阅读说明：本技术 一种环形泰勒虫ta04380截短蛋白及其应用 (Cyclic Theileria taenii TA04380 truncated protein and application thereof ) 是由 刘军龙 曹天行 李志� 王锦明 李有全 关贵全 罗建勋 殷宏 于 2021-07-29 设计创作，主要内容包括：本发明属于分子生物学领域,具体涉及一种环形泰勒虫TA04380截短蛋白及其应用。本发明首先公开了一种环形泰勒虫TA04380截短蛋白,并公开了编码所述环形泰勒虫TA04380截短蛋白的基因；其次,本发明发现,所述的环形泰勒虫TA04380截短蛋白与东方泰勒虫、中华泰勒虫、双芽巴贝斯虫以及牛巴贝斯虫等阳性血清不发生抗原交叉反应,可用于热带泰勒虫病的诊断靶标抗原,具有潜在应用；并且可用于热带泰勒虫病的特异性检测,具有重要的科学和应用价值。(The invention belongs to the field of molecular biology, and particularly relates to a loop theileria TA04380 truncated protein and application thereof. The invention firstly discloses a theileria annulata TA04380 truncated protein and discloses a gene for coding the theileria annulata TA04380 truncated protein; secondly, the invention discovers that the truncated protein of the theileria annulata TA04380 does not have antigen cross reaction with positive serum such as theileria orientalis, theileria sinensis, Babesia gemmifera, Babesia bovis and the like, can be used as a diagnosis target antigen of tropical theileriosis, and has potential application; and can be used for specific detection of theileriosis tropicalis, and has important scientific and application values.)

1. The theileria annulata TA04380 truncated protein is characterized in that the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID NO. 1.

2. The application of the theileria annulata TA04380 protein or the theileria annulata TA04380 truncated protein as a target antigen for diagnosing the tropical theileria diseases, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID No. 1.

3. The application of the theileria annulata TA04380 protein or the theileria annulata TA04380 truncated protein in preparing the kit for detecting the antibody of the theileria tropicalis is disclosed, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID No. 1.

4. A test strip coated with a theileria annulata TA04380 protein or a theileria annulata TA04380 truncated protein is disclosed, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown in SEQ ID NO. 1.

5. The kit for detecting the antibody against theileriosis tropicalis is characterized by comprising a theileria annulata TA04380 protein or a theileria annulata TA04380 truncated protein, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID No. 1.

6. A gene encoding the truncated theileria annulata TA04380 protein of claim 1, wherein the gene sequence is as shown in SEQ ID No. 2.

7. A recombinant vector comprising the gene of claim 6.

8. A recombinant cell comprising the gene of claim 6.

9. A method for preparing the theileria annulata TA04380 truncated protein of claim 1, wherein the method comprises the following steps: the gene of claim 6 is constructed in a prokaryotic expression vector, and is transferred into a host cell to be expressed to obtain the annular theileria TA04380 truncated protein.

10. The method of claim 9, comprising the steps of:

(1) construction of recombinant vectors: constructing the gene of claim 6 in pET-30a (+) prokaryotic expression vector to obtain recombinant vector pET30a-TA 04380;

(2) construction and inducible expression of recombinant cells: transforming the recombinant vector pET30a-TA04380 in the step (1) into Rosetta (DE3) competent cells, and screening to obtain recombinant cells;

(3) expression and purification: and (3) inducing the recombinant cell expression by IPTG, and purifying to obtain the circular theileria TA04380 truncated protein.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a theileria annulata TA04380 truncated protein, a gene, a vector and a host cell containing the gene, a test strip containing the theileria annulata TA04380 truncated protein, a production method of the theileria annulata TA04380 truncated protein, and application of the theileria annulata TA04380 truncated protein in preparation of a tropical theileria antibody detection kit.

