H. pylori cag a antibody test strip and application thereof

Abstract

The application relates to a Helicobacter pylori CagA antibody detection test paper and application thereof, and belongs to the technical field of biology. The application provides a Helicobacter pylori CagA antibody detection test paper, wherein a fusion protein is used as a detection antigen of the Helicobacter pylori CagA antibody, the fusion protein comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, or a derivative polypeptide with more than 80% homology with the amino acid sequence shown in SEQ ID NO. 1 and capable of being specifically combined with the Helicobacter pylori CagA antibody; the fusion protein used by the test paper can be specifically combined with the Helicobacter pylori CagA antibody, and has a very high application prospect in Helicobacter pylori CagA antibody detection; the fusion protein used by the test paper has a high expression amount, a high uniformity and a high purity, and is stable and not easy to be denatured.

Description

Technical Field

[0001] This invention relates to a Helicobacter pylori CagA antibody test strip and its application, belonging to the field of biotechnology. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori Helicobacter pylori (H. pylori) is a Gram-negative bacterium that is S-shaped or curved. In 1982, Barry J. Marshall and J. Robin Warren isolated and cultured Helicobacter pylori from human gastric mucosal specimens, thus successfully revealing its potential pathogenic mechanism. They were awarded the 2005 Nobel Prize in Physiology or Medicine for this achievement. In 1994, Helicobacter pylori was classified as a Group 1 carcinogen by the World Health Organization.

[0003] Helicobacter pylori infection is a long-term and chronic process. Once infected, the body generally cannot clear it spontaneously, leading to lifelong infection. Only through eradication therapy or severe intestinal metaplasia of the gastric mucosa, making bacterial colonization difficult, will Helicobacter pylori disappear from the body automatically. Studies have shown that long-term Helicobacter pylori infection can cause chronic gastritis, and as the infection deepens and the condition worsens, some patients may develop duodenal ulcers or even gastric cancer. Therefore, detecting Helicobacter pylori antibodies in a patient's blood in vitro to infer whether the patient has been infected or is currently infected with Helicobacter pylori, and then providing timely intervention for those infected, is crucial for protecting the patient's health. Summary of the Invention

[0004] To address the above problems, this invention provides a Helicobacter pylori CagA antibody detection strip, wherein the strip uses a fusion protein as the detection antigen for Helicobacter pylori CagA antibodies; the fusion protein: (a) A polypeptide with the amino acid sequence shown in SEQ ID NO.1; And / or, (b) a derivative polypeptide that has more than 80% homology with the amino acid sequence shown in SEQ ID NO.1 and is capable of specifically binding to Helicobacter pylori CagA antibody.

[0005] In one embodiment of the present invention, the test strip includes a chromatography membrane; one end of the chromatography membrane is connected to an adsorption pad; the adsorption pad carries the immunolabeled fusion protein and the immunolabeled first antibody; a detection line and a control line are sequentially arranged on the chromatography membrane along the liquid chromatography direction; the detection line is coated with the fusion protein; the control line is coated with a second antibody; the second antibody is capable of specifically binding to the first antibody.

[0006] In one embodiment of the present invention, the loading of the fusion protein on the colloidal gold adsorption pad is 0.1~1 μg / cm³.2 .

[0007] In one embodiment of the present invention, the loading of the fusion protein on the colloidal gold adsorption pad is 0.5 μg / cm³. 2 .

[0008] In one embodiment of the present invention, the loading amount of the first antibody on the colloidal gold adsorption pad is 0.1~5 μg / cm³. 2 .

[0009] In one embodiment of the present invention, the loading of the first antibody on the colloidal gold adsorption pad is 0.3 μg / cm³. 2 .

[0010] In one embodiment of the present invention, the detection line is formed by coating the detection line location with a fusion protein solution of concentration of 0.5~1.5 mg / mL at a liquid volume of 0.5~1.5 μL / cm.

[0011] In one embodiment of the present invention, the detection line is formed by coating the detection line location with a fusion protein solution of concentration of 1 mg / mL at a liquid volume of 1 μL / cm.

[0012] In one embodiment of the present invention, the control line is formed by coating the control line location with a second antibody solution of a concentration of 1~2 mg / mL at a liquid volume of 1~2 μL / cm.

[0013] In one embodiment of the present invention, the control line is formed by coating the control line location with a second antibody solution of concentration of 1.5 mg / mL at a liquid volume of 1.5 μL / cm.

