AU2020101749A4 - Recombinant protein for rapid detection of Toxoplasma gondii, preparation method and use thereof - Google Patents

Abstract

The present invention belongs to the technical field of bioengineering, and relates to a method for preparing a Toxoplasma gondii recombinant antigen and diagnostic use thereof. In the present invention, dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 are analyzed and selected to construct a prokaryotic expression vector and achieve fusion expression by genetic engineering technology; the prokaryotic expression vector is transformed into Escherichia coli BL21(DE3) to screen out a recombinant expression strain; fusion protein G267 is expressed and purified by the expression strain; a colloidal gold method for diagnosing T. gondii is established by the fusion protein. 1/1 - -C line -- T line 1:32000 1:16000 1:8000 1:4000 12000 FIG. 1

Description

1/1

- -C line -- T line

1:32000 1:16000 1:8000 1:4000 12000

FIG. 1

RECOMBINANT PROTEIN FOR RAPID DETECTION OF TOXOPLASMA GONDII, PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

The present invention relates to the technical field of bioengineering, and in particular to a method for preparing a Toxoplasma gondii recombinant antigen and use thereof in the rapid detection of T. gondii.

BACKGROUND

Toxoplasmosis is an infection in human and animals caused by T. gondii. T. gondii is a small parasite with simple structure. Cats and other felids are definitive hosts of T. gondii. T. gondii is parasitic in small intestinal epithelial cells of these animals and forms oocysts, which are passed out of the body in feces; if other mammals and birds eat oocysts, these animals are infected, and oocysts develop into encysts in body tissues thereof. Both oocysts and encysts are different developmental stages of T. gondii. T. gondii is not "picky", but the parasite only reproduces in other animals but definitive hosts in an asexual manner, and but fails to spread offspring thereof to the outside. Toxoplasmosis is a zoonosis, which is most harmful to pregnant women and fetuses. Therefore, early diagnosis of toxoplasmosis is extremely important.

So far, there are a plurality of methods for detecting toxoplasmosis in China:

1. Direct microscopic examination: Extract blood, bone marrow or cerebrospinal fluid (CSF), hydrothorax and ascites, sputum, bronchoalveolar lavage fluid (BALF), aqueous humor, or amniotic fluid from a patient to make a smear; alternatively, make lymph node, muscle, liver and placenta biopsies; after Wright-Giemsa staining, microscopically find trophozoites or encysts, but positive rate is not high.

2. Animal inoculation or tissue culture: Inoculate a body fluid sample or a tissue suspension into a mouse intraperitoneally to induce an infection and find out pathogens; if passage 1 is negative, three passages should be needed in a blind manner. Alternatively, T. gondii is separated and identified by tissue (monkey or porcine kidney cells) culture. The method is time-consuming, tedious, and inconvenient for early rapid screening, except for excellent specificity.

3. DNA hybridization: Chinese researchers first used a 32P-labeled probe containing a T. gondii specific DNA sequence to molecularly hybridize to peripheral blood cells or tissue DNAs of a patient, and specific hybridizing bands or dot blots indicated a positive reaction. The method features excellent sensitivity and high specificity, but fails to achieve large-scale promotion in community-level medical institutions due to the limitation of the instrument.

I

4. Intradermal test: Using a peritoneal fluid of an infected mouse or an embryonic fluid of a chicken embryo as an antigen, delayed tuberculin reaction occurs usually, which can be used in epidemiological investigation. The method is not widely used so far.

5. Serological test: In recent years, serological diagnosis has been developed, by which T. gondii infection can be determined by detecting T. gondii antigens in blood, CSF, and urine. The method is highly sensitive and easy to operate and provides a basis for early diagnosis. It should be noted that the sensitivity and specificity of the serological diagnosis are closely related to the use in detecting antigenic activity and specificity thereof. So far, however, most of T. gondii antigens used in clinical diagnosis are extracted naturally, which tend to cause false positive detection due to low purity and poor specificity. Therefore, preparation of T. gondii recombinant antigens and clinical diagnosis are particularly important.

SUMMARY

To solve the above problems in the prior art, the present application provides a recombinant protein for rapid detection of T. gondii and use thereof. An objective of this invention is to provide a complementary DNA (cDNA) sequence encoding T. gondii fusion protein G267.

