CN113201546A - Clonorchis sinensis specific antigen VAL28 and medical application - Google Patents
Clonorchis sinensis specific antigen VAL28 and medical application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/43559—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from trematodes
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention relates to a clonorchis sinensis specific antigen gene VAL28 and medical application, which is a new gene protein; the colloidal gold test strip prepared based on the clonorchis sinensis VAL28 protein has the advantages of high stability, strong specificity, good sensitivity, low detection cost and the like, is convenient to use, simple to operate, simple and clear in detection result, and suitable for clinical rapid diagnosis, solves the problems of short time for clinical detection and diagnosis of diseases, large number of samples to be detected and the like, and simultaneously can reduce the disease burden caused by clonorchis sinensis to a certain extent by a good detection method.
Description
Technical Field
The invention relates to a clonorchis sinensis specific antigen gene VAL28, and a colloidal gold immunochromatographic test paper for detecting clonorchis sinensis infection, which is developed based on VAL28 as an antigen, can be used for serum detection of clonorchis sinensis infection, and belongs to the technical field of immunology.
Background
Clonorchiasis sinensis, also known as hepatism, is prepared from clonorchis sinensis (Clonorchis sinensis)Clonorchis sinensis) Parasitic diseases mainly caused by hepatobiliary diseases in mammals including human beings are common food-borne zoozoonosis. The epidemic area of clonorchis sinensis overlaps with the epidemic area of viral hepatitis, and has a great threat to the global public health safety. The risk of the clonorchis sinensis infected person suffering from hepatobiliary duct cancer is 4.5 times that of the normal person, and the risk of suffering from cirrhosis and liver cancer is also increased by times. Therefore, the serological detection and epidemiological investigation of the clonorchis sinensis infection serum can effectively reduce the risk of clonorchis sinensis diseases, is beneficial to reducing the disease burden caused by the clonorchis sinensis diseases, and has important significance for the healthy development of people and animals.
At present, the diagnosis method for clonorchiasis sinensis is mainly a fecal ovum examination method, and diagnosis is made by observing morphological characteristics of worms under a microscope. The etiology diagnosis method has the advantages of rapidness, simplicity and convenience, and has certain disadvantages, for example, because the eggs of the clonorchis sinensis and the eggs of the epididymis sinensis are relatively similar, the requirements on the experience and the specialty of operators are higher, and false detection or missing detection is easy to occur in the case of mild infection. The immunological diagnosis method is an important supplement of the etiology diagnosis method, has the advantages of high sensitivity, strong specificity, good repeatability, suitability for detecting a plurality of basic units of samples and the like, is widely applied, and is an important supplement of the etiology diagnosis method.
Complete exposure of the VAL protein to the host's immune system is important for the correlation between the parasite and the host and is considered a potentially good antigen. The research utilizes NCBI to compare and analyze the clonorchis sinensis VAL28 gene sequence, and the result shows that the clonorchis sinensis VAL28 gene sequence has no homology with other parasite gene sequences uploaded in the gene bank at present, and the gene is preliminarily shown to have better specificity. At present, no report is available about the preparation of an immune colloidal gold test strip based on the clonorchis sinensis VAL28 as an antigen to detect the clonorchis sinensis infection.
Disclosure of Invention
The clonorchis sinensis specific antigen gene VAL28, the medical application and the colloidal gold immunochromatographic test paper for detecting clonorchis sinensis infection, which is prepared based on the gene as the antigen, can be used for detecting clonorchis sinensis infection serum.
The Clonorchis sinensis specific antigen gene VAL28 (Clonorchis sinensis venom allergen-like protein 28, CSVAL28 for short) (GenBank: MH 172541.1) has the gene sequence as follows: shown as SEQ No. 1.
The amplified clonorchis sinensis specific antigen gene VAL28 has target segment size of 495bp, and the target segment has signal peptide eliminated. The protein expressed by the gene contains SCP structural domain analyzed by SMART online software, and the physicochemical property of the protein is predicted by a ProtParam database of ExPASy, wherein the protein contains 165 amino acids and has an isoelectric point of 8.80.
The invention relates to a method for obtaining clonorchis sinensis specific antigen gene VAL28, which comprises the following steps:
according to the gene sequence of clonorchis sinensis VAL28, specific primers are designed, and the target fragment of clonorchis sinensis VAL28 is amplified through polymerase chain reaction to obtain clonorchis sinensis specific antigen gene VAL 28.
