CN112442133A - Babesia canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Babesia canis BcMSA1-BcSA1 recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence shown in SEQ ID NO.1 or consists of the amino acid sequence shown in SEQ ID NO. 1. The invention further discloses a gene of the recombinant protein, a vector and a host cell containing the gene, a preparation method of the recombinant protein and application of the recombinant protein in detecting an antibody of the Babesia canis. The recombinant protein is used for detecting the canine Babesia antibody, is convenient and rapid, has high sensitivity, no cross reaction with other pathogens, strong specificity, great clinical significance and wide application prospect.

Description

Babesia canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof

Technical Field

The invention belongs to the field of animal virus antibody detection, and particularly relates to a Babesia canis BcMSA1-BcSA1 recombinant protein, and a preparation method and application thereof.

Background

Babesia (Babesia) belongs to the genus Babesia, which includes more than 90 species of Babesia, and it has been reported that various species of Babesia infect dogs, mainly Babesia westermani (b.vogeli), Babesia gibsoni (b.gibsoni), Babesia canis (b.canis), Babesia rochei (b.rossi), and Babesia gemmifera (b.bigemina). Wherein the size of the Babesia canis is 3-5 μm, and the Babesia canis exists in a single or paired form.

Babesia can cause tissue and organ damage, can damage red blood cells of dogs, urine of sick dogs can turn red, symptoms such as high fever, anemia and rapid emaciation can also appear, and the sick dogs are at risk of death if not diagnosed and treated in time. In general, the latent period of the disease is usually 7 to 8 days, and more usually 10 days or more. Therefore, the ability to quickly and easily diagnose a dog for babesia is an important means of reducing dog mortality.

At present, the more direct clinical diagnosis method is an immunological method such as a blood smear staining method after venous blood collection, an Indirect fluorescent antibody test (IFA), an Enzyme-Linked immunosorbent assay (ELISA) and the like, and a PCR (polymerase chain reaction) method is also a detection method which is commonly used for the diagnosis of Babesia and is most sensitive and high in specificity for the diagnosis of Babesia. However, the methods such as the immunofluorescence method, the enzyme-linked immunosorbent assay (ELISA), the Polymerase Chain Reaction (PCR) technology and the like require the use of designated instruments and equipment, have corresponding test conditions and skills, and are difficult to popularize at the basic level. The colloidal gold labeling immunoassay method is a novel analysis technology which is started and developed rapidly in recent years, and has the characteristics of rapidness, simplicity, low cost, no pollution, no need of training, suitability for field detection compared with the traditional method, short color development time, no need of expensive instruments and the like, and wide market prospect and application value.

However, current detection methods and tools for detecting babesiosis infection in serological tests are still subject to major limitations.

Disclosure of Invention

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention provides a Babesia canis BcMSA1-BcSA1 recombinant protein, which comprises an amino acid sequence shown in SEQ ID NO. 1.

In the present invention, the recombinant protein is also called fusion protein or recombinant fusion protein, and is an expression product obtained by recombining two genes by a DNA recombination technique.

The BcMSA1(Babesia. canis Merozoite Surface Antigen 1, Babesia fission Surface Antigen 1) and BcSA1(Babesia. canis Secreted Antigen 1) proteins are the main immunogen proteins of the canine Babesia, and can induce and initiate the immune system of the body to generate immune response. The BcMSA1 and the main epitope of the BcSA1 protein are fused together for expression and antibody detection, so that the diagnostic sensitivity can be improved, and the cross reaction with other pathogens can be reduced.

In some embodiments of the present invention, preferably, the recombinant protein consists of the amino acid sequence shown in SEQ ID No. 1.

In a second aspect, the present invention provides a gene encoding the recombinant protein according to the first aspect of the present invention, which comprises the nucleotide sequence shown in SEQ ID NO. 2.

The gene sequence is used for expressing the recombinant protein in the escherichia coli, and codons are optimized according to the preference of the escherichia coli to the codons. The frequency of usage of synonymous codons by different species is different and this codon preference has an impact on the translation process. If a mRNA has many rare codons clustered, this will have a negative effect on the rate of ribosome movement and greatly reduce the protein expression level. The gene sequence is subjected to codon optimization, and the method is suitable for escherichia coli expression and can improve the protein expression efficiency.