Background

The annular theileriosis is also called Tropical theileriosis (Tropical theileriosis), which is a hematozoonosis that is caused by infection of bovine lymphocytes, dendritic cells, macrophages and erythrocytes by the annular theileria transmitted by hyalomma glaucoides to cause symptoms such as enlargement of lymph nodes and anemia of animals. The vitreous pedicularis, the small vitreous pedicularis and the Asian vitreous pedicularis have been proved to be the main transmission vectors of the annular theileria. The tropical theileriosis is mainly distributed in northern Africa, coastal areas of Mediterranean sea, Asia and other parts, and is mainly distributed in Xinjiang and northern areas in China, the tropical theileriosis clinically shows high fever, anemia, inappetence, emaciation and body surface lymph node swelling, and is usually passed through in an acute way, the infection rate and the death rate are high, the productivity and the health of livestock are seriously damaged, and the tropical theileriosis is one of important diseases affecting the development of the cattle industry. Hybrid cattle breeds, in contrast, are more susceptible to the disease than cattle of the local breed. The OIE International pestilence Bureau ranks the disease as a B-type epidemic disease, and the OIE International pestilence Bureau ranks the disease as a second-type epidemic disease.

The current routine diagnosis of theileriosis tropicalis relies mainly on the microscopic examination of blood smears and lymph node biopsy smears. In most cases, pathogens are not observed when parasitemia tends to be low. In addition, it is difficult to distinguish other species of theileria in mixed infections. Aiming at the condition of mixed infection, the application of molecular biology diagnosis technology can well distinguish different pathogens, but the methods have the defects of strong dependence on corresponding instruments, time consumption, labor consumption and the like.

Serological diagnostic methods, such as enzyme-linked immunosorbent assay (ELISA), have the characteristics of programmable operation, standardized result judgment and the like, and are widely applied to epidemiological investigation of various diseases. However, there is no serological detection method with good specificity for theileriosis annulata, and most of the currently known theileria annulata proteins have obvious cross reaction with other theileria or Babesia positive serum, such as the annular theileria MPSP protein, the annular theileria Dna J protein, the annular theileria TA13815 protein and the like, so that the serological detection method has poor specificity. Therefore, a circular Theileria annulata antigen which can be purified and expressed, has high specificity and good reactogenicity is found, and the circular Theileria annulata antigen plays an important role in diagnosis and epidemiological investigation of theileria tropicalis.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide a theileria annulata antigen with high specificity and good reactogenicity, which is used for specific diagnosis and epidemiological investigation of theileria tropical. The method specifically comprises the following steps:

in a first aspect, the invention provides a theileria annulata TA04380 truncated protein, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID NO. 1.

In a second aspect, the invention provides an application of a theileria annulata TA04380 protein or a theileria annulata TA04380 truncated protein as a target antigen for diagnosing tropical theileriosis, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID No. 1.

In a third aspect, the invention provides an application of a theileria annulata TA04380 protein or a theileria annulata TA04380 truncated protein in preparation of a kit for detecting an antibody against theileriosis tropicalis, wherein an amino acid sequence of the theileria annulata TA04380 truncated protein is shown as SEQ ID No. 1.

In a fourth aspect, the invention provides a test strip coated with a theileria annulata TA04380 protein or a theileria annulata TA04380 truncated protein, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown in SEQ ID No. 1.

Preferably, the test strip comprises a colloidal gold test strip and a colloidal selenium test strip.

In a fifth aspect, the invention provides a kit for detecting an antibody against theileriosis tropicalis, which contains theileria annulata TA04380 protein or theileria annulata TA04380 truncated protein, wherein the amino acid sequence of the theileria annulata TA04380 truncated protein is shown in SEQ ID No. 1.

Preferably, the kit for detecting the antibody against theileriosis tropicalis comprises an enzyme label plate coated with the annular theileria TA04380 protein or the annular theileria TA04380 truncated protein, or comprises a test strip coated with the annular theileria TA04380 protein or the annular theileria TA04380 truncated protein.

In a sixth aspect, the invention provides a gene encoding the truncated protein of theileria annulata TA04380 described in the first aspect, and the sequence of the gene is shown as SEQ ID NO. 2.

