Peptide Fragment, Monoclonal Antibody, Colloidal Gold Test Strip And Detection Method Thereof Used For Detection Of Stichopus Oligopeptide

Abstract

The invention belongs to the field of biotechnology, in particular, relates to peptide fragment, monoclonal antibody, colloidal gold test strip and detection method thereof used for detection of stichopus oligopeptide. Sequences of the peptide fragments are KIVPGVPD and GRDGDQGPV. After identification, the peptides KIVPGVPD and GRDGDQGPV are newly discovered oligopeptides, which are exclusive peptide fragments of Stichopus oligopeptide.

Claims

1. A peptide fragment used for preparing a colloidal gold test strip for detection of stichopus oligopeptide, the peptide fragment comprising SEQ ID NO: 1 or SEQ ID NO: 3.

2. A colloidal gold test strip, comprising a sample pad, a gold label pad, an NC membrane, an absorbent pad and a PVC bottom plate, wherein the gold label pad is coated with a monoclonal antibody of SEQ ID NO: 1 or SEQ ID NO: 3 labeled with colloidal gold.

3. The colloidal gold test strip according to claim 2, wherein the NC membrane sequentially comprises a detection line and a quality control line according to the direction of sample chromatography, the aforesaid detection line is coated with a conjugate of peptide fragment SEQ ID NO: 1 or SEQ ID NO: 3 and bovine serum albumin; and the quality control line is coated with sheep anti-mouse secondary antibody.

4. The colloidal gold test strip according to claim 2, wherein the monoclonal antibody is prepared according to the following method: take chloroauric acid solution and heat it, add trisodium citrate after boiling, and continue to boil for more than 5 minutes after the solution turns to wine red; After cooling to room temperature, add ultrapure water to obtain a colloidal gold solution; Adjust the pH value of the obtained colloidal gold solution to 7.8-8.5 with borate buffer, stir the colloidal gold solution and add monoclonal antibody, react for 20-40 minutes, add BSA, and centrifuge after stirring.

5. A method for detection of stichopus oligopeptide by using the colloidal gold test strip according to claim 2, wherein it comprises the following steps: 1) dissolve a sample in distilled water and drop it on the sample pad; 2) observe whether that detection line changes from colorless to red after the quality control line develops color; If the detection line turns red, the sample to be detected is not stichopus oligopeptide; If the detection line does not change color, the sample to be detected is stichopus oligopeptide.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 is a structural schematic diagram of a colloidal gold test strip prepared by using specific peptide fragments of the present invention.

[0024] In which: 1. Sample pad; 2. gold label pad; 3. NC membrane; 4. detection line; 5. quality control line; 6. absorbent pad; 7. PVC bottom plate.

[0025] FIG. 2 is a schematic diagram of the detection results of the colloidal gold test strip prepared by using specific peptide fragments of the present invention.

DESCRIPTION OF THE EMBODIMENTS

[0026] The invention is described in detail in combination with the drawings and specific embodiments, but it should not be understood as a limitation of the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1

[0027] Sequence Source and Alignment Information of Specific Peptide Fragments

[0028] 1. Sources of Stichopus-Specific Oligopeptide KIVPGVPD

[0029] Stichopus-specific oligopeptide KIVPGVPD, whose amino acid sequence is as shown in SEQ ID NO. 1, is derived from protein 2 alpha fibrillar collagen, whose Accesion number on NCBI is PIK60694, and whose amino acid sequence is as shown in SEQ ID NO. 2. After NCBI blast comparison, the peptide fragment was not consistent with any other species, and it is further identified as a new oligopeptide by online database BIOPEP and EROP-Moscow.

[0030] 2. Sources of Stichopus-specific oligopeptide GRDGDQGPV

[0031] Stichopus-specific oligopeptide GRDGDQGPV has an amino acid sequence as shown in SEQ ID NO. 3 and is derived from protein alpha-2 collagen. Its Accesion number on NCBI is PIK60696, and its amino acid sequence is shown in SEQ ID NO. 4. After NCBI blast comparison, the peptide fragment was not consistent with any other species, and it is further identified as a new oligopeptide by online database BIOPEP and EROP-Moscow.