In a third aspect, the present invention provides an expression vector comprising the gene of the second aspect of the present invention.

In some embodiments of the invention, the expression vector is pET30a, which is kanamycin resistant and the expressed fusion protein has a histidine (His) tag.

In a fourth aspect, the present invention provides a host cell comprising an expression vector according to the third aspect of the invention.

Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

In some embodiments of the invention, the host cell is a prokaryotic host cell. Preferably, the host cell is escherichia coli, more preferably, escherichia coli is BL 21. The expression by using the escherichia coli has the advantages of short period, low cost, large expression amount and the like.

In a fifth aspect, the present invention provides a method for producing a recombinant protein according to the first aspect of the present invention, comprising the step of inducing the host cell according to the fourth aspect of the present invention to express the protein.

Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

In some embodiments of the invention, the host cell is a prokaryotic host cell. Preferably, the host cell is escherichia coli, more preferably, escherichia coli is BL 21. The expression by using the escherichia coli has the advantages of short period, low cost, large expression quantity and the like.

In some embodiments of the invention, the step of inducing E.coli to express the protein is:

s1, culturing the Escherichia coli in LB medium containing 50. mu.g/mL of kanamycin at 37 ℃,

s2, when the culture solution OD600 of the escherichia coli is 0.5-0.7, the induction expression is carried out by IPTG with the final concentration of 1mM, and the induction conditions are as follows: the rotation speed is 200rpm at 25 ℃ for 4 h; by using the induction condition, the recombinant protein can be more slowly expressed, and sufficient time is provided for the formation of space conformation, which plays an important role in the function of the recombinant protein.

S3, centrifuging the culture solution at 4 ℃ and 7000rpm for 10min, and collecting thalli;

s4, crushing the thallus by using a Buffer Binding Buffer;

s5, carrying out ultrasonic disruption on thalli under the conditions of: 600w, ultrasonic for 2s, and interval of 5s, and 80-120 times in total;

s6, centrifuging at 12000rpm at 4 ℃ for 30min, and collecting supernatant, wherein the recombinant protein is in the supernatant.

Preferably, the induction is performed in step S2 at an OD600 of the E.coli culture broth of 0.6.

Preferably, in step S5, the ultrasonication is performed 100 times. By adopting the crushing method, the condition that the recombinant protein is lost due to over violent crushing is avoided.

In some embodiments of the invention, further comprising the step of purifying the recombinant protein. The recombinant protein can be purified by various methods, such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography. In some embodiments of the invention, the method of affinity chromatography is selected such that higher purity can be achieved in a single purification step due to the addition of the His-tag to the recombinant protein.

In some embodiments of the invention, the supernatant containing the recombinant protein is passed through a Ni column and then eluted with an Elution Buffer to obtain the desired protein.

Preferably, the formulation of the Elution Buffer solution Elution Buffer is as follows: 50mM Tris, 0.2M NaCl, 0.5M Imidazole, pH 8.0.

The sixth aspect of the invention provides the use of a recombinant protein according to the first aspect of the invention in the preparation of a kit for detecting babesia canis antibodies.

The seventh aspect of the present invention provides a kit for detecting babesia canis antibodies, comprising the recombinant protein according to the first aspect of the present invention.

Further, the kit also comprises mouse IgG and goat anti-mouse IgG.

In some embodiments of the invention, canine babesia antibodies are detected using a double antigen sandwich gold-labeling method.

In some embodiments of the invention, the kit comprises a double antigen sandwich gold-labeled test strip, and the reagent method of the test strip is as follows:

s1, preparing a recombinant protein colloidal gold compound and a mouse IgG colloidal gold compound respectively;

s2, mixing the recombinant protein colloidal gold compound and the mouse IgG colloidal gold compound to prepare a gold-labeled pad;

s3, marking on a nitrocellulose membrane by using the recombinant protein as a detection line and using goat anti-mouse IgG as a quality control line;

s4, mounting filter paper, a polyester plate containing a nitrocellulose membrane, a gold label pad and a sample pad on a bottom plate, wherein a part of the filter paper is overlapped and pressed on the polyester plate, a part of the polyester plate is overlapped and pressed on the gold label pad, a part of the gold label pad is overlapped and pressed on the sample pad, a test area and a quality control area are respectively arranged on the polyester plate, the test area is provided with a test line (T line), the quality control area is provided with a quality control line (C line), the test line is close to the gold label pad, and the quality control line is close to the filter paper, thus preparing the test strip.

