CN106978425A - The albumen of the sugared 5 phosphoric acid reduction isomerase genes of the deoxidation D of Babesia orientalis 1 wood paulownias and its coding - Google Patents

Abstract

The present invention discloses a Babesia orientalis 1-deoxy-D-xylose-5-phosphate reductoisomerase gene, which has a nucleotide sequence as shown in SEQ ID NO: 1. The present invention also discloses the gene The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The recombinant protein disclosed by the invention can effectively detect the presence of the babesiosis orientalis antigen in the blood of infected buffaloes, has good reactogenicity, and is helpful for controlling the prevalence and spread of the babesiasiasis in buffaloes in my country.

Description

Babesia orientalis 1-deoxy-D-xoutunose-5-phosphate reductoisomerase gene and its encoded protein

technical field

The invention belongs to the field of molecular biology and relates to a new gene and its recombinant protein, specifically referring to the recombination of the Babesia orientalis 1-deoxy-D-xylose-5-phosphate reductoisomerase (BoDXR) gene and its encoding Protein, the present invention also relates to the application of recombinant protein.

Background technique

Babesiosis, also known as pear body disease, tick fever or Texas fever, is caused by a variety of tick-borne, intraerythrocytic protozoa of the genus Babesia. Clinically, it mainly causes fever, hemoglobinuria, hemolytic anemia and death in various vertebrates. The pathogen belongs to Apicomplexa, Piroplasma, Babesco, Babesia of the genus Babesia, which was first discovered in 1888 by Victor Babes in bovine erythrocytes suffering from hemoglobinuria and red water fever symptoms .

Currently, about half of the world's cattle industry is threatened by Babesia, causing serious economic losses to the livestock industry in tropical and subtropical countries. According to current research reports, in Hubei, Fujian, Jiangxi, Anhui, Zhejiang, Hunan, Guangxi, Yunnan, Guizhou and other provinces in southern my country, the main cause of buffalo disease is Babesia orientalis. The parasite is strictly host specific, infecting only buffalo. Its vector (Rhizocephalus falciparum) is a three-host tick with one generation per year that transmits Babesia orientalis via eggs. Babesia orientalis mainly breaks out in summer and autumn, causing significant economic losses to animal husbandry.

Based on the above, it can be seen that once a certain area becomes an epidemic area, it is difficult to cure and eliminate Babesia orientalis disease. Therefore, to control the spread and prevalence of the disease, diagnosis and vaccine are particularly important. To date, there are no diagnostic kits for the detection of babesiosis orientalis. Therefore, screening and identification of Babesia orientalis antigens with good immunogenicity and reactogenicity is one of the solutions.

It is known that mammals use the mevalonate pathway to synthesize the isoprenoid compounds required for life, while Babesia orientalis uses the non-mevalonate pathway to synthesize isoprenoids, which is similar to the synthesis of mammals The pathways are different, so the 1-deoxy-D-xoutonose-5-phosphate reductoisomerase gene was cloned in Babesia orientalis, the prokaryotic expression vector was constructed, polyclonal antibodies were prepared, and the antibodies with good immunity were screened and identified. The antigenic and reactogenic Babesia orientalis antigens are of great significance for the clinical detection of Babesia orientalis infection.

Contents of the invention

The first object of the present invention is to obtain the 1-deoxy-D-liponate-5-phosphate reductoisomerase gene from Babesia orientalis.

The second object of the present invention is to efficiently express the Babesia orientalis 1-deoxy-D-woodoic acid-5-phosphate reductoisomerase gene in an Escherichia coli expression system to obtain a recombinant protein.

The third object of the present invention is to provide the use of the above-mentioned recombinant protein in the preparation of a detection kit for Babesia orientalis.

To achieve the above object, the present invention adopts the following technical solutions:

(1) Acquisition of Babesia orientalis 1-deoxy-D-woodoic acid-5 phosphate reductoisomerase gene: using the polymerase chain reaction (PCR) method, the whole genome and total RNA of Babesia orientalis The reverse-transcribed cDNA is used as a template, and the target fragment of 1-deoxy-D-woodoic acid-5-phosphate reductoisomerase is obtained by PCR, and the open reading frame (ORF) of the whole gene nucleotide sequence is as shown in the sequence table SEQ ID NO: 1 shown.

(2) A recombinant protein encoding the 1-deoxy-D-woodoic acid-5-phosphate reductoisomerase gene, the amino acid sequence of which is shown in SEQ ID NO: 2 in the sequence table.

(3) The preparation method of the recombinant protein is as follows: the open reading frame (ORF) of Babesia orientalis 1-deoxy-D-woodoic acid-5 phosphate reductoisomerase shown in the sequence table SEQ ID NO: 1 and Escherichia coli expression vector constructed recombinant expression plasmid pE32a-BoDXR, electrotransformed into BL21(DE3), antibiotic ampicillin selected highly resistant transformants, and induced with 0.2mmol/L IPTG at 37°C for 4h. The above-mentioned IPTG-induced expression bacterial liquid is collected, and the protein isolated and purified is the recombinant protein encoding the 1-deoxy-D-woodoic acid-5-phosphate reductoisomerase gene.