In a seventh aspect, the present invention provides a recombinant vector comprising the gene of the above sixth aspect.

In an eighth aspect, the present invention provides a recombinant cell comprising the gene of the above sixth aspect.

In a ninth aspect, the present invention provides a method for preparing the theileria annulata TA04380 truncated protein, wherein the method comprises the following steps: the gene of the sixth aspect is constructed in a prokaryotic expression vector and transferred into a host cell to be expressed to obtain the annular theileria TA04380 truncated protein.

Preferably, the method comprises the steps of:

(1) construction of recombinant vectors: constructing the gene in the sixth aspect into a pET-30a (+) prokaryotic expression vector to obtain a recombinant vector pET30a-TA 04380;

(2) construction and inducible expression of recombinant cells: transforming the recombinant vector pET30a-TA04380 in the step (1) into Rosetta (DE3) competent cells, and screening to obtain recombinant cells;

(3) expression and purification: and (3) inducing the recombinant cell expression by IPTG, and purifying to obtain the circular theileria TA04380 truncated protein.

The invention has the beneficial effects that: the invention provides a loop theileria TA04380 truncated protein, and the loop theileria TA04380 truncated protein is obtained through expression and purification; the theileria annulata TA04380 truncated protein has good biological activity, has no cross-reactivity with other piriformis positive serum, can effectively detect a corresponding antibody against theileria annulata in the blood of the cattle, and can be used as a candidate target molecule for serological diagnosis and detection of theileria tropicalis; the circular Theileria TA04380 truncated protein can be used for preparing a kit for detecting the antibody of theileria tropicalis; the ELISA kit prepared by the truncated protein of the theileria annulata TA04380 can be used for detecting the antibody of theileria tropicalis, and has high sensitivity, specificity and accuracy and wide application value.

Drawings

FIG. 1 is an amplification electrophoretogram of a truncated protein gene fragment of theileria annulata TA04380, wherein M: DNA molecular weight standard; 1: a target gene amplified from the theileria annulata cDNA;

FIG. 2 is an electrophoretogram of the double restriction enzyme products of the recombinant vector pET30a-TA04380 of the present invention, wherein M: DNA molecular weight standard; 1: the product of double enzyme digestion of the pET30a-TA04380 recombinant vector;

fig. 3 is an electrophoretogram of the expression product of the truncated protein of theileria annulata TA04380, wherein M: protein molecular mass standard; 1: (ii) ultrasonicated precipitation; 2: (iii) sonicated supernatant; 3: purified target protein;

figure 4 is an identification of the reactogenicity of the taylophilus annulatus TA04380 truncated protein, where M: protein molecular mass standard; 1: a circular theileria spp positive serum; 2: gemma babesia positive serum; 3: babesia bovis positive serum; 4: a positive serum of theileria sinensis; 5: (ii) oriental theileria positive serum; 6: healthy bovine serum;

FIG. 5 indirect ELISA assay results.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention firstly discovers that the annular theileria TA04380 protein can not be directly expressed and purified by directly using the annular theileria TA04380 gene, and the 1bp-636bp nucleotide sequence of the annular theileria TA04380 is selected and constructed in a pET-30a (+) prokaryotic expression vector and is transferred into a Rosetta (DE3) competence, so that the annular theileria TA04380 truncated protein can be obtained by induced expression. The theileria annulata TA04380 truncated protein has good biological activity, has no cross-reactivity with other piriformis positive serum, can effectively detect a corresponding antibody against theileria annulata in bovine blood, and can be used as a candidate target molecule for serological diagnosis and detection of tropical theileriosis; and the circular Theileria TA04380 truncated protein can be used for preparing a kit for detecting the antibody of theileria tropicalis.

The present invention will be described in detail with reference to examples.

Example 1 purification of merozoites of theileria annulata

(1) The cattle with the annular Theileria infection rate of about 10% are well kept, carotid artery bleeding is carried out, blood flows into a conical flask prepared in advance and containing 20% of sodium citrate and glass beads, and the conical flask is shaken continuously to prevent blood coagulation.