When the kit is used, a biological sample is dripped to a sample pad, and the detection result is judged after the kit is placed at room temperature for 10min, wherein the judgment standard is as follows:

two strips appear, wherein one strip is positioned in a quality control area, and the other strip is positioned in a test area and is positive;

secondly, only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;

and thirdly, the quality control line has no strip, which indicates that the test strip is damaged, and the test strip is replaced with a new test strip for retesting no matter whether the detection line has the strip or not.

In some embodiments of the invention, a positive canine babesia antibody test indicates that the biological sample from the individual contains an canine babesia antibody, meaning that the individual has or has been infected with canine babesia.

In some embodiments of the invention, the biological sample is serum or plasma, or any other body fluid that may contain antibodies.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the BcMSA1 and BcSA1 proteins can induce and start an immune system of an organism to generate immune response, so that the BcMSA1 and BcSA1 fusion proteins are used for antibody detection, the detection sensitivity can be improved, cross reaction with other pathogens can be reduced, the specificity is strong, and the method has great clinical significance and wide application prospect.

The colloidal gold-labeled immunoassay method adopted by the invention is a novel analysis technology, has the characteristics of rapidness, simplicity, convenience, low cost, no pollution and no need of training, is more suitable for field detection compared with the traditional method, has the advantages of short color development time, no need of expensive instruments and the like, and has wide market prospect and application value.

Drawings

FIG. 1 shows the results of gel electrophoresis of the purification of Babesia canis BcMSA1-BcSA1 fusion protein. 1: loading the sample after cell disruption; 2: flow through; 3: 50mM Imidazole elution; 4: 0.5M Imidazole.

FIG. 2 shows a reagent diagram of a test strip according to one embodiment of the present invention. 1: a sample pad; 2: a gold label pad; 3: NC film; 31: a detection line (T-line); 32: a quality control line (line C); 4: filtering paper; 5: a base plate.

FIG. 3 is a diagram showing the results of detection using the test strip according to one embodiment of the present invention. T: detection line, C: and (4) quality control line.

Fig. 4 shows the overall results of the test strip of the present invention on clinical canine serum samples.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 construction of Babesia canis BcMSA1-BcSA1 fusion protein Gene expression vector

The Babesia canis BcMSA1 Gene was designed based on the protein sequence of NCBI Gene bank: KR 134351. According to the hydrophilicity and hydrophobicity to protein (https://web.expasy.org/protscale/) After analysis, a BcMSA1(1-70aa) sequence is selected from the region with the predicted higher hydrophilic content for fusion. The BcSA1 Gene was designed based on the protein sequence of NCBI Gene bank: KR 134352. According to the hydrophilicity and hydrophobicity to protein (https://web.expasy.org/protscale/) After analysis, BcSA1(10-250aa) sequences are selected from the regions with high predicted hydrophilic content for fusion.

The amino acid sequence of the recombinant protein fusion protein BcMSA1-BcSA1 is as follows (SEQ ID NO. 1):

MMLLFALSTLVTFAFCDGENTILLSNVEFHTPVSSVKLLKEYSSNQESMAVIMMLTEMPNTSGKLTDGKVHPHNFILIFQLLATMGNAQSTSSQENSRDGLREVLEYTNQLHNNYGSAVRKVTDKLKNEIDVYCKSTDDKGYYFANGSFGYFRKALNDSFNFRFQLLSNYNDYRKYKTRFQDTDDEAEKHVKYLKENLFDLFGTLSYMYFQCSHKCQKYNGGKWEEQSMNQSGSEVSKWLMGSNSAATDSVHFLGRDFSTSELTNIKGKELADKDRASLSDLIKYSGRGNLQHALFWMLFIGPWVDGKTGH

since the frequency of usage of synonymous codons is different for different species, this codon preference has an impact on the translation process. If a mRNA has many rare codons clustered, this will have a negative effect on the rate of ribosome movement and greatly reduce the protein expression level.