[0014] In one embodiment of the present invention, the immunolabeling is colloidal gold labeling, colloidal arsenic labeling, colloidal carbon labeling, colored latex labeling, or fluorescent latex labeling.

[0015] In one embodiment of the present invention, the first antibody is a rabbit IgG antibody; the second antibody is a goat anti-rabbit IgG antibody.

[0016] In one embodiment of the present invention, the test strip further includes a base plate, a sample pad, and an absorbent pad; the base plate is provided with the sample pad, the adsorption pad, the chromatography membrane, and the absorbent pad in sequence along the liquid chromatography direction.

[0017] The present invention also provides a method for detecting Helicobacter pylori CagA antibodies, wherein the method uses the above-mentioned Helicobacter pylori CagA antibody test strip to detect the sample to be tested.

[0018] In one embodiment of the present invention, the sample to be tested is whole blood or serum.

[0019] This invention also provides the application of the above-described test strip or method in detecting Helicobacter pylori CagA antibodies.

[0020] The technical solution of this invention has the following advantages: This invention provides a Helicobacter pylori CagA antibody detection strip. The strip uses a fusion protein as the detection antigen for Helicobacter pylori CagA antibodies. The fusion protein includes a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, or a derived polypeptide with more than 80% homology to the amino acid sequence shown in SEQ ID NO.1 and capable of specifically binding to Helicobacter pylori CagA antibodies. The fusion protein used in the strip can specifically bind to Helicobacter pylori CagA antibodies and has extremely high application prospects in the detection of Helicobacter pylori CagA antibodies. The fusion protein used in the strip has a high expression level, high uniformity and purity, and is stable and not easily denatured. Attached Figure Description

[0021] Figure 1 Agarose gel electrophoresis results of the amplification products.

[0022] Figure 2 Results of SDS-PAGE gel electrophoresis analysis of purified proteins.

[0023] Figure 3 Schematic diagram of the overall structure of the Helicobacter pylori CagA antibody test strip.

[0024] Figure 4 : Positive sample test result.

[0025] Figure 5 Negative sample test result.

[0026] Figure 3 The components are: base plate 1, sample pad 2, adsorption pad 3, chromatography membrane 4, absorbent pad 5, detection line T, and control line C. Detailed Implementation

[0027] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0028] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0029] Example 1: Fusion Protein and its Preparation This embodiment provides a fusion protein that can specifically bind to Helicobacter pylori CagA antibody; the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0030] The preparation process of the fusion protein is as follows: 1. Preparation of recombinant bacteria A gene encoding the fusion protein HP2 with the nucleotide sequence shown in SEQ ID NO.2 was synthesized; the synthesized gene was amplified by PCR using upstream and downstream primers, and restriction enzyme sites were added. Bam HⅠ / Xho I. Obtain the amplification products (the amplification products are recovered by agarose gel electrophoresis; the agarose gel electrophoresis results of the amplification products are shown in the figure). Figure 1 The amplification product was mixed with pM1-C2 plasmid (purchased from Ubisoft) and restriction endonuclease was used. Bam HⅠ / Xho I. (Purchased from NEB) After enzyme digestion and ligation, the ligation product was obtained; the ligation product was transformed into E. coli ( Escherichia coli DH5α (purchased from Beijing Qingke Biotechnology Co., Ltd.) was used to obtain the transformation product. The transformation product was plated on LB solid medium (purchased from Solarbio) containing 100 μg / mL ampicillin and incubated upside down in a 37℃ incubator for 12 h to obtain transformants. Transformants were picked and inoculated into 5 mL of LB liquid medium (purchased from Solarbio) containing 100 μg / mL ampicillin and cultured in shake flasks at 37℃ and 180 rpm for 12 h. The plasmid was then extracted for enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant strain DH5α / pMA1-C2- was obtained. hp2 .

[0031] 2. Fermentation of recombinant bacteria Pick the recombinant bacteria DH5α / pMAl-C2- obtained in step 1 hp2 Single colonies were inoculated into 500 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 180 rpm for 12 h to obtain the culture medium. The entire culture medium was then transferred to 1000 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 220 rpm for 4 h until OD reached the target concentration. 600 When the concentration of the molecule is 0.8, the temperature is lowered to 16℃ and cultured with shaking at 180 rpm for 1 h. After 1 h, IPTG (final concentration 1 mM) is added and fermented at 16℃ and 180 rpm for 12 h to obtain the fermentation broth.