(2) The blood is divided into 50mL centrifuge tubes, centrifugal for 10min at 3500rpm, the supernatant is discarded, and 1 XPBS is added for washing once;

(3) centrifuging the liquid at 3500rpm for 10min, discarding supernatant, removing leukocyte layer as much as possible, adding 2 times volume of 1 × PBS, mixing, adding into leukocyte filter, and filtering (this step requires ice operation);

(4) subpackaging the filtered erythrocyte suspension in a 50mL centrifuge tube, centrifuging at 3500rpm for 10min, discarding the supernatant, resuspending the precipitated erythrocytes with 3 times of 7% glycerol (prepared by 1 × PBS) for precipitation, shaking for several times, standing at room temperature for 20min, centrifuging at 3500rpm for 10min, discarding the supernatant;

(5) pouring all erythrocyte precipitates into a conical flask, adding 1 XPBS with 20 times volume, quickly shaking the conical flask, and standing at room temperature for 5min to lyse erythrocytes;

(6) split charging the lysate into 50mL centrifuge tubes, centrifuging at 12000rpm for 30min, discarding the supernatant, combining all precipitates into 50mL centrifuge tubes, washing with 1 XPBS for several times until the supernatant is clear, and split charging, wherein the supernatant in the step can also be split charged into 50mL centrifuge tubes for later use;

(7) the sediment can be subpackaged into 1.5mL of EP tubes for freezing storage at-70 ℃, and part of liquid can be taken for blood smear analysis.

Example 2 extraction of Total RNA of theileria annulata and Synthesis of Single-stranded cDNA

(1) Taking 200 mu L of merozoite purified in example 1, adding 1mL of Trozol, and repeatedly blowing to crack cells;

(2) centrifuging at 12000rpm at 4 deg.C for 10min, and transferring the supernatant to a new centrifuge tube;

(3) adding 200 mu L chloroform into the supernatant, covering a centrifugal tube cover, violently shaking L5 s, and incubating for 5min at room temperature of 15-30 ℃ to completely separate nucleoprotein in the sample;

(4) the above liquid was centrifuged at 12000rpm for 15min at 4 ℃ and the mixture was divided into three layers: transferring the upper water phase to a new centrifuge tube carefully, adding 500 μ L isopropanol, reversing and mixing, and incubating at 15-30 deg.C for 10 min;

(5) centrifuging the mixed liquid at 4 ℃ and 12000rpm for 10min, removing the supernatant, adding 1mL of 75% ethanol to wash total RNA of the strip, and gently reversing the heavy suspension and precipitation;

(6) centrifuging at 4 ℃ and 7500rpm for 5min, removing supernatant, and drying on an ultra-clean workbench for 5-8 min;

(7) finally 20. mu.L RNase-Free dH was added₂O-soluble RNA, 55 ℃EIncubating at 60 deg.C for 10min, and immediately performing reverse transcription or storing at-70 deg.C;

(8) removing genome DNA reaction: 5 XgDNA Eraser Buffer, 2. mu.L; gDNA Eraser, 1. mu.L; total RNA, 1. mu.g; RNase-Free Water to 10. mu.L; reacting at 42 ℃ for 2 min;

(9) reverse transcription reaction: 10 mu L of the reaction solution in the step (8); PrimeScript RT Enzyme Mix, 4. mu.L; RT Primer Mix, 0.5. mu.L; 5 XPrimeScript Buffer, 1. mu.L; RNase-Free dH₂O, 4.5 μ L; total, 20 μ L; reacting at 37 deg.C for 15min, and storing at 85 deg.C for 5s and-20 deg.C.

Example 3 cloning of the Theileria annulata TA04380 Gene and bioinformatic analysis thereof

1. Bioinformatics analysis

The transmembrane region distribution and signal peptide sequence of the TA04380 gene-encoded protein were first analyzed using TMHMM (http:// www.cbs.dtu.dk/services/TMHMM /) and SignaiP online software, resulting in the absence of signal peptide and the presence of four transmembrane regions in the region 212aa-413 aa.