Embodiment 2

[0032] Preparation of Colloidal Gold Test Strip

[0033] 1. Colloidal Gold Test Strip Prepared by Peptide Fragment KIVPGVPD

[0034] Dissolve 10 mg of BSA in 0.01 mol/L PBS (pH 7.4)(solution 1). Dissolve 4 mg of purified KIVPGVPD peptide fragment in 0.01 mol/L PBS (pH 7.4)(solution 2). Dissolve 4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 0.5 ml of 0.01 mol/L PBS (pH 7.4)(solution 3). Add solution 2 to solution 1, mix well in ice bath for 30 min, centrifuge to remove precipitation, add supernatant droplets into solution 3, continue ice bath for 15 min, and stir at room temperature for 5 min. Dialysis in 0.01 mol/L PBS (pH7.4) for 24 h, during this period, change the dialysate twice, and carry out centrifugation to remove precipitation to obtain the conjugate of the peptide fragment KIVPGVPD and bovine serum albumin; Immunize BALB/C mice with KIVPGVPD-BSA; The spleen lymphocytes of immunized mice and myeloma cell (SP2/0) is fused under the promotion of PEG, and the hybridoma cell line stably secreting KIVPGVPD-BSA monoclonal antibody is screened; The hybridoma cell line is grown in increments, and the obtained culture solution is purified by the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which is frozen and stored at −20° C. for later use.

[0035] Put an Erlenmeyer flask containing 100 ml of 0.01% chloroauric acid solution on a magnetic stirrer to heat and stir. Quickly add 2 ml of 1% trisodium citrate after boiling, and continue to boil for 5 minutes after the solution turns to wine red. After cooling to room temperature, make up to 100 ml with ultrapure water to obtain a colloidal gold solution with a gold particle diameter of about 10 nm. Adjust the pH value of the obtained 100 ml colloidal gold solution to 8.2 with 20 mol/L borate buffer, slowly add 45 μg of monoclonal antibody KIVPGVPD-BSA per ml of colloidal gold solution while stirring on a magnetic stirrer, and react for 30 minutes. Add 10% BSA to a final concentration of 1%, and gently stir for 10 minutes. Centrifuge at 45,000 rpm for 30 min at 4° C., discard the supernatant and the resulting precipitate is the purified colloidal gold-labeled monoclonal antibody. Spray this colloidal gold-labeled monoclonal antibody on the gold label pad, spray the KIVPGVPD-BSA conjugate on the NC film as a test line, spray goat anti-mouse IgG on NC membrane as a quality control line, then adhere the sample pad (glass fiber), gold label pad (glass fiber), NC film (including quality control line and test line) and the absorbent pad to a PVC bottom plate in sequence.

[0036] 2. Colloidal Gold Test Strip Prepared by Peptide GRDGDQGPV

[0037] Dissolve 10 mg of BSA in 0.01 mol/L PBS (pH 7.4)(solution 1). Dissolve 4 mg of purified GRDGDQGPV peptide fragment in 0.01 mol/L PBS (pH 7.4)(solution 2). Dissolve 4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 0.5 ml of 0.01 mol/L PBS (pH 7.4)(solution 3). Add solution 2 to solution 1, mix well in ice bath for 30 min, centrifuge to remove precipitation, then drop it into solution 3, continue ice bath for 15 min, and stir at room temperature for 5 min. Dialysis in 0.01 mol/L PBS (pH7.4) for 24 h, during this period, change the dialysate twice, and carry out centrifugation to remove precipitation to obtain the conjugate of the peptide fragment GRDGDQGPV and bovine serum albumin; Immunize BALB/C mice with GRDGDQGPV-BSA; The spleen lymphocytes of immunized mice and myeloma cell (SP2/0) is fused under the promotion of PEG, and the hybridoma cell line stably secreting GRDGDQGPV-BSA monoclonal antibody is screened; The hybridoma cell line is grown in increments, and the obtained culture solution is purified by the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which is frozen and stored at −20° C. for later use.