(4) Recombinant protein purification treatment: Induce expression in 1000ml of bacterial solution according to the above method, transfer the expressed bacterial solution to a centrifuge tube and centrifuge at 8000r/min for 10min at 4°C to collect the bacteria. Take 30mL buffer A (PH7.8) (Tris.base 50mM EDTA 0.5mM NaCl 50mM glycerol 5% DTT 5mM) to resuspend the bacteria and precipitate, then use a pressure breaker to crush 3 times; 4°C, 10000r/min centrifuge 5min, discard the supernatant , the precipitate was collected, washed three times with PBS, and the protein was extracted by inclusion body purification.

(5) Preparation of polyclonal antibody: In order to prepare the antiserum of BoDXR recombinant protein, five 4-week-old healthy SPF Kunming mice were immunized with the purified recombinant protein. After the immunization, the purified 100ug recombinant protein was emulsified with complete Freund's adjuvant, and injected subcutaneously. After the adjuvant was emulsified, multi-point subcutaneous injection was used to boost the immunization, and the antiserum was collected 7 days after the last immunization.

(6) The recombinant protein obtained by the present invention has biological activity and good reactogenicity. The prepared polyclonal antibody was used as the primary antibody, and then the antiprotozoa of Babes orientalis and healthy buffalo erythrocytes were used as the antigen, and the HRP-labeled goat anti-mouse IgG was used as the secondary antibody for Western blot analysis to detect the expression of the recombinant protein. Reactogenicity, the results show that only Babesia orientalis antigen has a specific reaction band, and healthy buffalo red blood cells have no reaction, so the prepared recombinant protein can be used to effectively detect Babesia orientalis.

Beneficial effects of the present invention:

The recombinant protein of Babesia orientalis 1-deoxy-D-woodoic acid-5 phosphate reductoisomerase encoded by the present invention can effectively detect the presence of Babesia orientalis antigen in the blood of infected buffaloes, and has a good response It can help to control the prevalence and spread of buffalo babesiosis in my country.

Description of drawings

Fig. 1 is the electrophoresis diagram of the amplification of the 1-deoxy-D-xytoside-5-phosphate reductoisomerase gene of Babesia orientalis. 1: Target gene amplified from Babesia orientalis gDNA; 2: Target gene amplified from Babesia orientalis cDNA; 3: Negative control M: DNA molecular weight standard.

Fig. 2 is the electrophoresis diagram of PCR amplification of the recombinant plasmid of the invention. The primers used are the universal upstream primer T7 on the carrier and the downstream primer BoDXR-R2 of the target gene, 1 and 2 are the target fragments amplified by the two picked positive clones; M: DNA molecular weight standard.

Fig. 3 is the electrophoresis diagram of the product of double enzyme digestion of the recombinant plasmid of the present invention. 1 and 2 are target fragments digested with expression primers amplified using BsaI XhoI (purchased from NEB Biological Company); M: DNA molecular weight standard.

Fig. 4 is the SDS-PAGE electrophoresis result of the qualitative analysis of the recombinant expression product of Babesia orientalis 1-deoxy-D-xoutonose-5-phosphate reductoisomerase. 1, 3: pET32a-BoDXR expression product not induced by IPTG; 2, 4: induced pET32a-BoDXR; M: protein molecular mass standard.

Figure 5 is the SDS-PAGE electrophoresis result of the recombinant expression product of Babesia orientalis 1-deoxy-D-xoutonia-5-phosphate reductoisomerase, Figure 5A is the result of SDS-PAGE electrophoresis for soluble analysis, and Figure 5B is the result of purification SDS-PAGE electrophoresis results of the product. 1: pET32a-BoDXR expression product not induced by IPTG; 2: pET32a-BoDXR expression product induced by IPTG; 3: supernatant of lysate of expressing bacteria; 4: inclusion body of lysate of expressing bacteria; 5: purified Recombinant expression product; M: protein molecular mass standard.

Fig. 6 is the Western blot identification of the recombinant protein of Babesia orientalis 1-deoxy-D-xoutonose-5-phosphate reductoisomerase. 6A identifies the correct expression of BoDXR; 6B identifies the quality of the prepared polyclonal antibody; 6C and 6D identify the expression of BoDXR protein in vivo and in vitro is the same; 1: BoDXR is not induced by IPTG; 2: purified rBoDXR protein; 3: B. orientalis lysate; 4: Buffalo healthy red blood cells.

detailed description

The present invention will be described in detail below in conjunction with examples.

Example 1 Cloning of Babesia orientalis 1-deoxy-D-woodoic acid-5-phosphate reductoisomerase gene

1: Purification of Babesia orientalis and extraction of whole insect antigen

Purification of Babesia orientalis: put freshly collected infected erythrocytes (infection rate is about 5-10%) into a sterilized RNase-free centrifuge tube containing EDTAk anticoagulant, and transfer the cells to a 15mL centrifuge tube In the middle, centrifuge at 3000rpm for 6min to remove the supernatant and white flocs (leukocytes) on the surface; add 1xPBS with 3 times the volume of blood cells, mix well, centrifuge at 3000rpm for 6min, remove the supernatant, repeat this operation twice until the supernatant is colorless Transparent; then add 5 times the volume of erythrocyte lysate, mix well, let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, remove the supernatant, and collect the precipitate. Add 3 times the volume of 1xPBS to the precipitate, mix well, blow repeatedly with a 5mL syringe 10 times, then filter the solution with a sterilized 2um filter, after filtration, centrifuge at 12000rpm for 5min, collect the precipitate, repeat this step until the supernatant Colorless and transparent; the precipitate was washed once with sterilized RNase-free PBS, and 1/10 volume of sterilized RNase-free PBS solution was added, and this was the purified leukocyte-free Babesia orientalis body.