2. Construction of cloning vector PGEM-Teasy-TA04380

According to the published TA04380 nucleotide sequence (NCBI: XM-947519.1) of theileria annulata on NCBI/GenBank, 1 pair of amplification primers are designed for the TA04380 gene (1bp-636bp) by using Primer 5.0 software, and the upstream and the downstream carry EcoR I and Hind III restriction sites respectively:

TA 04380-F: 5 '-3' ccgGAATTCatgttaaaattgtacaaaacagtaaaatatg (shown in SEQ ID NO. 3); TA 04380-R: 5 '-3' cccAAGCTTcaagtttaagcagtcatgaacagtcaaaaac (shown in SEQ ID NO. 4);

performing PCR amplification by taking the annular theileria cDNA as a template, wherein the total reaction volume is 50 mu L: EX taq DNA polymerase, 25 μ L; TA04380-F (10. mu.M), 2. mu.L; TA04380-R (10. mu.M), 2. mu.L; cDNA, 2. mu.L; RNase-free Water, 19. mu.L; the reaction conditions are as follows: at 95 ℃ for 3 min; at 95 ℃ for 30 s; 30s at 55 ℃; 72 ℃ for 1 min; 35 cycles; 72 ℃ for 10 min; storing at 4 ℃.

Carrying out nucleic acid gel electrophoresis on the target fragment obtained by amplification, wherein the result is shown in figure 1, and a 636bp (shown in SEQ ID NO. 2) target band is obtained by amplification; purifying by a PCR product purification kit, connecting with pGEM-T Easy vector for 2h at room temperature, transforming to 50 mu L DH5 alpha competent cells, carrying out ice bath for 30min, carrying out heat shock for 90s, then carrying out ice bath for 2min, quickly adding 500 mu L SOC culture medium for 1h at 37 ℃, coating on an LB plate containing ampicillin for culture, selecting a single colony for culture, carrying out PCR identification, expanding and culturing a positive colony, extracting a plasmid, sending to a biological engineering (Shanghai) member company Limited for sequencing, and obtaining a gene sequence (shown in SEQ ID NO. 2) for expressing the TA04380 truncated protein of theileria annulata.

Example 4 construction of recombinant prokaryotic expression vectors and inducible expression of fusion proteins thereof

Construction of pET30a-TA04380 recombinant expression plasmid

The positive plasmid pGEM-T easy-TA04380 and the expression vector pET30a (+) were double-digested with restriction enzymes EcoR I and Hind III, respectively. Total reaction 50 μ L: the endonucleases EcoR I and Hind III were 2. mu.L, 10 XBuffer, 5. mu.L each; pGEM-T easy-TA04380 plasmid, 2. mu.g; RNase-free Water to 50. mu.L; reacting at 37 ℃ for 2 h;

after the completion of the digestion reaction, the digestion effect was observed by 1.5% agarose gel electrophoresis, and the digestion product was recovered. The target gene fragment after the gel recovery and the pET-30a (+) vector were mixed in proportion and ligated overnight at 16 ℃. The ligation product is transformed into DH5 alpha competent cells and spread on an LB plate containing kanamycin, a monoclonal bacterium is selected to be subjected to PCR identification, a positive plasmid is sent to the company Limited in the Biotechnology engineering (Shanghai) for sequencing, after the positive plasmid pET30a-TA04380 is subjected to double enzyme digestion by EcoR I and Hind III, the linear fragment of pET30a can be seen to be 5244bp, and the target band of TA04380 is 636bp (as shown in FIG. 2, and the gene sequence is shown as SEQ ID NO. 2).