The invention relates to a colloidal gold immunochromatographic test strip for detecting clonorchis sinensis infection, which is prepared based on clonorchis sinensis VAL28 protein and comprises the following components in parts by weight: the method comprises the steps of performing protein purification on clonorchis sinensis VAL28, firing a colloidal gold solution by using a trisodium citrate reduction method, determining conditions such as the pH value of the colloidal gold solution and the labeling amount of SPA protein, assembling a sample pad, a nitrocellulose membrane (NC membrane), a gold-labeled pad, a PVC bottom plate, absorbent paper and the like, and determining the optimal coating amount of a detection line (clonorchis sinensis VAL28 protein) and a quality control line (rabbit anti-SPA protein) of the assembled test strip to prepare the clonorchis sinensis infection diagnosis and detection colloidal gold test strip.
The invention has the advantages that:
provides a new specific antigen gene VAL28 of clonorchis sinensis, which is a new gene protein; the colloidal gold test strip prepared based on the clonorchis sinensis VAL28 protein has the advantages of high stability, strong specificity, good sensitivity, low detection cost and the like, is convenient to use, simple to operate, simple and clear in detection result, and suitable for clinical rapid diagnosis, solves the problems of short time for clinical detection and diagnosis of diseases, large number of samples to be detected and the like, and simultaneously can reduce the disease burden caused by clonorchis sinensis to a certain extent by a good detection method.
Description of the drawings:
FIG. 1 shows PCR amplification of the VAL28 gene (M: DL2000 marker; 1: VAL28 PCR product);
FIG. 2 is a diagram showing the double restriction enzyme identification of the pMD-18T-VAL28 cloning vector (M: DNA marker (DL: 5000);
1: the product of double enzyme digestion of the pMD-18T-VAL28 cloning vector; 2: pMD-18T-VAL28 cloning vector);
FIG. 3 shows the double restriction enzyme identification of pET-28a-VAL28 expression vector (M: DNA marker (DL 10000) 1: double restriction enzyme product of pET-28a-VAL28 expression vector; 2: pET-28a-VAL28 expression vector);
FIG. 4 shows a diagram of the purification of recombinant pET-28a-VAL28 protein (M: protein marker; 1: purified recombinant pET-28a-VAL28 protein);
FIG. 5 and FIG. 6 are Western blotting analysis of recombinant pET-28a-VAL28 protein (M: protein marker; 1: Clonorchis sinensis negative serum; 2: Clonorchis sinensis positive serum);
FIG. 7 is a diagram of a colloidal gold solution;
FIG. 8 is a transmission electron microscope image of colloidal gold particles;
FIG. 9 is a transmission electron micrograph of colloidal gold particles labeled SPA protein;
FIG. 10 is a graph showing the determination of the optimum pH of a colloidal gold solution;
FIG. 11 is a graph showing the determination of the optimal labeling amount of SPA protein;
FIG. 12 is a graph showing the determination of the optimal coating concentration of protein on the detection line (T line);
FIG. 13 is a graph showing the determination of the optimal coating concentration of the control line (line C) protein;
FIG. 14 is a graph showing the results of a specificity test;
FIG. 15 is a graph showing the results of a sensitivity test;
FIG. 16 is a graph showing the results of a reproducibility test;
fig. 17 is a partial actual sample detection diagram.
Detailed Description
The present invention is further illustrated by the following examples, which do not limit the present invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.
Example 1
Acquisition and identification of clonorchis sinensis specific antigen gene
Amplification of clonorchis sinensis VAL28 Gene
According to the gene sequence of clonorchis sinensis VAL28 (GenBank: MH 172541.1), the specific primer sequences are designed as follows:
F:CGCGGATCCTTTGTGAAGCTGCATGATC；
R:CCCAAG CTTCTTCAAGTGGCCTGGAC (underlined is the cleavage site and underlined is the protective base). The target fragment of clonorchis sinensis VAL28 is amplified by polymerase chain reaction. The result shows that a 513bp fragment (including a VAL28 target fragment, an enzyme cutting site and a protective base) is successfully amplified. (as shown in FIG. 1)
Construction of pMD-18T-VAL28 cloning vector and pET-28a-VAL28 expression vector
The recombinant plasmid pMD-18T-VAL28 was identified by double digestion with BamHI and HindIII enzymes, separated and identified by 1% agarose gel electrophoresis. As a result, the pMD-18T-VAL28 cloning vector was successfully constructed (as shown in FIG. 2).
The recombinant plasmid pET-28a-VAL28 was subjected to double enzyme digestion and identification by using BamHI and HindIII enzymes, and separated and identified by 1% agarose gel electrophoresis. The results showed that the pET-28a-VAL28 expression vector was successfully constructed (as shown in FIG. 3).