Extraction of holozoan antigens: Add 1/10 volume of RAPI cell lysate to the parasites obtained in the upward step, and then ultrasonically disrupt for 30 minutes, then centrifuge at 12000 rpm for 20 minutes, and the supernatant is the obtained holozoan antigens.

2: Extraction of the whole genome of Babesia orientalis

Take 200 μL of freshly collected Orient Babes blood containing anticoagulant and place it in a 1.5 mL polypropylene centrifuge tube, and extract it according to the instructions of Tiangen Blood Genomic DNA Extraction Kit, and store the extracted whole genome at -20 degrees. 3: Extraction of total RNA from Babesia orientalis

All utensils used for RNA extraction were treated with 0.1% DEPC water and baked at 150°C for 3 hours; plastic utensils were soaked in 0.1% DEPC water overnight and sterilized at 121°C for 30 minutes; metal utensils were soaked in 1mol/L NaOH for 2h, After thoroughly rinsing with 0.01% DEPC water, dry at 37°C; each reagent was prepared with 0.01% DEPC water without ribonuclease (RNase). RNA was extracted from 400 μL whole blood of Babesia orientalis. Total RNA was extracted according to the instructions of Trizol reagent. Extracted total RNA samples were frozen at -80°C for future use.

4: Synthesis of total cDNA of Babesia orientalis

Using the reverse transcription kit of Beijing Quanshijin Company, the total RNA of Babesia orientalis extracted was used as a template for reverse transcription. The specific operation steps are as follows:

①First-strand cDNA synthesis and gDNA removal

Anchored Oligo (dT) ₁₈ Primer 0.5ug/uL) 1ul

Total RNA 4ul

RNase-free water _dH2O 3ul

After mixing, perform denaturation and annealing reactions under the following conditions on a PCR instrument:

65°C for 5 minutes, ice bath on ice for 2 minutes

② Prepare the following reverse transcription reaction solution in the above-mentioned Microtube tube.

2xES Reaction Mix 10ul

EasyScript RT/RI Enzyme Mix 1uL

gDNA Remover 1uL

③After mixing, carry out the reverse transcription reaction on the PCR instrument according to the following conditions:

42℃ 30min

↓

85℃ 5min

↓

After processing, keep on ice.

The cDNA obtained by reverse transcription was stored at -20°C for future use.

5: Cloning and Sequence Analysis of Babesia orientalis 1-deoxy-D-xyloose-5-phosphate reductoisomerase

1) Primer design

According to the 1-deoxy-D-xylose-5-phosphate reductoisomerase (DXR) sequence information of known apicomplexan parasites submitted on NCBI/GenBank, according to the 1-deoxy-D-xylose- For the conservation of the 5-phosphate reductoisomerase sequence, use the BLASTp method to obtain a sequence with the highest homology in the Babesia orientalis genome database, and then use clonemanager software to separate the cDNA sequence open reading frame (ORF) For the upstream and downstream regions, design a pair of primers (BoDXR-F1, BoDXR-R1);

BoDXR-F1:5'-ATGAATGCAGCAGTGAGTTTTTTATG-3'

BoDXR-R1: 5'-TTAGTATGTGAAGCATTAATATATGTGATAG-3'.

2) PCR amplification: PCR amplification was performed using Babesia orientalis gDNA and cDNA as templates respectively, the reaction system was 25 μL, and the PCR reaction system was as follows:

PCR reaction conditions: the reaction conditions are 94°C for 5min; 94°C for 30sec, 55°C for 30sec, 68°C for 1min and 50sec; 68°C for 10min; 34 cycles.

3) PCR product identification: After the amplification is completed, take 8μ of the PCR product plus 2uL of 6×nucleic acid loading buffer for spotting, 0.8% agarose gel, 1×TAE buffer, 120V, electrophoresis for 20 minutes to observe the results, The results showed that the target band amplified with gDNA was different from the target band amplified with cDNA, indicating that the gene contained introns (see Figure 1).

4) According to the instructions of the Beijing Quanjin DNA Agarose Gel Recovery Kit, the amplified gDNA and cDNA were cut and recovered by gel cutting.