Inducible expression of TA04380 truncated protein

After transformation of the recombinant plasmid pET30a-TA04380 into Rosetta (DE3) competent cells, single colonies were picked for overnight activation as described in 1: inoculating 100% of the strain into fresh LB culture medium containing kanamycin, and obtaining bacterial liquid OD₆₀₀When the value reaches 0.6-0.8, adding 1mM IPTG to the final concentration, inducing expression at 37 ℃ for 8h, collecting all bacteria liquid, centrifuging at 12000rpm for 5min, discarding supernatant, adding PBS to resuspend the bacteria, and repeatingAnd (3) carrying out ultrasonication (2 s of ultrasonication, 3s of interval and 150W) for 40min after freeze thawing until the mixture is clear and transparent. After the lysate after ultrasonication was centrifuged at 12000rpm for 5min, the supernatant and the precipitate were subjected to SDS-PAGE, respectively. The results showed that the TA04380 truncated protein was mainly expressed in the form of inclusion bodies, the molecular weight of the truncated protein was about 32.7kDa, and the results were consistent with the expectations (as shown in FIG. 3, the protein sequence is shown in SEQ ID NO. 1).

Purification of TA04380 recombinant protein

(1) Equilibration of nickel affinity chromatography column: first, the nickel column was washed three times with 5mL of 1 × PBS, and the nickel column was denatured with 5mL of Denaturing Binding Buffer (pH 7.8);

(2) binding the recombinant protein to a nickel affinity chromatography column: after the sonicated pellet was dissolved in 9mL of Denaturing Binding Buffer (pH 7.8) and bound to Ni-NTA agarose at 4 ℃ overnight, the flow-through was collected on a column.

(3) Washing the hybrid protein: adding 5mL of a degrading Binding Buffer (pH 7.8), a degrading Washing Buffer 1(pH 6.0) and a degrading Washing Buffer 2(pH 5.3) into the Ni-NTA Column, Washing the nickel affinity chromatography Column for three times, and collecting three flow-through liquids;

(4) eluting the target protein: to the nickel affinity column, 10mL of a Denaturing Elution Buffer (pH 4.0) was added and the column was passed through, and all the effluent was collected in a 1.5mL centrifuge tube (see FIG. 3).

Example 5 reactogenicity characterization of the truncated TA04380 protein

The purified truncated protein was subjected to SDS-PAGE, transferred to PVDF membrane using an electrotransfer at 25V for 25min and incubated overnight at 4 ℃ with 5% BSA blocking agent, and then incubated with 5% BSA at a rate of 1: diluting at 50 proportion, adding circular Theileria sinensis positive serum, Oriental Theileria sinensis positive serum, Chinese Theileria sinensis positive serum, Babesia gemmifera positive serum, Babesia bovis positive serum and healthy bovine serum, and incubating at room temperature for 1 h. Wash 4 times with TBST, 5min intervals each time. AP-labeled rabbit anti-bovine IgG (1: 8000) was added as a secondary antibody and incubated at room temperature for 1h, washed 4 times with TBST, with 5min intervals, and observed using BCIP/NBT color reagent. The western blotting result is shown in fig. 4, which shows that the truncated protein of theileria annulata TA04380 has specific reaction to the positive serum of theileria annulata TA04380, and the band is single, but has no reaction to the positive serum of other theileria annulata; the T.annulata TA04380 truncated protein has good specificity and reactogenicity, and can be used for specificity detection of T.annulata.

Example 6TA04380 truncated protein for Indirect ELISA diagnosis of Toileriosis annulata

(1) Antigen coating: the purified annular theileria reinhardtii MPSP protein, SPAG protein, TA13815 protein and TA04380 truncated protein are diluted to 20 mu g/mL by carbonate buffer solution, each well is 100 mu L, and the protein is coated overnight at 4 ℃.

(2) Primary antibody incubation: after three washes with PBST, the serum dilutions were run at 1: 100 dilutions of the circular theileria, the oriental theileria, the Chinese theileria, the gemma babesi, the bovine babesia positive serum and the healthy bovine negative serum, 100 μ L per well with serum dilution as a blank control, and incubation at 37 ℃ for 1 h.