[0032] 3. Purification and identification of fusion proteins The fermentation broth was centrifuged at 8000 rpm for 10 min to collect the bacterial cells. 50 mL of PBS buffer (pH 7.4, 0.01 M, Thermo Fisher Scientific) was added to the bacterial cells for resuspending. The resuspending was then disrupted three times using a high-pressure homogenizer at 800 bar to obtain the disruption solution. The disruption solution was centrifuged at 12000 rpm for 40 min, and the supernatant was collected. The supernatant was purified using an MBP column (purchased from Beijing Bio-Rad Laboratories) to obtain the purified protein. (The purified protein was analyzed by SDS-PAGE gel electrophoresis; the results are shown in [link to analysis]). Figure 2 ); The MBP column purification process is as follows: (1) Drop the preservation solution from the MBP column, add 10 mL of water to wash the MBP column, and repeat twice; (2) Add 10 mL of MBP equilibration solution to equilibrate the column, and repeat twice; (3) Add the supernatant to the MBP column; (4) Wash the column twice with 10 mL of equilibration buffer to remove any extraneous proteins; (5) Elute the protein with 4 mL of elution buffer and collect it.

[0033] The purified protein was further purified using the Dextrilin Sepharose High Performance (Lot: 10288766) kit (purchased from GE) to obtain the fusion protein. The protein concentration was measured to be 2.6 mg / mL using a UV spectrophotometer.

[0034] Example 2: Colloidal gold test strip for detecting Helicobacter pylori CagA antibody and its preparation like Figure 3 As shown, this embodiment provides a Helicobacter pylori CagA antibody test strip, which includes a base plate 1; the base plate 1 is provided with a sample pad 2, an adsorption pad 3, a chromatography membrane 4, and an absorbent pad 5 sequentially along the liquid chromatography direction; the adsorption pad 3 carries the colloidal gold-labeled fusion protein of Example 1 and colloidal gold-labeled rabbit IgG antibody; the chromatography membrane 4 is provided with a detection line T and a control line C sequentially along the liquid chromatography direction; the detection line T is coated with the fusion protein of Example 1; the control line C is coated with goat anti-rabbit IgG antibody; The colloidal gold-labeled fusion protein was loaded onto the colloidal gold adsorption pad at a loading rate of 0.5 μg / cm³. 2 The loading capacity of the colloidal gold-labeled rabbit IgG antibody on the colloidal gold adsorption pad was 0.3 μg / cm³. 2The detection line is formed by coating the detection line position with a fusion protein solution labeled with colloidal gold at a concentration of 1 mg / mL at a liquid volume of 1 μL / cm; the control line is formed by coating the control line position with a goat anti-rabbit IgG antibody solution labeled with colloidal gold at a concentration of 1.5 mg / mL at a liquid volume of 1.5 μL / cm.

[0035] The preparation method of the Helicobacter pylori CagA antibody test strip is as follows: 1. Mark 1.1 Burning gold 1) Measure 792 mL of process water into a triangular flask using a graduated cylinder. Use a pipette to add 8 mL of 2% (w / v, g / 100 mL) aqueous solution of chloroauric acid (purchased from Aladdin Company) into the triangular flask to make the final concentration of chloroauric acid reach 0.02% (w / v). Shake well and heat on a 2000-watt electric furnace until it boils completely. 2) After boiling completely, quickly add 15 mL of 1% (w / v) trisodium citrate (purchased from Aladdin Company) solution to the Erlenmeyer flask; continue heating. At this time, it can be observed that the pale yellow chloroauric acid aqueous solution quickly turns gray after the addition of trisodium citrate, then turns black, and then gradually stabilizes into red. The whole process takes 2-3 minutes. After 10 minutes, the deep red color no longer changes, so stop heating. 3) Cool the colloidal gold to room temperature (25°C) and dissolve it in process water to the initial volume. Set aside. The colloidal gold in this bottle is clear and transparent, resulting in a double gold solution.

[0036] 1.2 Adjusting pH 1) Take a 10 mL test tube and use a pipette to take 1 mL of the double gold solution prepared in step 1.1 into the test tube; 2) Take 1 μL of 0.2 mol / L K2CO3 and 1 μL of antigen (the antigens are the fusion protein purified in Example 1 and rabbit IgG with a stock solution concentration of 16.8 mg / mL purchased from Sigma) into a test tube, shake gently, and observe the color change. If the color changes, gradually increase the amount of K2CO3 until the color does not change (in this experiment, the pH of the fusion protein is set to 0.6%, and the pH of the rabbit IgG is set to 0.8%).