5) Cloning, screening and sequencing of the target gene

Take 1 μL (50ng/uL) of the pEASY-Blunt vector and 4 μL of the target fragment recovered from the gel, mix them in a PCR tube, react in a PCR instrument at 25 degrees for 20 minutes, take 5 μL of the ligation product, and add it to 50 uL of Escherichia coli Trans1-T1 under sterile conditions For the competent cells, use a pipette to gently blow and mix, place in an ice bath for 30 minutes, then heat shock for 90 seconds in a water bath at 42°C, and then immediately cool in an ice bath for 2 minutes, and be careful not to shake. Then add 500 μL of LB culture solution preheated to 37°C to the competent cells, and shake gently at 180 rpm at 37°C for 45 minutes to restore the drug resistance of the bacteria. After recovery of drug resistance, centrifuge at 5000rpm for 3min, discard part of the supernatant in an ultra-clean table, leave 100uL of the supernatant to resuspend the precipitate, take 80uL of the resuspension and spread it evenly on the LB agar plate containing ampicillin (AMP) (100μg/mL), After standing at 37°C for half an hour, invert the plate and continue to incubate at 37°C for 10 hours, until a single white colony can be picked with the naked eye. Pick two colonies and inoculate them in LB culture medium containing 100μg/mL AMP, shake vigorously (180rpm) for 10-12 hours, amplify the target gene with the universal primers on the carrier, and take out the clones that are positive by PCR. Sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing analysis, the results showed that the length of the fragment amplified by gDNA was 1554bp, and the length of the fragment amplified by cDNA was 1371bp, and the target gene amplified by cDNA was analyzed and had a complete open read frame (ORF), the sequence of its open reading frame is as shown in the sequence listing SEQ ID NO: 1.

Serial number: SEQ ID NO: 1

Sequence length: 1371bp

Sequence type: cDNA

Source: Babesia orientalis

Sequence features: correct open reading frame (ORF): 1371bp; determined positions: start and stop codons exist: ATG, position 1; TGA, position 1371.

6) The sequence ORF obtained in step 5 above has a full length of 1371 bp, and the encoded protein has the amino acid sequence shown in SEQ ID NO: 2 in the sequence table. The protein contains 456 amino acids with a molecular weight of 50KD. The determined amino acid sequences were analyzed using BLAST (Basic Local Alignment Search Tool) software on the website of the National Center for Biotechnology Information (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov). Using the Megalign software in the DNAstar software package, the nucleotide sequence similarity between different species was compared by the ClustalW method. The results showed that the full-length ORF coding region sequence of Babesia orientalis 1-deoxy-D-xyloose-5-phosphate reductoisomerase was obtained.

Example 2: Construction of recombinant prokaryotic expression vector and induced expression in Escherichia coli

1) Design primers for the expression of 1-deoxy-D-xautonose-5-phosphate reductoisomerase by means of homologous recombination:

Design expression primers (BoDXR-F2, BoDXR-R2) according to the ORF sequence of the 1-deoxy-D-xautonose-5-phosphate reductoisomerase gene, and add the downstream primers used to amplify the pET32a target gene in front of the upstream primers The reverse complementary sequence is used as a homology arm, and the downstream primer is added to the reverse complementary sequence of the upstream primer used to amplify the pET32a target gene as a homology arm. details as follows:

BoDXR-F2: 5'GCCATGGCTGATATC GGATCC ATGGTTGGAAACCACCGC 3'

BoDXR-R2: 5'GTGGTGGTGGTGGTG CTCGAG TCACTTTCGACCACTGATGC 3'

pET32aF:CTCGAGCACCACCACCACCAC

pET32aR:GGATCC GATATCAGCCATGGC

2) Construction of recombinant expression plasmids

Use expression primers (BoDXR-F2, BoDXR--R2) to carry out PCR amplification on the recombinant plasmid pEASY-Blunt-BoDXR to obtain the target fragment, and at the same time use (pET32aF pET32aR) primers to amplify the expression vector pET32a, perform PCR reaction, and run DNA gel to obtain Target gene. Use Beijing Quanjin DNA Agarose Gel Recovery Kit to recover the target fragment. The concentrations of the recovered products were measured with a spectrophotometer. The results showed that the recovery concentration of pET32a was 50 ng/mL, and the recovery product concentration of cDNABoDXR target gene was 45 ng/mL. According to the instructions of Vazyme's one step Cloning Kit, the target fragment was ligated with the vector fragment.

(1) Connection program

37°C for 30min

(2) Connection system

3) Conversion

After the ligation, the ligation product was placed on ice for 5 min, and then 10 uL of the ligation product was added to 100 uL of Escherichia coli DH5a competent, gently blown and mixed with a pipette, and placed in an ice bath for 30 min. Water bath at 42°C, heat shock for 90s, then immediately cool in ice bath for 2min, be careful not to shake. Transfer the bacterial liquid to 500 μL of LB culture medium preheated to 37°C, shake gently at 180 rpm at 37°C for 45 minutes to restore the resistance of the bacteria, take 150 μL of the bacterial liquid and spread it on a medium containing ampicillin (AMP) (100 μg/mL) on LB agar plates. Invert the plate and incubate in a constant temperature incubator at 37°C for 12-16 hours, pick a single colony and inoculate it in LB culture solution containing 180 μg/mL AMP, shake vigorously (230 rpm) for 10-12 hours and then identify. At the same time, the pET32a empty plasmid vector without adding any foreign gene was transformed as a control.

4) Identification of positive clones

Use the universal primer upstream of the vector and the downstream primer of the target gene to identify whether the positive clone is inserted correctly.

Take 1ul of the bacterial solution for PCR, the reaction system is 25ul, as follows:

PCR reaction conditions: the reaction conditions are 95°C for 5min; 94°C for 30sec, 57°C for 30sec, 72°C for 1min and 40sec; 72°C for 10min; 35 cycles.