(3) And (3) secondary antibody incubation: after three washes with PBST, 1: mu.L of 5000-diluted rabbit anti-bovine IgG secondary antibody (HRP) was incubated at 37 ℃ for 1 h.

(4) Color development: after three PBST washes, 100. mu.L of TMD substrate was added to each empty chamber and developed for 10min in the dark, and the reaction was stopped by adding 100. mu.L of sulfuric acid (2M).

(5) Reading: readings were taken at a wavelength of 450 nm.

The result is shown in fig. 5, the annular theileria MPSP protein can detect various positive serums of the annular theileria, the double-bud Babesia and the Chinese theileria as positive serums, has poor specificity, can detect the positive serums of healthy bovine serum and has low accuracy; the SPAG protein of the theileria annulata can detect various positive serums of the theileria annulata, the Theileria orientalis and the Babesia gemmifera as positive and has poor specificity; the TA13815 protein can be detected as positive to multiple positive serums of the annular theileria and the bovine babesia and has poor specificity; the TA04380 truncated protein can only detect positive serum of theileria annulata, and has good specificity.

In conclusion, the theileria annulata TA04380 truncated protein can specifically detect the positive serum of theileria annulata, can be used as a target antigen for serological diagnosis and detection of theileria tropicalis, and can realize specific detection of theileria tropicalis; has high sensitivity, specificity and accuracy.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> circular theileria TA04380 truncated protein and application thereof

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Ser His Phe Asn Ser Ile Asn Tyr Thr Ser Pro Asn Leu Pro Thr Thr

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Leu Ser Asn Ile Pro Phe Leu Val Lys Pro Cys Phe Val Phe Ser Asp

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Asn Ser Ser Ile Lys Tyr Gly Leu Asn His Pro Tyr Leu Arg Phe Phe

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

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Thr Thr Asn Glu Glu Asn Ile Asn Asn Glu Glu Asp Ala Glu Ile Ala

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Gln Asn Asp Val Asn Asp Arg Phe Val Asn Ser Asn Phe Ser Pro Asp

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Asp Leu Asn Glu Lys Val Tyr Asn Val Lys Asn Lys Thr Gly Asn Glu

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Leu Ile Glu Glu Leu Glu Glu Asp Leu Lys Ile Ser Gly Glu Ser Leu

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Gly Phe Glu Glu Leu Phe Glu Ile Pro Asp Tyr His Gln Tyr Leu Met

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Thr Arg Ile Met Asp Leu Asn Gly Asn Lys Arg Ser Ile Leu Gln Arg

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Phe Leu Pro Val Glu Tyr Val Gln Gln Met Phe Leu Thr Val His Asp

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Cys Leu Asn Leu

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atgttaaaat tgtacaaaac agtaaaatat gtaaataata attattttgt tataaataat 60

gttttctgtc ctggaaatac aattaaaaga caaatttcac actttaattc aataaattat 120

acatcaccta accttcctac aactctttct aatatacctt tcttagttaa accttgtttt 180

gtatttagtg ataatagctc tataaaatat ggcctcaatc acccttattt gagattcttc 240

tcaaacctta atgattcaag tatttactca aaccaaaata ataccaatac aaccaatgaa 300

gaaaatataa ataatgaaga agatgctgaa atagcccaga atgatgtaaa tgaccgattt 360

gtcaactcca attttagccc ggatgatctc aatgaaaagg tgtataatgt taaaaacaag 420

actggaaatg aattaattga ggaattggaa gaggacttga aaatttcagg agaaagtttg 480

ggttttgagg aattatttga aattccagac taccaccaat atctgatgac tagaatcatg 540

gacttgaatg gtaataaaag gagtatttta cagcgttttt tacccgtaga atatgtccag 600

cagatgtttt tgactgttca tgactgctta aacttg 636

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ccggaattca tgttaaaatt gtacaaaaca gtaaaatatg 40

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cccaagcttc aagtttaagc agtcatgaac agtcaaaaac 40