Another objective of the invention is to provide a recombinant Escherichia coli specifically expressing the T. gondii fusion protein G267. The strain can specifically express the T. gondii fusion protein G267 after induction with isopropyl-p-D-thiogalactopyranoside (IPTG), providing a specific antigen for serological diagnosis.

Still another objective of the invention is to provide a method for expressing and purifying the T. gondii fusion protein G267.

Yet another objective of the present invention is to provide an accurate, rapid, and simple method for diagnosing T. gondii.

The technical solutions of the present invention are as follows:

A nucleotide has a nucleotide sequence as shown in SEQ ID No: 1.

A fusion protein has an amino acid sequence as shown in SEQ ID No: 2.

Preferably, the above fusion protein is encoded by the above nucleotide sequence.

Preferably, dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 are constructed into a prokaryotic expression vector and fusion expression is achieved by genetic engineering technology.

A plasmid vector includes the nucleotide sequence according to claim 1.

A recombinant expression strain is provided, where the plasmid vector according to claim 5 is transformed into E. coli and screened to obtain the recombinant expression strain.

Preferably, the E. coli is BL21(DE3).

A method for detecting a T. gondii antibody with the fusion protein includes the following steps:

(1) labeling colloidal gold particles with the fusion protein;

(2) coating a nitrocellulose membrane with the fusion protein; and

(3) assembling a T. gondii colloidal gold test product and using the product in sample detection.

The present invention is implemented through the following experimental schemes:

(1) Presently known gene sequence of T. gondii is aligned to related sequences of other pathogenic microorganisms; dominant epitopes of T. gondii are artificially designed and simulated through a computer; based on both sensitivity and specificity, dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 are finally determined and integrated as target sequences.

(2) A recombinant prokaryotic expression vector, pET-28a(+)-PGRA, is constructed by inserting the dominant epitopes of P24, P30, GRA2, and GRA6 between multiple cloning sites BamHI and EcoRI of a prokaryotic expression vector, pET-28a(+).

(3) The recombinant prokaryotic expression vector pET-28a(+)-PGRA in step (2) is transformed into E. coli BL21(DE3) competent cells, and a monoclonal strain is obtained by kanamycin resistant screening; after induction, the strain specifically expresses the dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 and a fusion protein G267 is obtained.

(4) The fusion protein G267 expressed in step (3) is purified. Purification of the fusion protein G267 specifically includes the following steps:

picking up a pET-28a(+)-PGRA recombinant strain monoclone from a kanamycin resistant Luria-Bertani (LB) agar plate to an LB liquid medium, adding kanamycin to a final concentration of 50 pg/mL, culturing on a constant temperature shaker for 12 h at 37°C; diluting the strain 1:100 with the LB liquid medium supplemented with 50 pg/mL kanamycin, dispensing into a bacteria culture bottle, and culturing on the constant temperature shaker at 37°C until an OD600 of 0.7; adding IPTG to a final concentration of 1.0 mmol/L, inducing for 4 h, and then conducting sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); after induction, centrifuging and sonicating the strain to extract inclusion bodies, purifying the inclusion bodies with polyethylene glycol (PEG), dialyzing, and storing at -20°C for use.

(5) The fusion protein G267 purified in step (4) is used to label colloidal gold particles and coat a nitrocellulose membrane to make a test strip; a serum sample is tested with a colloidal gold strip, and a linear use range thereof is determined according to color changes in colloidal gold.

The present invention has the following beneficial technical effects:

1. The prokaryotic expression vector pET-28a(+) related to the invention is one of the most commonly used prokaryotic expression vectors in the field of genetic engineering, without biological risk. The recombinant prokaryotic expression vector pET-28a(+)-PGRA constructed on this basis does not have any biological risk as well. The E. coli strain used to construct fusion protein G267 is BL21(DE3), which is one of the most commonly used expression strains in the field of molecular biology and does not have biological risk. The whole preparation process is highly safe, without such potential risk as T. gondii contamination and spreading.

2. In the present invention, the dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 are fused by molecular biological technology, and the fusion protein G267 is obtained after induced expression and purification. The fusion protein has abundant epitopes and high specificity; the fusion protein, as a colloidal gold-labeled antigen and a coating an antigen, is assembled into a colloidal gold strip, which is suitable for large-scale clinical diagnosis of T. gondii.