After PCR, run agarose gel electrophoresis, the result shows (as shown in Figure 2), for the bacterial solution identified as positive by PCR, resuscitate and shake 5mL of bacteria, continue to shake at 37 degrees for 8h, and then extract according to Beijing Quanshijin Biological Co., Ltd. The plasmid was extracted according to the instruction manual of the plasmid kit, and the concentration of the plasmid was measured with a spectrophotometer, and the result was 170 ng/mL, and then double-enzyme digestion was carried out with BsaI XhoI (NEB Company) enzyme for identification.

Enzyme digestion system: BsaI 1uL

After 1 hour at 37°C, DNA gel was run for identification. The results showed that the enzyme excised two bands of expected size, which were 5755bp and 1482bp respectively (Figure 3).

The amplified positive clones were taken and sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing analysis. The 1371bp cDNA sequence was obtained for sequence comparison, indicating that the prokaryotic recombinant expression plasmid pET32a-BoDXR has been successfully constructed.

5) Construction and small-scale induction of pET32a-BoDXR expression vector

The pET32a-BoDXR-DH5a plasmid (1 μL) was aseptically transformed into Escherichia coli BL21 competent cells (100 μL), and the specific operation method was as above step 3). Pick two pET32a-BoDXR/BL21(DE3) monoclonal colonies and add them to 5mL LB medium containing AMP, culture at 37°C, 180rpm for about 8h, then transfer to 4mLLB containing AMP at 1:100, continue at 37°C , cultured at 180rpm for about 3h, until the OD600 was about 0.5, took 2mL from each bottle and placed it in a new shaker flask without adding inducer, and added 0.8mmol/L IPTG to the other 2mL to continue induction at 37°C for 3h, and then centrifuged at 12000rpm for 2min Collect the bacteria, add 40uLddH ₂ O 10uL1M DTT.50uL 2xSDS loading buffer to each tube, cook in boiling water for about 10 minutes, when the protein changes from viscous to fluid, it is completely denatured, and then quickly put it on ice for 5 minutes , followed by running SDS-PAGE gel to identify whether it was expressed, the result showed that the recombinant BoDXR was successfully expressed in Escherichia coli (as shown in Figure 4).

6) Solubility analysis of expression products of recombinant proteins

Recovery step 4) For the successfully expressed bacterial liquid, induce the expressed bacterial liquid according to the above method, take 1.5 mL and centrifuge at 8000r/min for 2 minutes to collect the bacterial cells, resuspend the bacterial cells with 500 μL of PBS solution, centrifuge again to collect the bacterial cells, and wash the bacterial cells repeatedly 2 -3 times to remove the medium residue.

Place 500 μL of bacterial cell solution in each tube on the ice-water mixture for crushing with an ultrasonic breaker, 15 s/time, with an interval of 30 s, and break each tube 8-10 times until the liquid is clear and translucent.

Centrifuge the crushed sample at 12000r/min for 10min at 4°C. Take 40 μL supernatant solution to a 1.5mLEP tube, add 50 μL 2×SDS sample buffer, and 10 μL DTT solution, vortex and mix well; add 40 μL sterilized ddH ₂ O, 50 μL 2×SDS sample buffer , and 10 μL DTT solution, vortexed well. All the samples were boiled in boiling water for 10 min, and immediately transferred to ice for 5 min. The processed samples were detected by SDS-PAGE electrophoresis to observe the results. After analysis, the protein was expressed in the precipitate. As shown in Figure 5A.

7) Mass purification and extraction of expression products

Purification and extraction of BoDXR recombinant protein: The bacterial culture (500 mL) with a large amount of induced expression was centrifuged in a 500 mL centrifuge tube at 8000 r/min for 10 min at 4°C. After the bacterial pellet was resuspended in 50mL PBS, it was crushed with an ultrasonic breaker until the liquid was clear; 4°C, centrifuged at 12000r/min for 10min, discarded the supernatant, and the pellet was resuspended in 30mL of PBS, centrifuged at 12000r/min for 10min, washed three times repeatedly, and the last time Add 19.7mL of BufferA, 300uL of 20% SKL (sodium lauryl sarcosine), 100uL of 1M DTT to the precipitate, and stir vigorously with a shaker to dissolve it. Let stand at room temperature for 2 hours, centrifuge at 12000r/min for 20min at 4°C, transfer the supernatant to a clean centrifuge tube, add 210uL of 20% PEG4000, 420uL of 50mM oxidized glutathione and 420uL of 100mM reduced glutathione to the supernatant, After recovery at room temperature for 2 hours, the supernatant was put into a dialysis bag and dialyzed in PBS for 2 days. The protein obtained after dialysis was concentrated with sucrose. The results of gel run identification after concentration are as shown in number 5 in Figure 5B.

Embodiment 3: the preparation of polyclonal antibody

(1) Determination of protein concentration by BCA method: Take 10 μL of 5 mg/ml protein standard product and add it to 90 uLPBS standard diluent, dilute to a final concentration of 0.5 mg/ml standard protein; Add 8, 12, 16, and 20 μL to the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μL; dilute the samples with PBS 20 times, 10 times, 5 times, and 1 times respectively to the sample wells of the 96-well plate The medium to final volume is 20 μL; add 200 μL BCA working solution to each well, and place at 37°C for 30 min; measure A562. Calculate the protein concentration of the sample according to the standard curve.