Expression and purification of recombinant pET-28a-VAL28 protein
Respectively culturing the pET-28a bacterial liquid and the recombinant expression vector pET-28a-VAL28 bacterial liquid which is successfully sequenced in an LB culture medium, adding one thousandth of IPTG (isopropyl-beta-thiogalactoside) for inducing for 5 hours when the pET-28a bacterial liquid and the recombinant expression vector pET-28a-VAL28 bacterial liquid grow to OD600=0.6, completing expression and purification, and then completing the identification of the protein through SDS-PAGE analysis. The results showed that a clear band of interest was visible at about 22kDa, indicating that the recombinant pET-28a-VAL28 protein was successfully purified (as shown in FIG. 4).
Western blotting analysis of recombinant pET-28a-VAL28 protein
By using a Western blotting detection method, a dog serum sample without infecting the clonorchis sinensis and a dog positive serum sample infecting the clonorchis sinensis are detected by taking the recombinant pET-28a-VAL28 protein as an antigen. The result shows that no band is generated when detecting negative serum (as shown in figure 5), and a clear band is generated at about 22kDa when detecting positive serum of clonorchis sinensis infected dogs (as shown in figure 6), which indicates that the recombinant pET-28a-VAL28 protein has good immunogenicity.
Example 2
Firing of colloidal gold solutions
The colloidal gold solution is fired by a trisodium citrate reduction method, and whether the color of the colloidal gold particle solution is changed or not, and whether precipitation and aggregation are generated or not is observed by naked eyes. The results showed that the colloidal gold solution was wine-red in color, and no aggregation and precipitation occurred. (as shown in FIG. 7)
Identification of colloidal gold particles
Then, the diameter and dispersion of the colloidal gold particles can be observed by shooting a transmission electron microscope. The results show that the particle size of the colloidal gold particles is about 40nm, the size is uniform, and the distribution is uniform (as shown in figure 8); gold particles labeled SPA protein, surrounded by a halo stain (as shown in figure 9).
Determination of the optimum pH
Respectively adding 0.2mol/LK in different volumes2CO3Adding the mixture into 1mL of colloidal gold solution in sequence, adding 20 mu g of SPA protein into each tube for over-labeling, slowly mixing the mixture uniformly, standing the mixture for 30min, adding 100 mu L of 10% NaCl into each tube, standing the mixture for 2h, and observing color change. The results show that when 2. mu.L of 0.2mol/LK was added2CO3At this point, the color no longer changes, and the solution reaches an optimal pH (as shown in FIG. 10).
Determination of the optimal amount of protein marker
Adding 1mL of colloidal gold solution into 6 tubes of 1.5mLEP, adjusting the pH value of the colloidal gold solution to the optimal value, sequentially adding 0, 2, 4, 6, 8 and 10 mu L of SPA protein into the EP tube, uniformly mixing, standing at room temperature for 30min, adding 100 mu L of 10% NaCl into each tube, standing for 1h, and observing color change. The results show that when 5. mu.g of SPA protein was added, the color did not change any more, and 20% more was added to the amount of the best label, that is, 6. mu.g was the amount of the best label of SPA protein (as shown in FIG. 11).
Preparation and identification of gold-labeled antibody
(1) According to the determined best K2CO3Adjusting the colloidal gold solution to the optimal pH value, and adding the optimal SPA protein labeling amount for labeling;
(2) adding 100 μ L of 10% BSA (dissolved in 0.02M Tris-HCl) into each tube, and blocking for 10-15 min;
(3) centrifuging for 30min, and centrifuging at 4 deg.C;
(4) discarding the supernatant, adding 10% volume of re-dissolving solution for re-suspension, and storing at 4 ℃ for later use;
(5) and observing by a transmission electron microscope. The results showed a halo of dye around the gold particle that labeled SPA protein (as shown in FIG. 9).
Assembly of test strips
(1) Attaching the NC film to the PVC bottom plate (taking care to keep the NC film clean and not to have scratches);
(2) pressing a gold label pad 2mm above the NC film;
(3) pressing the sample pad 2mm above the gold label pad;
(4) pressing the water absorption paper on the NC film for 2 mm;
(5) and cutting the test strips, wherein the width of each test strip is 4 mm.
Determination of optimal coating amount of detection line (T line) and quality control line (C line)
Diluting the T-line coated protein at different concentrations, observing the color development condition of the T line, and determining the optimal coating concentration of the T-line protein, wherein the result shows that the optimal coating concentration of the T line is 0.5 mug/muL (as shown in figure 12);
the C-line protein was diluted at different concentrations, and the color development of the C-line was observed to determine the optimal coating concentration of the C-line protein, which indicated that the optimal coating concentration of the T-line was 0.25. mu.g/. mu.L (FIG. 13).