[0037] 1.3 Labeling of rabbit IgG (pH: 0.8%, final concentration 20 μg / mL) 1) Take 60 mL of the double gold solution prepared in step 1.1 and place it in a clean small beaker. Stir it at a constant speed with a magnetic stirrer. Add 480 μL of 0.2 mol / L K2CO3 and 71.5 μL of rabbit IgG solution prepared in step 1.2 and stir for 15 min. Add 300 μL of 10% (w / v) milk powder and stir for 15 min. 2) Fill the cleaned centrifuge tubes with the solution prepared in step 1), weigh and balance them with an electronic balance, centrifuge at 12000 rpm for 15 min in a low-temperature high-speed centrifuge, remove and aspirate the supernatant, discard it, collect the precipitate, and reconstitute the precipitate with 3 mL of 5% (v / v) TuIgG diluent (purchased from Sigma) to obtain the labeled rabbit IgG solution.

[0038] 1.4 Fusion protein labeling (pH: 0.6%, final concentration of fusion protein: 8 μg / mL) 1) Take 50 mL of the double gold solution obtained in step 1.1 and place it in a clean small beaker. Stir it evenly with a magnetic stirrer. Add 300 μL of 0.2 mol / L K2CO3 (50 mL) Add 153.8 μL of the fusion protein solution (8 μg / mL) / 2.6 mg / mL obtained in step 1.2 and stir for 15 min. Then add 250 μL of 10% (w / v) milk powder and stir for 15 min. 2) After filling and equilibrating the solution into clean centrifuge tubes, centrifuge at 12000 rpm for 15 min in a low-temperature high-speed centrifuge. Remove the supernatant, discard it, and collect the precipitate. Reconstitute the precipitate with 5% (v / v) TuIgG diluent (purchased from Sigma) to 2 mL, add 400 μL of labeled rabbit IgG solution, and bring the volume up to 3 mL with the reconstitution solution (0.01 M PBS buffer containing 2% (w / v) BSA and 0.02% (w / v) thimerosal) to obtain the labeled fusion protein solution. 3) Take one colloidal gold adsorption pad, sputter gold wire concentration of 3.0 μL / cm, dry in the drying room for 4 hours, and store.

[0039] 2. Wrapped 2.1 T-line Example: Prepare 100 μL of T line with a membrane concentration of 0.8 mg / mL and a fusion protein concentration of 2.6 mg / mL.

[0040] Amount of fusion protein: (100 μL) The fusion protein solution obtained in step 1.2 (0.8 mg / mL / 2.6 mg / mL) was 30.8 μL. Alcohol: 100μL / 10, which is 10 μL; Base solution (0.01 M PBS buffer + 2% (w / v) trehalose): 100 μL - amount of fusion protein - amount of alcohol, totaling 59.2 μL.

[0041] The above solutions were sequentially sampled into 500 μL EP tubes and mixed using a vortex mixer.

[0042] 2.2 Line C Example: Prepare 100 μL of C line with a coating concentration of 0.8 mg / mL and goat anti-rabbit IgG (purchased from Sigma) concentration of 12.8 mg / mL.

[0043] Amount of antigen: (100 μL) (1.5 mg / mL) / 12.8 mg / mL, which equals 11.7 μL; Alcohol: 100μL / 10, which is 10 μL; Base solution (0.01M PBS buffer + 2% (w / v) trehalose): 100 μL - amount of fusion protein - amount of alcohol, totaling 59.2 μL.

[0044] The above solutions were sequentially sampled into 500 μL EP tubes and mixed using a vortex mixer.

[0045] 2.3 Processing The prepared C and T line solutions are coated on a coating machine, and after coating, they are placed in a 4°C refrigerator for no less than 4 hours. They are then soaked in the five-item treatment solution for 30 minutes, coated with film, and dried under drying conditions.

[0046] 3. Composite A handle paper is attached to one side of the membrane C line, overlapping the membrane surface by 1 mm. Gold is cut into strips and attached to one side of the membrane T line. The HP carrier is attached, and an adhesive tape is attached to the gold. The strips are then cut into 3.0 mm wide test strips to obtain the Helicobacter pylori CagA antibody test strip.