(2) 5 healthy Kunming mice with SPF level were purchased, and blood was collected by tail docking as negative serum before the first immunization.

(3) Preparation of polyclonal antibody of mouse origin: the initial immunization dose is to inject 100ug of protein emulsified in Freund's complete adjuvant to each Kunming mouse, the inoculation volume is 200uL, and the injection site is injected subcutaneously at multiple points on the back and abdomen; the second immunization: About 14 days after the initial immunization, the dose is 200uL/cattle (recombinant protein content 50ug), plus Freund's incomplete adjuvant. The injection site was subcutaneously injected at multiple points on the back and abdomen; the third immunization was carried out 14 days after the second immunization, the injection dose and site were the same as the second immunization, and some blood was collected from the tail 7-14 days after the immunization for antibody detection Titer, the fourth immunization was carried out 7 days after the third immunization, the blood was collected 7 days after the fourth immunization, the serum was separated, and stored at minus 20 degrees.

Example 4: Identification of reactogenicity of recombinant protein

Perform SDS-PAGE electrophoresis on the purified recombinant protein. According to the size of the target protein band and the corresponding position of the control protein marker, cut the electrophoresis-completed SDS-PAGE gel into the required size, and rinse it with water; Cut 6 pieces of filter paper of the same size and 1 piece of PVDF membrane, soak the filter paper in the electrotransfer buffer for 5 minutes, soak the PVDF membrane in methanol solution for 15 seconds, rinse with water, and soak in the electrotransfer buffer for 5 minutes; Place 3 pieces of filter paper, SDS-PAGE gel, PVDF membrane and the remaining 3 pieces of filter paper in sequence on the negative plate of the electrotransfer instrument, and align them completely. In each step of making the electrotransfer sandwich, gently remove the air bubbles with a glass rod; cover carefully The positive plate of the electroporation instrument, calculate the electroporation area, adjust the electroporation instrument to a steady flow, and perform electroporation at 1mA/cm2, the electroporation time depends on the molecular size of the target protein; the PVDF membrane after electroporation is quickly rinsed twice with TBST solution and rinsed with 5% Block with skimmed milk powder at room temperature for 1-2h or overnight at 4°C; blot dry the blocking solution on the PVDF membrane, rinse with TBST 5 times for 3 minutes each time, then properly dilute the primary antibody, act at room temperature for 1-2h or incubate overnight at 4°C ;Put the PVDF membrane in a new sterile plate, rinse 5 times with PBST, 5min each time; properly dilute the secondary antibody (generally 1:1000), act at room temperature for 1h, rinse 5 times with PBST, 5min each time ; Perform DAB color development or ECL chemiluminescence color development on the PVDF membrane, and observe the color development at the size of the target protein, as shown in Figure 6A/6B/6C/6D.

The number 1 represents that the above sample is the lysate of recombinant pET32a-BoDXR bacterial fluid that has not been induced by IPIG; the number 2 represents the protein extract of recombinant pET32a-BoDXR bacterial liquid induced by IPTG; the number 3 represents the processed Babesia orientalis antigen; the number 4 represents the healthy red blood cells of buffalo.

Figure 6A The primary antibody is a mouse His-tag, and the secondary antibody is a goat anti-mouse IgG polyclonal antibody.

Figure 6B/6C/6D The primary antibody is a mouse polyclonal antibody prepared with recombinant protein pET32a-BoDXR, and the secondary antibody is a goat anti-mouse IgG polyclonal antibody.

The results of Western blot showed that the purified recombinant protein BoDXR had a specific reaction with the Babesia orientalis holoantigen at 68kDa, but had no reaction with healthy red blood cells (see Figure 6C and 6D). It shows that the protein expressed in vitro is consistent with the naturally expressed protein in Babesia orientalis.

<110> Huazhong Agricultural University

<120> Babesia orientalis 1-deoxy-D-xoutonose-5-phosphate reductoisomerase gene and its encoded protein