3. The fusion protein G267 used in the present invention is obtained by culture of E. coli, induction, and purification, and is suitable for large-scale production due to simple and practical production process and low production cost.

4. The present invention provides a novel method for differential diagnosis of T. gondii, which is easy and feasible to use and operate, and is extremely suitable for large-scale clinical diagnosis of T. gondii.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates how to use a colloidal gold strip prepared in Example 14 of the present invention.

DETAILED DESCRIPTION

The present invention will be described in detail below in conjunction with examples. Detailed text descriptions of the design idea of the present invention are made in the following examples, but these text descriptions just briefly illustrates, but not limits, the design idea of the invention, and any combination, addition, or modification without departing from the idea of the invention shall fall within the scope of the present invention.

Example 1: Selection of dominant epitopes of T. gondii

With T. gondii as a target antigen, hydrophilicity and antigenicity of an epitope sequence thereof were analyzed by biology software DNAssist2.0, and dominant epitopes of P24, P30, GRA2, and GRA6 were selected. Meanwhile, sequence alignment results showed that sequences of dominant epitopes of P24, P30, GRA2, and GRA6 selected were broad-spectrum, which are common epitopes of all T. gondii proteins; moreover, dominant epitopes of P24, P30, GRA2, and GRA6 were not significantly homologous to other protein sequences and only present in a protein sequence of T. gondii.

Example 2: Tandem dominant epitopes of T. gondii

Sequences of dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 were linked to obtain an amino acid sequence of a recombinant protein. Specific sequence thereof is shown in SEQ ID No: 2.

Example 3: Optimization of a nucleotide sequence encoding the recombinant protein

In order to increase the expression of recombinant protein in E. coli the on the premise of unchanged amino acid sequence of the recombinant protein, a nucleotide sequence encoding the recombinant protein was converted into the corresponding nucleotide sequence according to the codon usage of E. coli. Specific sequence is shown in SEQ ID No: 1. Moreover, nucleotide sequences corresponded to restriction sites BamHI and EcoRI were added into the upstream and downstream of the nucleotide sequence, which was synthesized by Hangzhou Goodhere Biotechnology Co., Ltd. A synthesized target gene was linked to a pMD20-T vector (Takara Biotechnology (Dalian) Co., Ltd.).

Example 4: Construction of a recombinant protein expression vector

Target gene-containing pMD20-T vector and pET-28a(+) vector (Novagen, Germany) were double digested with restriction endonucleases BamHI and EcoRI (Takara Biotechnology (Dalian) Co., Ltd.) for 12 h at 37°C, respectively; each digested product was electrophoresed on 1% agarose gel, and the target gene and the pET-28a(+) vector were recovered by cutting gel (all gel extraction kits used in the present invention were purchased from Ningbo ZD Biotechnology Co., Ltd.). Recovered target gene and pET-28a(+) vector were linked in a given ratio by T4 ligase (Takara Biotechnology (Dalian) Co., Ltd.) for 12 h at 4°C; ligation product was transformed into DH5a competent cells (Hangzhou Goodhere Biotechnology Co., Ltd.) and plated on a kanamycin

(50 pg/mL) resistant LB plate; after culturing for 12 h at 37°C, a monoclonal strain was picked up from the plate to a kanamycin (50 pg/mL) resistant LB liquid medium; after culturing on a constant temperature shaker for 12 h at 37°C, plasmids were extracted by a plasmid purification kit (all plasmid purification kits used in the present invention were purchased from Ningbo ZD Biotechnology Co., Ltd.), and a correct recombinant expression vector was obtained after double enzyme digestion with BamHI and EcoRI.

Example 5: Construction of a recombinant protein expression strain

Well-constructed recombinant expression vector was transformed into F coli BL21(DE3) competent cells, and the cells were plated on a kanamycin (50 pg/mL) resistant LB plate and cultured overnight at 37C. The next day, a monoclonal strain was picked up from the plate to a kanamycin (50 pg/mL) resistant LB liquid medium; after culturing on a constant temperature shaker for 8 h at 37°C, IPTG (final concentration: 1.0 mmol/L) was added for induced expression for 4 h to prepare a protein electrophoresis sample. Results of 13.5% polyacrylamide gel electrophoresis (PAGE) indicated that a recombinant protein was expressed successfully and a recombinant protein expression strain was obtained.

