control center for prevention and control of parasitic diseases, university of Fudan

<120> Babesia microti 2D41 antigen protein and application thereof

<130> CPC-NP-18-101029

<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 (6)

1. An isolated gene having the sequence set forth in SEQ ID NO: 7 is shown in the specification; the separated gene is used for expressing the babesia microti 2D41 antigen protein.

2. The babesia microti 2D41 antigen protein expressed by the isolated gene of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO: shown in fig. 8.

3. The use of an isolated gene according to claim 1 for the preparation of a medicament for the prevention of babesiosis in a field mouse.

4. The use of the babesia microti 2D41 antigen protein of claim 2 in the preparation of a medicament for preventing babesia microti disease.

5. The use of claim 3 or 4, wherein the medicament is a vaccine.

6. A vaccine comprising a polypeptide as set forth in SEQ ID NO: 8, and 2D41 antigen protein of Babesia microti.

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