<160> 3

<210> 1

<211> 1371

<212> cDNA

<213> Babesia orientalis

<400> 1

ATGGTTGGAA ACCACCACAG GGGCTCCATT TTATATATGT GGTGTGCAGT GTCGCTGGTG 60

GCACTGCCGC AAACCACCGG ATTTCGATAC TTCTCCCAATA CTAGTCCATC GTTGAGAAGC 120

AGACCTTTTAT TGGCGAATCC TATAAAGGTG GCTGTTATTG GGAGCACTGG AAGCATTGGG 180

ACTCAGACCC TGGATATTAT ACGCCGCATC AATAATACAG CTGATGAACC TAAATTCAAG 240

GTTGTCGCTT TAACAGCCAA TCGTGACATT TCTGGTTTAG CGAAGCTAGC AGCAGAATTT 300

ACGCCCCACA TTTTGAATAT TAATTATGGT GGTGAGGAAC TCCGGCAAAC TGTGCCAAAT 360

AGCTGCGACA TTACCACTGG GAAAGAGGGC GTCTTAAACA TTTGTAAATC CTTCGACTTT 420

GACTTGTTGG TAATGGGGAT ATCGGGCTGT GCTGGCATAG AACCAACCAT CGCAGCAGCA 480

CAATCCGGTA AAAGGATGGC GTTAGCTAAC AAAGAATCTG TGGTATCTGC AGGGAGCCTG 540

TTGCGAAAGG CCATCAACAA AAGTAAATGC GAGCTTATTC CTATTGACTC CGAGCACAAT 600

GCAATATACC AATGTCTTAC GTCATCTGCA CGTGATTTGT TAGAGCAAAC AAAATTGAGA 660

GATACGAGGC CCTTGTGTGG TATTTCTAAA AACGTTTTAG ATTCAGTAAA ACGCCTTATC 720

ATCACCAGCA GTGGTGGTCC GTTTAGAGAT ACAAACCCGT CGTTGATGAA GACATTGCGC 780

CTAAAGGATG CTCTTAAGCA TCCTGTTTGG AGTATGGGTG CTAAAATAAC CATTGACTCG 840

TCCACCATGA TGAACAAGGG ATTAGAGGTA ATAGAGGCTC ACGAACTGTT CGGGATCCCA 900

TATGATAGTA TAAAAGTACT CATTCACAAG GAATGTATCG TACATTCAAT GGTGCAATTT 960

GTTGATAACT CGGTTCTGGC ACAATTATAC AACCCTGATA TGAGGCTCCC AATAGCATAT 1020

GCACTCAACT GGCCGGACAG GCTTGCTAAT ACCTTACAAG AATTGAACCT CGTTGAACAG 1080

ACGCTTACTT TCGCTGATCC TGACTTACAG AAGTTTCCTT GCCTTAAATT GGCTTATGAA 1140

GTTGGGAAGA TGGGAGGACT CTACCCCACA GTTTTAAATG CTGCAAATGA GCAGGCAAAT 1200

GAACTACTAA GGCGCGATGC AATTGCACAT TGTGAAATAT ACGATCTTGT AAAGCGTGCA 1260

ATTGATGCTT TCCAACATCC AAGTATTAGC CAACCTGGCA TTAAGGATAT AATGGAAGCC 1320

GATAGCTGGG GAAAGAAACA TGTGATGGAC TGCATCAGTG GTCGAAAGTG A 1371

<210> 2

<211> 456

<212> RNA

<213> Babesia orientalis

<400> 2

Met Val Gly Asn His Arg Arg Trp Ser Val Leu Tyr Met Trp Cys 15

Ala Val Ser Leu Val Ala Leu Pro Gln Thr Thr Gly Phe Arg Tyr 30

Ser Pro Asn Thr Ser Pro Ser Leu Arg Ser Arg Pro Leu Leu Ala 45

Asn Pro Ile Lys Val Ala Val Ile Gly Ser Thr Gly Ser Ile Gly 60

Thr Gln Thr Leu Asp Ile Ile Arg Arg Ile Asn Asn Thr Ala Asp 75

GAA CCT AAA TTC AAG GTT GTC GCT TTA ACA GCC AAT CGT GAC ATT 270

Glu Pro Lys Phe Lys Val Val Ala Leu Thr Ala Asn Arg Asp Ile 90

Ser Gly Leu Ala Lys Leu Ala Ala Glu Phe Thr Pro His Ile Leu 105

Asn Ile Asn Tyr Gly Gly Glu Glu Leu Arg Gln Thr Val Pro Asn 120

Ser Cys Asp Ile Thr Thr Gly Lys Glu Gly Val Leu Asn Ile Cys 135

Lys Ser Phe Asp Phe Asp Leu Leu Val Met Gly Ile Ser Gly Cys 150

Ala Gly Ile Glu Pro Thr Ile Ala Ala Ala Gln Ser Gly Lys Arg 165

Met Ala Leu Ala Asn Lys Glu Ser Val Val Ser Ala Gly Ser Leu 180

Leu Arg Lys Ala Ile Asn Lys Ser Lys Cys Glu Leu Ile Pro Ile 195

Asp Ser Glu His Asn Ala Ile Tyr Gln Cys Leu Thr Ser Ser Ser Ala 210

Arg Asp Leu Leu Glu Gln Thr Lys Leu Arg Asp Thr Arg Pro Leu 225

Cys Gly Ile Ser Lys Asn Val Leu Asp Ser Val Lys Arg Leu Ile 240

Ile Thr Ser Ser Gly Gly Pro Phe Arg Asp Thr Asn Pro Ser Leu 255

Met Lys Thr Leu Arg Leu Lys Asp Ala Leu Lys His Pro Val Trp 270

Ser Met Gly Ala Lys Ile Thr Ile Asp Ser Ser Thr Met Met Asn 285