Example 6: Determination of optimum inducer concentration

Recombinant protein G267 expression strain was streaked on an LB plate; after culturing overnight at 37°C, a single colony was picked up and inoculated into 5 mL of LB liquid medium, while adding kanamycin to a final concentration of 50 pg/mL; after culturing on a constant temperature shaker for 12 h at 37°C, the bacteria suspension was diluted 1:100 with the LB liquid medium supplemented with 50 pg/mL kanamycin, dispensed into five test tubes (5 mL per tube), and cultured on the constant temperature shaker at 37°C until an OD600 of 0.7; IPTG was added to final concentrations of 0.2, 0.5, 0.8, 1.0, and 1.5 mmol/L, respectively; after culturing for 3 h, aliquots of the bacteria suspension were subjected to SDS-PAGE, and an optimum IPTG induced concentration was determined as 1.0 mmol/L according to the expression of the fusion protein G267.

Example 7: Determination of optimum induction time

Recombinant protein G267 expression strain was streaked on an LB plate; after culturing overnight at 37°C, a single colony was picked up and inoculated into 5 mL of LB liquid medium, while adding kanamycin to a final concentration of 50 pg/mL; after culturing on a constant temperature shaker for 12 h at 37°C, the bacteria suspension was diluted 1:100 with the LB liquid medium supplemented with 50 pg/mL kanamycin, dispensed into five test tubes (5 mL per tube), and cultured on the constant temperature shaker at 37°C until an OD600 of 0.7; IPTG was added to a optimum induced concentration of 1.0 mmol/L; after induction culture for 3, 3.5, 4, 4.5, and 5 h, aliquots of the bacteria suspension were subjected to SDS-PAGE, and an optimum IPTG induction time was determined as 4 h according to the expression of the fusion protein G267.

Example 8: Abundant expression and purification of T. gondii fusion protein G267

A pET-28a(+)-PGRA recombinant strain monoclone was picked up from a kanamycin resistant LB plate to an LB liquid medium, while adding kanamycin to a final concentration of 50 pig/mL; after culturing on a constant temperature shaker for 12 h at 37°C, the strain was diluted 1:100 with the LB liquid medium supplemented with 50 pg/mL kanamycin, dispensed into a bacteria culture bottle, and cultured on the constant temperature shaker at 37°C until an OD600 of 0.7; IPTG was added to a final concentration of 1.0 mmol/L, followed by culture and induction for 4 h. Bacterial cells were collected after centrifugation, resuspended in a cell lysis buffer, frozen for 30 min at -70°C, and sonicated for 3 min; pellets were collected after centrifugation at 4°C, and a supernatant was discarded. The pellets were dissolved in 10 mL of denatured inclusion body solution under stirring; after centrifugation at 4°C, PEG4000, oxidized glutathione, and reduced glutathione were added to a supernatant to concentrations of 0.2% (W/V), 1 mmol/L, and 2 mmol/L, respectively; the mixture was allowed to stand for 30 min at 4°C, transferred into a dialysis bag with a molecular weight cutoff of 10-12 kD, and dialyzed in phosphate buffer saline (PBS, 10 mmol/L, pH 7.4) overnight. After dialysis, the dialysate was removed and dispensed immediately, and stored at -70°C for use.

Formula of the cell lysis buffer: Tris-Cl 50 mmol/L, pH 8.0

EDTA 1 mmol/L

NaCl 100 mmol/L

Denatured inclusion body solution: bacterial lysate + SKL 0.3% (W/V)

Example 9: Western Blot

Purified fusion protein G267 was electrophoresed on a 12% polyacrylamide gel and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. Primary antibodies were T. gondii negative and positive sera, respectively; secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody; substrate was luminol commonly used in Western Blot. X-ray films were exposed, developed, and fixed.

Formulas of related solutions were as follows:

1. Running buffer:

Glyl 8.8 g, Tris 3.03 g, SDS 1 g, diluting to 1,000 mL with double distilled water.

2. Transfer buffer:

Gly 2.9 g, Tris 5.8 g, SDS 0.37 g, methanol 200 mL, diluting to 1,000 mL with double distilled water.