[0038] Put an Erlenmeyer flask containing 100 ml of 0.01% chloroauric acid solution on a magnetic stirrer to heat and stir. Quickly add 2 ml of 1% trisodium citrate after boiling, and continue to boil for 5 minutes after the solution turns to wine red. After cooling to room temperature, make up to 100 ml with ultrapure water to obtain a colloidal gold solution with a gold particle diameter of about 10 nm. Adjust the pH value of the obtained 100 ml colloidal gold solution to 8.2 with 20 mol/L borate buffer, slowly add 45 μg of monoclonal antibody GRDGDQGPV-BSA per ml of colloidal gold solution while stirring on a magnetic stirrer, and react for 30 minutes. Add 10% BSA in gold standard solution to a final concentration of 1%, and gently stir for 10 minutes. Centrifuge at 45,000 rpm for 30 min at 4° C., discard the supernatant and the resulting precipitate is the purified colloidal gold-labeled monoclonal antibody. Spray this colloidal gold-labeled monoclonal antibody on the gold label pad, spray the GRDGDQGPV-BSA conjugate on the NC film as a test line, spray goat anti-mouse IgG on NC membrane as a quality control line, then adhere the sample pad (glass fiber), gold label conjugate pad (glass fiber), NC film (including quality control line and test line) and the absorbent pad to a PVC bottom plate in sequence.

Embodiment 3

[0039] Detecting Stichopus Oligopeptide by Colloidal Gold Immunoassay

[0040] S1. Dissolve the sample with distilled water to 1 mg/ml, and add 3 drops to the sample pad of the colloidal gold test strip prepared in Embodiment 2;

[0041] S2. After the quality control line develops color, observe whether the detection line changes from colorless to red; if the detection line turns red, the sample to be tested is not stichopus oligopeptide; if the detection line does not change color, the sample to be tested is stichopus oligopeptide.

Embodiment 4

[0042] Sensitivity Detection of Colloidal Gold Test Strip

[0043] According to the method described in Embodiment 3, stichopus oligopeptide with 8 gradients, respectively 0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml, 100 μg/ml, and 1000 μg/ml are detected with the detection strip described in Embodiment 2, and the detection line is observed with naked eyes three times. Results are as shown in icon 1, it's found that a deep red band appears on the detection line when the concentration of stichopus oligopeptide is 0 μg/ml. With the increase of the concentration, the color of the detection line gradually weakens. When the concentration of stichopus oligopeptide increases to 20 μg/ml, there is no red band on the detection line. When the concentration of stichopus oligopeptide increases to 1000 μg/ml, there is no red band on the detection line and red band appears on the quality control line. Therefore, it is judged that the detection sensitivity of the test strip is 20 μg/ml.

TABLE-US-00001 TABLE 1 Sensitivity detection results of colloidal gold test strips in the present invention Stichopus oligopeptide/ Parallel sample μg/ml 1 2 3 0 − − − 5 − − − 10 − − − 20 + + + 40 + + + 80 + + + 1000 + + + Note: “+” means positive, “−” means negative

Embodiment 5

[0044] Specificity Detection of Colloidal Gold Test Strip

[0045] Prepare stichopus oligopeptide, soybean oligopeptide, marine fish oligopeptide and oyster oligopeptide respectively into 1 mg/ml samples, and detect them according to the method described in Embodiment 3. The results are as shown in Table 2. The results show that there is no color in the detection line of stichopus oligopeptide samples, and while a red band appears in the quality control line. Red bands appear in the sample detection lines of soybean oligopeptide, marine fish oligopeptide and oyster oligopeptide, and red bands also appear in the quality control line, which indicates that the test strip has good specificity.

TABLE-US-00002 TABLE 2 Specificity detection results of colloidal gold test strips in the present invention Stichopus Soybean Marine fish Oyster Sample oligopeptide oligopeptide oligopeptide oligopeptide Result + − − − Note: “+” means positive, “−” means negative

[0046] It should be noted that the colloidal gold labeled monoclonal antibody in Embodiment 2 may also be a mixture of a specific anti-KIVPGVPD monoclonal antibody and a specific anti-GRDGDQGPV monoclonal antibody; preferably, the mass ratio of the specific anti-KIVPGVPD monoclonal antibody to the specific anti-GRDGDQGPV monoclonal antibody is 1:1.

[0047] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected. For the sake of avoiding elaboration, the present invention describes the preferred embodiments.

[0048] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments once those skilled in the art have become aware of the basic inventive concepts. As such, that append claims are intended to be interpreted as including the preferred embodiments and all alterations and modification falling within the scope of the invention.

[0049] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.