Lys Gly Leu Glu Val Ile Glu Ala His Glu Leu Phe Gly Ile Pro 300

Tyr Asp Ser Ile Lys Val Leu Ile His Lys Glu Cys Ile Val His 315

Ser Met Val Gln Phe Val Asp Asn Ser Val Leu Ala Gln Leu Tyr 330

Asn Pro Asp Met Arg Leu Pro Ile Ala Tyr Ala Leu Asn Trp Pro 345

Asp Arg Leu Ala Asn Thr Leu Gln Glu Leu Asn Leu Val Glu Gln 360

Thr Leu Thr Phe Ala Asp Pro Asp Leu Gln Lys Phe Pro Cys Leu 375

Lys Leu Ala Tyr Glu Val Gly Lys Met Gly Gly Leu Tyr Pro Thr 390

Val Leu Asn Ala Ala Asn Glu Gln Ala Asn Glu Leu Leu Arg Arg 405

Asp Ala Ile Ala His Cys Glu Ile Tyr Asp Leu Val Lys Arg Ala 420

Ile Asp Ala Phe Gln His Pro Ser Ile Ser Gln Pro Gly Ile Lys 435

Asp Ile Met Glu Ala Asp Ser Trp Gly Lys Lys His Val Met Asp 450

Cys Ile Ser Gly Arg Lys 456

<210> 3

<211> 1554

<212>g DNA

<213> Babesia orientalis

<400> 3

ATGGTTGGAA ACCACCGCAG GTGGTCCGTT TTATATATGT GGTGTGCAGT GTCGCTGGTG 60

GCGCTGCCGC AAACCACCGG ATTTCGTAAG TTCACAAAAT GGCTGTTTAT AACTCGGCAG 120

GATACACTCC CAACACTAGT CCATCGTTAA GAAGCCCACC TTTGTAAGTC ATTTTATAGG 180

TTCGACCATT ACATAATCTT CAGATTGGCG AATCCTATAA AGGTGGCTGT TATTGGGAGC 240

ACTGGAAGCA TTGGAACGCA GACCCTGGAT ATTATACGCC GCATCAATAA TACAGCTGGT 300

GAACCTAAAT TCAAGGTTGT GGCTTTAACA GCCAATCGTG ACATTTCTGG TTTAGCGAAC 360

CTAGCAGCAG AATTCACGTG AGTTGCATTT GCATAGAACA AAACACAAT AGGCCCCACA 420

TTTTGAATAT TAATTATGGT GCTGAGGAAC TCCGGAAAAC TGTGCCAAAC AATTGCGACA 480

TTACCACGGG GAAGGAGGGT CTCTTAAACA TTTGTAAATC CTTCGACTAT GACTTGTTGG 540

TAATGGGGAT ATCGGGATGT GCTGGCATAG AACCAACCAT CGCAGCAGCA CAATCCGGTA 600

AAAGGATGGC TTTGGCTAAC AAAGAATCTG TGGTATCTGC AGGTAAATCA GTTGTTTCAT 660

GTGATCTTAA TTGCCTGTAG GGAGCCTGTT GCGAAAGGCC ATCAACGAAA GTAAATGCGA 720

GCTGATTCCT ATTGACTCCG AGCACAATGC AATATACCAA TGTCTTACGT CATCTGCACG 780

TGATTTGTTA GAGCAAACCA AATTGAGAGA TCCGAGGCCC TTGTGTGGTA TTTCTAAAAA 840

CGTTTTAGAT TCAGTAAAAC GCCTTATCAT CACCAGCAGT GGTGGTCCTT TTAGAGATAC 900

AAGCCCGTCT TTGATGAATA CAATGCGCCT AAAGGATGCT CTTAAACATC CTGTCTGGAG 960

TATGGGTGCT AAAATAACCA TTGACTCCTC CACCATGATG AACAAGGGAT TAGAGGTAAT 1020

AGAGGCTCAC GAACTGTTTG GGATCCCATA TGATAGTATA AAAGTACTCA TTCACAAGGA 1080

ATGTATCGTA CATTCAATGG TGCAATTTGT TGATAACTCG GTTCTGGCAC AATTATACAA 1140

CCCTGATATG AGGCTCCCAA TAGCATATGC ACTCAACTGG CCGGACAGGC TTGCTAATAC 1200

CCTGCAAGAA TTGAACCTCG TTGAACAGAC TCTTACTTTC GCTGATCCTG ACTTACAGAA 1260

GTTCCCCTGC CTTAAATTGG CTTATGAAGT TGGGAAGATG GGAGGACTGT ACCCCACAGT 1320

TTTAAATGCT GCAAATGAGC AGGCAAATGA ACTACTAAGG CACGATGCAA TTGCACATTG 1380

TGAAATATAC GATCTTGTAA AGCGTGCCAT TGATGCTTTC CAACATCCAA ATTTAAACCA 1440

ACCTGGCATT GAGGTATGCT ATATAATATA CGATTTTATC ATTTGCAGGA TATAATGGAA 1500

GCCGATAGCT GGGGAAAGAA ACATGTGATG GACTGCATCA GTGATCGAAA GTGA 1554

Claims (3)

1. The Babesia orientalis 1-deoxy-D-xoutunose-5-phosphate reductoisomerase gene, which has the nucleotide sequence shown in SEQ ID NO:1. 2. the protein encoded by the Babesia orientalis 1-deoxy-D-xoutunose-5-phosphate reductoisomerase gene according to claim 1, the protein has amino acids as shown in the sequence table SEQ ID NO:2 sequence. 3. the application of the albumen described in claim 2 in the preparation of Babesia orientalis detection kit.