3. Wash buffer:

Tris 3 g, NaCl 8 g, KC 0.2 g, Tween-20 1 mL, diluting to 1,000 mL (pH 7.8).

4. Fluorogenic substrate:

Product from Pierce Inc. (Product code: Prod#34075).

Example 10: Preparation of rabbit polyclonal antibody

A male New Zealand white rabbit was selected; the white rabbit was primed and intradermally injected into multiple sites with 300 pg of fusion protein G267 emulsified by Freund's complete adjuvant (FCA) (1 mL per rabbit in total). After 20 days, the rabbit was boosted, during which the rabbit was intradermally injected into multiple sites with 150 pg of fusion protein G267 emulsified by Freund's incomplete adjuvant (FIA) (1 mL per rabbit in total). Thereafter, the rabbit was boosted every 15 days, for five times; the method was conducted as did in the second boosting. Before the fifth boosting, 5 mL of blood was drawn from the ear artery and purified to obtain a G267 polyclonal antibody, and a titer thereof was enzyme-linked immunosorbent assay (ELISA). After 10 days, 100 mL of blood was drawn from the carotid artery, and the rabbit was sacrificed. The ELISA specifically included the following steps:

diluting the fusion protein G267 with coating buffer (final concentration: 1 g/mL); adding the fusion protein G267 (100 pL/well) into an ELISA plate (Wuxi Guosheng Bioengineering Co., Ltd.), coating for 12 h at 4°C, and then washing the ELISA plate with wash buffer once on DEM-3 Microplate Washer (Da An Gene Co., Ltd. of Sun Yat-Sen University); blocking with blocking buffer (200 pL/well) for 1 h at 37C, and washing the plate on the microplate washer once; incubating with 1 g/mL G267 polyclonal antibody (100 pL/well) for 35 min at 37C, and washing the plate with wash buffer three times; incubating with HRP-conjugated goat anti-rabbit secondary antibody (100 pL/well) for 30 min at 37C, washing the plate with wash buffer four times; adding 50 pL each of chromogen solutions A and B, developing in the dark for 10 min at 37C, stopping the reaction with stop solution (50 pL/well), and reading optical density (OD) values after zeroing a blank well on a microplate reader (Thermo) at 450 nm. Formulas of related solutions were as follows: coating buffer: Na2CO3 1.59 g, NaHCO3 2.93 g, diluting to 1,000 mL (pH 9.6) with double distilled water; blocking buffer: Na2HPO4-12H20 2.68 g, NaH2PO4-2H20 0.39 g, NaCI 8.5 g, bovine serum albumin (BSA) 20 g, diluting to 1,000 mL (pH 7.4) with double distilled water; wash buffer: Na2HPO4-12H20 2.68 g, NaH2PO4-2H20 0.39 g, NaCl 8.5 g, Tween-20 0.5mL, diluting to 1,000 mL (pH 7.4) with double distilled water; chromogen solution A: dissolving 200 mg of tetramethylbenzidine (TMB) in 100 mL of absolute ethanol, and diluting to 1,000 mL with double distilled water; chromogen solution B: citric acid 2.1 g, Na2HPO4-12H20 71 g, diluting to 1,000 mL with double distilled water; when in use: 1 mL of chromogen solution A + 1 mL of chromogen solution B + 0.4 pL of 30% H202; and stop solution: 2 M H2SO4, 21.7 mL of concentrated H2SO4, diluting to 1,000 mL with double distilled water.

Example 11: Purification of rabbit polyclonal antibody

Agarose affinity matrix Protein G Sepharose Column (GenScript (Nanjing) Co., Ltd.) was equilibrated with 50 mL of equilibrium buffer PBS (10 mg/mL, pH 7.4), and OD value was zeroed on a protein nucleic acid detector (Shanghai Huxi Analysis Instrument Factory). Serum was centrifuged for 5 min at 12,000 rpm; a supernatant was collected, filtered through a 0.45 pm filter, loaded, and washed with PBS until the OD value was 0; next, the supernatant was eluted with 0.1 M glycine (pH 3.0), and effluent was collected and neutralized with 500 mM Tris-HCl (pH 8.5) to around pH 7.0; thus, T. gondii rabbit polyclonal antibody was obtained.