In the invention, escherichia coli is used as an expression system, in order to obtain higher expression efficiency and higher expression quantity, codon optimization is carried out during the expression of foreign protein, and the foreign protein is reversely translated into a nucleotide sequence, wherein the obtained nucleotide sequence is as follows (SEQ ID NO. 2):

ATGATGCTGCTGTTTGCGCTGAGCACCCTGGTGACCTTTGCGTTTTGCGATGGCGAAAACACCATTCTGCTGAGCAACGTGGAATTTCATACCCCGGTGAGCAGCGTGAAACTGCTGAAAGAATATAGCAGCAACCAGGAAAGCATGGCGGTGATTATGATGCTGACCGAAATGCCGAACACCAGCGGCAAACTGACCGATGGCAAAGTGCATCCGCATAACTTTATTCTGATTTTTCAGCTGCTGGCGACCATGGGCAACGCGCAGAGCACCAGCAGCCAGGAAAACAGCCGCGATGGCCTGCGCGAAGTGCTGGAATATACCAACCAGCTGCATAACAACTATGGCAGCGCGGTGCGCAAAGTGACCGATAAACTGAAAAACGAAATTGATGTGTATTGCAAAAGCACCGATGATAAAGGCTATTATTTTGCGAACGGCAGCTTTGGCTATTTTCGCAAAGCGCTGAACGATAGCTTTAACTTTCGCTTTCAGCTGCTGAGCAACTATAACGATTATCGCAAATATAAAACCCGCTTTCAGGATACCGATGATGAAGCGGAAAAACATGTGAAATATCTGAAAGAAAACCTGTTTGATCTGTTTGGCACCCTGAGCTATATGTATTTTCAGTGCAGCCATAAATGCCAGAAATATAACGGCGGCAAATGGGAAGAACAGAGCATGAACCAGAGCGGCAGCGAAGTGAGCAAATGGCTGATGGGCAGCAACAGCGCGGCGACCGATAGCGTGCATTTTCTGGGCCGCGATTTTAGCACCAGCGAACTGACCAACATTAAAGGCAAAGAACTGGCGGATAAAGATCGCGCGAGCCTGAGCGATCTGATTAAATATAGCGGCCGCGGCAACCTGCAGCATGCGCTGTTTTGGATGCTGTTTATTGGCCCGTGGGTGGATGGCAAAACCGGCCAT

a recombinant gene sequence is synthesized by the company of Biotechnology engineering (Shanghai) and is connected with pET30a plasmid to form a recombinant expression vector.

Example 2 expression of Babesia canis BcMSA1-BcSA1 fusion proteins

The babesia BcMSA1-BcSA1 fusion gene plasmid was transformed into Escherichia coli BL21, spread on LB plate containing 50. mu.g/mL kanamycin (Shanghai, cat # K0408), cultured overnight at 37 ℃, a single colony was picked up, cultured with 300mL of LB medium containing the same concentration of kanamycin at 37 ℃ until OD600 reached about 0.6, and induced to express with IPTG (Shanghai, cat # IB0168) at a final concentration of 1mM under the induction conditions: 25 ℃ and a rotation speed of 200rpm for 4 h. After induction, the culture broth was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.

Example 3 purification and renaturation of Babesia canis BcMSA1-BcSA1 fusion proteins

Crushing the thallus by 50mL of loading Buffer Binding Buffer (50mM Tris, 0.2M NaCl, pH8.0); then carrying out ultrasonic crushing for 100 times under the conditions of 600w, 2s of ultrasonic treatment and 5s interval; finally, the supernatant is collected by centrifugation at 12000rpm for 30min at 4 ℃, and the target protein is in the supernatant. Then, the mixture was further purified by Ni column chromatography, and the desired protein was eluted with Elution Buffer (50mM Tris, 0.2M NaCl, 0.5M Imidazole, pH 8.0). The target protein was detected by PAGE gel electrophoresis, and the results are shown in FIG. 1.