Example 3
Determination of colloidal gold test strip performance
Specificity test
The assembled test strip is used for respectively detecting a clonorchis sinensis positive sample (a), a clonorchis sinensis negative sample (b), an oriental epididymis positive sample (c), a fasciola hepatica positive sample (d), a canine toxoplasma gondii positive serum (e), a neospora caninum positive serum (f) and a bilocular trematoda positive serum (g), and observing whether the detection line develops color. The results show that the method has better specificity (as shown in figure 14).
Sensitivity test
Under the premise of determining the optimal coating concentration of the detection line and the quality control line, carrying out multiple dilution on the clonorchis sinensis positive serum, observing the color development condition of the T line of each test strip, and judging the sensitivity of the prepared colloidal gold test strip; the results show that the sensitivity is 1:200 (as shown in FIG. 15).
Repeatability test
Three batches of colloidal gold test strips are used for respectively detecting clonorchis sinensis negative serum and positive serum, the color change of the detection line is observed, and whether the repeatability of the test strips is good or not is judged. The results show that the method has good reproducibility (as shown in fig. 16).
Shelf life test
And assembling the test strip according to determined conditions, and sealing, drying and storing at room temperature, 4 ℃ and 37 ℃. The tests were carried out after 7d, 15d, 30d, 60d, respectively. Observing the change of the color depth of the test strip to determine the optimal preservation time. The results showed that the coloring effect was still satisfactory when the composition was stored for one month (see Table 1).
TABLE 1 shelf life test
Note: "+ + + +" indicates that the bands are particularly clear; "+ +" indicates clear bands; "+" indicates that the band is lighter in color.
Example 4
Detection of actual samples
103 dog serum samples are collected and detected, 11 dog sera are shown to be positive sera, and the positive rate is 10.67%. A portion of the sample (as shown in figure 17).
Sequence listing
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<211> 28
<212> DNA
<213> Clonorchis sinensis (Clonorchis sinensis)
<400> 2
cgcggatcct ttgtgaagct gcatgatc 28
<210> 3
<211> 26
<212> DNA
<213> Clonorchis sinensis (Clonorchis sinensis)
<400> 3
cccaagcttc ttcaagtggc ctggac 26
Claims (3)
1. A clonorchis sinensis specific antigen gene VAL28 has a gene sequence as follows: shown as SEQ No. 1.
2. The method for obtaining clonorchis sinensis specific antigen gene VAL28 according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
according to the gene sequence of clonorchis sinensis VAL28, specific primer sequences are designed as follows:
F:CGCGGATCCTTTGTGAAGCTGCATGATC；
R:CCCAAG CTTCTTCAAGTGGCCTGGAC；
amplifying a target fragment of clonorchis sinensis VAL28 by polymerase chain reaction;
carrying out double enzyme digestion identification on the recombinant plasmid pMD-18T-VAL28 by using BamHI and HindIII, and carrying out electrophoresis separation and identification on 1% agarose gel to construct a pMD-18T-VAL28 cloning vector;
carrying out double enzyme digestion identification on the recombinant plasmid pET-28a-VAL28 by using BamHI and HindIII, and carrying out electrophoresis separation and identification on 1% agarose gel to construct an expression vector pET-28a-VAL 28;
respectively culturing the pET-28a bacterial liquid and the recombinant expression vector pET-28a-VAL28 bacterial liquid which is successfully sequenced in an LB culture medium, adding one thousandth of IPTG (isopropyl-beta-thiogalactoside) for inducing for 5 hours when the pET-28a bacterial liquid and the recombinant expression vector pET-28a-VAL28 bacterial liquid grow to OD600=0.6, completing expression and purification, and then completing the identification of the protein through SDS-PAGE analysis.
3. The colloidal gold immunochromatographic test strip for detecting clonorchis sinensis infection, which is prepared by using the clonorchis sinensis specific antigen gene VAL28 of claim 1, is characterized in that:
the method comprises the steps of performing protein purification on clonorchis sinensis VAL28, firing a colloidal gold solution by using a trisodium citrate reduction method, determining conditions such as the pH value of the colloidal gold solution and the labeling amount of SPA protein, assembling a sample pad, a nitrocellulose membrane (NC membrane), a gold-labeled pad, a PVC bottom plate, absorbent paper and the like, and determining the optimal coating amount of a detection line (clonorchis sinensis VAL28 protein) and a quality control line (rabbit anti-SPA protein) of the assembled test strip to obtain the clonorchis sinensis VAL 28.
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