Example 12: Labeling colloidal gold particles with fusion protein G267

Ten milliliters of 0.01% colloidal gold solution was mixed well with 20 pL of 0.2 mol/L potassium carbonate solution; the mixture was reacted with 100 pg of T. gondii fusion protein G267 for 2 h at room temperature, blocked with 1 mL of 10% BSA for 2 h, and centrifuged (for 20 min at 7,500 rpm); a supernatant was discarded, and pellets were dissolved fully in 1 mL of reconstituted solution; the reconstituted solution was uniformly sprayed to glass fibers (6 mm wide) at 10 pl/cm using a platform dispenser (Shanghai Jinbiao Biologic Technology Co., Ltd.), and the glass fibers were allowed to stand in an electrothermal thermostatic incubator (Shanghai Yiheng Technology Instrument Co., Ltd.) for 30 min at 37°C.

Formulas of related solutions were as follows:

0.01% colloidal gold solution: 1 mL of 1% chloroauric acid solution and 1.4 mL of 1% citric acid solution were dissolved in ultrapure water, heated, and reacted, followed by diluting to 100 mL;

1% chloroauric acid solution: 1 g of AuCL3.HCl-4H2O powder was dissolved in ultrapure water and diluted to 100 mL;

1% citric acid solution: 1 g of citric acid crystal was dissolved in ultrapure water and diluted to 100 mL;

reconstituted solution: 6.057 g of Tris base was dissolved in 800 mL of double distilled water, adjusted to pH 8.0 with a moderate amount of HCl, and diluted to 1,000 mL with double distilled water.

Example 13: Preparation of T. gondii diagnostic test strip

After the T. gondii fusion protein G267 was diluted with coating buffer (to a final concentration of 1 mg/mL), a nitrocellulose membrane (Sartorius) was uniformly coated with it at 1 pl/cm on a platform dispenser (Shanghai Jinbiao Biologic Technology Co., Ltd.), and thus a T line was made. A nitrocellulose membrane (Sartorius) was uniformly coated with T. gondii rabbit polyclonal antibody (to a final concentration of 1 mg/mL) at 1 pl/cm on a platform dispenser (Shanghai Jinbiao Biologic Technology Co., Ltd.), and thus a C line was made. After dispensing and coating, nitrocellulose membranes were allowed to stand in an electrothermal thermostatic incubator (Shanghai Yiheng Technology Instrument Co., Ltd.) for 30 min at 37°C.

According to a conventional process, sample pad, glass fiber, nitrocellulose membrane, and filter paper were assembled successively on a polyvinyl chloride (PVC) base plate and cut into 4 mm wide strips; a reagent card cassette was disposed and compressed. Formula of related solution was as follows:

coating buffer: Na2HPO4-7H2 43.42g, NaH2PO4-H2O5.244 g, diluting to 1,000 mL (pH 7.4) with double distilled water.

Example 14: Operation and standard of the T. gondii diagnostic test strip

A mouse was immunized with T.gondii standard; serum was drawn and loaded (100 pL/well) after gradient dilution; after standing for 15 min at room temperature, as shown in FIG. 1, purplish red reaction bands darkened gradually with the increase of test sample concentration.

The experimental result shows that the fusion protein G267 prepared from dominant epitopes of T. gondii P24, P30, GRA2, and GRA6 in the present invention and the colloidal gold test strip established thereby enable rapid and effective differential diagnosis of T. gondii infection.

Example 15: Sensitivity and specificity tests for the test strip

Sensitivity test: According to the operation method of the T. gondii test strip, positive sera (clinically confirmed) after gradient dilution were tested by the test strip of the present invention; cutoffs (subject to a cutoff of 200: >200, positive; <200, negative) were read on an assay reader (Hangzhou Weizan Science and Technology Co., Ltd.), respectively, with a coincidence rate of 100%. Results are shown in Table 1.

Table 1: Tests for clinically positive samples

Serum 1 of a patient with toxoplasmosis

Dilution 1:2,000 1:1,000 1:500 1:100 1:20

Reading 123 249 506 2543 9863

Serum 2 of a patient with toxoplasmosis

Dilution 1:2,000 1:1,000 1:500 1:100 1:20

Reading 159 297 586 3029 12894

Serum 3 of a patient with toxoplasmosis

Dilution 1:2,000 1:1,000 1:500 1:100 1:20

Reading 99 212 449 2217 8239

Specificity test: According to the operation method of the T. gondii test strip, three other serum materials were tested by the test strip of the present invention, and response specificity was observed. Results showed that: 10 negative serum samples (clinically confirmed) tested by the test strip of the present invention obtained a coincidence rate of 100%; no positive result was observed in tests for 10 liver fluke-positive, 10 hepatitis A positive, and 10 hepatitis B positive serum samples, indicating that other diseases are hardly false positive to the test strip of the present invention, with excellent specificity.