As is clear from FIG. 1, the purified fusion protein was very pure, and the purified recombinant protein was dialyzed against dialysis buffer (50mM Tris, 0.2M NaCl, pH8.0) and the dialysis solution was changed every 12 hours for 3 times. The protein solution after dialysis was taken out, filtered through a 0.22 μm filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.

Example 4 detection of Babesia canis antibodies by double antigen sandwich gold-labeling method

Preparation of 1 double-antigen sandwich gold-labeled detection strip

1.1 firing of colloidal gold

1000mL of ultrapure water is added into a triangular flask, the triangular flask is heated to boiling on a magnetic heating stirrer, then 4mL of 10% chloroauric acid (sigma) is added, 6mL of 10% trisodium citrate solution is added, the heating and boiling are continued for 5min, then the triangular flask is cooled to room temperature, colloidal gold is filtered by a 0.22 mu m filter, and the triangular flask is placed at 4 ℃ for standby.

1.2 labeling of recombinant Babesia canis BcMSA1-BcSA1 fusion proteins

Putting 100mL of colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring₂CO₃Adjusting pH of the gold water to 10.5, stirring, adding 1.5mg purified recombinant canine Babesia BcMSA1-BcSA1 fusion protein, stirring at room temperature for 15min, adding 1mL 10% BSA solution, stirring at room temperatureAfter stirring for 15min, the mixture was centrifuged at 12000rpm for 10min, the supernatant was carefully aspirated and discarded, and the precipitate was diluted to 1mL with a gold-labeled diluent (20mM Tris, 1% BSA, 0.03% Proclin300, pH8.0), which was a labeled recombinant canine Babesia babesi BcMSA1-BcSA1 fusion protein colloidal gold complex.

1.3 murine IgG markers

Putting 100mL of colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring₂CO₃The pH of the gold solution was adjusted to 7.0, and after stirring, 1mg of mouse IgG (Hangzhou Longji Biotechnology Co., Ltd., cat # AS00901) was added, and after stirring at room temperature for 15min, 1mL of 10% BSA solution was added, and after stirring at room temperature for 15min, centrifugation was carried out at 12000rpm for 10min, the supernatant was carefully aspirated and discarded, and the precipitate was diluted to 1mL with a gold-labeled diluent (20mM Tris, 1% BSA, 0.03% Proclin300, pH8.0) to obtain a volume of 1mL, which was a labeled mouse IgG colloidal gold complex.

Diluting the gold-labeled compound by 100 times with a gold-labeled diluent, mixing with the diluted canine Babesia BcMSA1-BcSA1 fusion protein colloidal gold compound in the step 1.2, soaking the mixture in glass fibers, and drying the mixture at 37 ℃ for 4 hours to obtain the gold-labeled pad.

1.4 Point membranes of recombinant Babesia canis BcMSA1-BcSA1 fusion proteins

Diluting the purified BcMSA1-BcSA1 fusion protein to 0.9mg/mL by using a spotting diluent (50mM Tris, 2% sucrose, pH8.5) as a detection Line (Test-Line, T Line) of a colloidal gold Test strip, diluting goat anti-mouse IgG (Hangzhou Longji Biotechnology Co., Ltd., a cargo number: PS00901) to 0.3mg/mL by using the same diluent as a quality Control Line (Control-Line, C Line) of the colloidal gold Test strip, scribing the two diluted solutions onto a nitrocellulose membrane, and drying at 37 ℃ overnight.

1.5 Assembly of test paper strip for detecting Babesia canis antibody by double-antigen sandwich gold-labeling method

The gold label pad, the coated raw materials and a mounting base plate such as a nitrocellulose membrane (NC membrane) polyester plate, filter paper, a sample pad and the like are assembled into the test paper strip for detecting the canine Babesia antibody by the double antigen sandwich method. The specific installation manner is shown in fig. 2: the sample pad 1, the gold label pad 2, the NC membrane 3, and the filter paper 4 are mounted on the base plate 5, respectively. Wherein a part of the sample pad 1 is superposed and pressed on the gold label pad 2, a part of the gold label pad 2 is superposed and pressed on the NC membrane 3, and a part of the filter paper 4 is superposed and pressed on the NC membrane 3. The NC membrane 3 is divided into a test area and a quality control area, the test area is provided with a detection line 31(T line), the quality control area is provided with a quality control line 32(C line), the detection line 31 is close to the gold mark pad 2, and the quality control line 32 is close to the filter paper 4.