Sequence Listing

<110> Jiangsu Institute of Parasitic Diseases

<120> RECOMBINANT PROTEIN FOR RAPID DETECTION OF TOXOPLASMA GONDII, PREPARATION METHOD AND USE THEREOF

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ctgggcccgg tgaaactgag cgccgaagge ccgaccacca tgaccctggt gtgcggcaaa 360

gatggcgtga aagtgccgca ggataacaac cagtattgca gcggcaccac cctgaccggc 420

tgcaacgaaa aaagetttaa agatattctg cegaaactga gcgaaaaccc gtggggegge 480

ggcggettta aagtggccaa agaagccgcc ggccgcggca tggtgaccgt gggcaaaaaa 540

ctggccaacg tggaaagega tegcagcace accaccaccc aggeccegga tagcccgaac 600

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Gly Ala Pro Thr Gly Ala Pro Ala Leu Leu Leu Ile Ala Ile Leu Ala

1 5 10 15

Ser Ala Gly Ser Thr Ser Gly Val Gly Ala Val Ala Val Gly Gly Val

20 25 30

Ile Ala Thr Met Leu Gly Gly Gly Gly Ala Gly Ile Leu Leu Thr Val

35 40 45

Pro Ile Gly Leu Pro Pro Val Thr Thr Gly Thr Pro Val Val Gly Cys

50 55 60

Ile Leu Gly Ala Ala Ala Gly Ser Cys Met Val Thr Val Thr Val Gly

65 70 75 80

Ala Ala Ala Ser Ser Val Val Ala Ala Val Ala Ala Cys Ser Thr Gly

85 90 95

Ala Ala Ser Thr Leu Gly Pro Val Leu Leu Ser Ala Gly Gly Pro Thr

100 105 110

Thr Met Thr Leu Val Cys Gly Leu Ala Gly Val Leu Val Pro Gly Ala

115 120 125

Ala Ala Gly Thr Cys Ser Gly Thr Thr Leu Thr Gly Cys Ala Gly Leu

130 135 140

Ser Pro Leu Ala Ile Leu Pro Leu Leu Ser Gly Ala Pro Thr Gly Gly

145 150 155 160

Gly Gly Pro Leu Val Ala Leu Gly Ala Ala Gly Ala Gly Met Val Thr

165 170 175

Val Gly Leu Leu Leu Ala Ala Val Gly Ser Ala Ala Ser Thr Thr Thr

180 185 190

Thr Gly Ala Pro Ala Ser Pro Ala Gly Leu Ala Gly Thr Gly Val Pro

195 200 205

Val Gly Pro Gly Gly Ala Gly Gly Gly Gly Gly Ala Ile Gly Gly Gly

210 215 220

Gly Thr Ala Ala Ala Thr Ser Ser Val Gly Gly Pro Gly Ala Leu Val

225 230 235 240

Pro Ser Leu Ala Thr Gly Leu Ala His Ala Leu Ile Gly Ala Val

245 250 255

Claims (5)

What is claimed is:

1. A nucleotide, wherein the nucleotide has a nucleotide sequence as shown in SEQ ID No: 1.

2. A fusion protein, wherein the fusion protein has an amino acid sequence as shown in SEQ ID No: 2.

3. A plasmid vector, wherein the plasmid vector comprises the nucleotide sequence according to claim 1.

4. A recombinant expression strain, wherein the plasmid vector according to claim 3 is transformed into Escherichia coli (E. coli) and screened to obtain the recombinant expression strain.

5. A method for detecting a T. gondii antibody with the fusion protein according to claim 2, comprising the following steps:

(1) labeling colloidal gold particles with the fusion protein;

(2) coating a nitrocellulose membrane with the fusion protein; and

(3) assembling a T gondii colloidal gold test product and using the product in sample detection.