Further, the assembled test paper strip is cut into strips with the length of 3mm by a slitter, and then the strips are put into a specially made plastic card, so that the mature detection reagent card is formed.

Detection of Babesia antibody test paper strip/card by 2 double-antigen sandwich gold-labeled method

Adding 90 μ L of sample (canine serum, plasma) to be detected to sample application position (S), standing at room temperature for 10min, and determining the result according to the following criteria (as shown in FIG. 3):

two strips appear, wherein one strip is positioned in a quality control area, and the other strip is positioned in a test area and is positive;

secondly, only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;

and thirdly, the quality control line has no strip, which indicates that the test strip is damaged, and the test strip is replaced with a new test strip for retesting no matter whether the detection line has the strip or not.

3 detection result of test strip/card for detecting babesia canis antibody by double-antigen sandwich gold-labeling method

A total of 25 Babesia positive canine sera (sample No. 1-25) and 47 normal uninfected and non-immunized canine sera (sample No. 26-72) were tested, wherein the test results were positive on both lines T and C, and negative on only one line C.

The results are shown in table 1: 13 positive samples were detected in 25 positive sera, 2 negative samples (sample No. 9 and sample No. 17) were missed, and 1 false positive sample (sample No. 67) appeared in 47 negative sera.

TABLE 1 Babesia canis antibody test results

From this, it was found that the sensitivity and specificity of the sample detection were 92% and 97.9%, respectively, and the overall coincidence rate was 95.8%, as shown in fig. 4.

The results show that the recombinant canine babesia BcMSA1-BcSA1 fusion protein has very high sensitivity and specificity when used for detecting the canine babesia, can be used as a raw material for preparing a canine babesia antibody detection test strip, and can be widely applied to clinical detection.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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<120> Babesia canis BcMSA1-BcSA1 recombinant protein, and preparation method and application thereof

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gaatatagca gcaaccagga aagcatggcg gtgattatga tgctgaccga aatgccgaac 180

accagcggca aactgaccga tggcaaagtg catccgcata actttattct gatttttcag 240

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ctgcgcgaag tgctggaata taccaaccag ctgcataaca actatggcag cgcggtgcgc 360

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atgggcagca acagcgcggc gaccgatagc gtgcattttc tgggccgcga ttttagcacc 780

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Claims (10)

1. A Babesia canis BcMSA1-BcSA1 recombinant protein comprising SEQ ID NO.

1.

2. The recombinant protein according to claim 1, consisting of the amino acid sequence shown in SEQ ID No. 1.

3. A gene encoding the recombinant protein of claim 1 or 2, comprising the nucleotide sequence of SEQ id No. 2.

4. An expression vector comprising the gene of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. A method for producing a recombinant protein according to claim 1 or 2, comprising the step of inducing the host cell according to claim 3 to express the protein.

7. The method of claim 6, wherein the expression vector is pET30a and the host cell is E.coli.

8. The method of claim 7, wherein the step of inducing the host cell to express the protein comprises:

s1, culturing the Escherichia coli in LB medium containing 50. mu.g/mL of kanamycin at 37 ℃,

s2, when the culture solution OD600 of the escherichia coli is 0.5-0.7, the induction expression is carried out by IPTG with the final concentration of 1mM, and the induction conditions are as follows: the rotation speed is 200rpm at 25 ℃ for 4 h;

s3, centrifuging the culture solution at 4 ℃ and 7000rpm for 10min, and collecting thalli;

s4, crushing the thallus by using a Buffer Binding Buffer;

s5, carrying out ultrasonic disruption on thalli under the conditions of: 600w, ultrasonic for 2s, and interval of 5s, and 80-120 times in total;

s6, centrifuging at 12000rpm at 4 ℃ for 30min, and collecting supernatant, wherein the recombinant protein is in the supernatant.

9. Use of the recombinant protein of claim 1 or 2 in the preparation of a kit for detecting babesia canis antibodies.

10. A kit for detecting babesia canis antibody, comprising the recombinant protein of claim 1 or 2.

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