[0047] Example 3: Application of colloidal gold test strips for detecting Helicobacter pylori CagA antibodies 1000 negative samples and 100 positive samples (blood samples, from Henan Provincial Center for Disease Control and Prevention) were collected. The Helicobacter pylori CagA antibody test strip of Example 2 was used to detect Helicobacter pylori CagA antibodies in the samples. A positive result was indicated by the appearance of one band on both the test line and the control line; a negative result was indicated by the appearance of only one band on the control line. The test results are as follows: The negative compliance rate is greater than 95% ( Figure 4 (This refers to the test results of some negative samples); the positive detection rate is greater than 96% ( Figure 5 (Results of partial positive samples). It is evident that the accuracy of using the Helicobacter pylori CagA antibody test strip of Example 2 for detecting Helicobacter pylori CagA antibodies in samples is high. Using the Helicobacter pylori CagA antibody test strip of Example 2 to detect the presence of Helicobacter pylori antibodies in patient blood samples in vitro, in order to infer whether a patient has been infected or is currently infected with Helicobacter pylori, shows great promise for application.

[0048] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention. sequence list <110> Nantong Yishi Biotechnology Co., Ltd. <120> A Helicobacter pylori CagA antibody test strip and its application <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 676 <212> PRT <213> Artificial sequence <400> 1 Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val Asp 1 5 10 15 Asn Ala Val Ala Ser Tyr Asp Pro Asp Gln Lys Pro Ile Val Asp Lys 20 25 30 Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Gly Ile Ser Gln Leu Arg 35 40 45 Glu Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn Gln 50 55 60 Tyr Phe Ser Asp Phe Ile Glu Lys Ser Asn Asp Leu Ile Asn Lys Asp 65 70 75 80 Asn Leu Ile Asp Val Glu Ser Ser Thr Glu Ser Phe Arg Lys Phe Gly 85 90 95 Asp Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn Asp 100 105 110 Pro Ser Lys Ile Asn Thr Arg Ser Ile Arg Asn Phe Met Glu His Thr 115 120 125 Ile Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu Lys 130 135 140 Ser Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile Arg 145 150 155 160 Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu Arg 165 170 175 Gln Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu Asp 180 185 190 Ile Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys 195 200 205 Glu Ala Ile Asn Gln Glu Pro Val Pro His Val Gln Pro Asp Ile Ala 210 215 220 Thr Ser Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp Leu 225 230 235 240 Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met Glu 245 250 255 Met Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe 260 265 270 Asn Gln Leu Leu Ile His Asn Asn Ala Leu Ser Ser Val Leu Met Gly 275 280 285 Ser His Asp Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala Gly 290 295 300 Asn Gly Gly Phe Gly Asp Lys His Asp Trp Asn Ala Thr Val Gly Tyr 305 310 315 320 Lys Asp Gln Gln Gly Asn Asn Val Ala Thr Ile Ile Asn Val His Met 325 330 335 Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn 340 345 350 Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln 355 360 365 Arg Ala Leu Ser Gln Glu Glu Ile Leu Asn Lys Ile Asp Phe Met Glu 370 375 380 Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Asn Leu Ser Glu Lys Glu 385 390 395 400 Lys Glu Lys Phe Arg Asn Glu Ile Lys Asp Phe Gln Lys Asp Ser Lys 405 410 415 Pro Tyr Leu Asp Ala Leu Gly Asn Asp Arg Ile Thr Phe Val Ser Lys 420 425 430 Lys Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Asn Lys Gly Asp 435 440 445 Leu Ser Tyr Thr Leu Lys Asp Tyr Gly Lys Lys Ala Asp Lys Ala Leu 450 455 460 Asp Arg Glu Lys Asn Val Thr Leu Gln Gly Ser Leu Lys His Asp Gly 465 470 475 480 Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser Lys 485 490 495 Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu Glu 500 505 510 Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn Asn 515 520 525 Leu Ala Ile Thr Ser Val Val Arg Arg Asp Leu Glu Asp Lys Leu Ile 530 535 540 Ala Lys Gly Leu Pro Pro Gln Glu Ala Asn Lys Leu Val Lys Gly Phe 545 550 555 560 Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys 565 570 575 Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg 580 585 590 Ala Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu Arg Leu Glu 595 600 605 Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn Lys 610 615 620 Met Glu Ala Lys Ser Gln Ala Asn Ser Gln Lys Asp Glu Ile Phe Ala 625 630 635 640 Leu Ile Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala Ile Ala Tyr Thr 645 650 655 Gln Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glu Asn 660 665 670 Ile Asn Lys Asp 675 <210> 2 <211> 2043 <212> DNA <213> Artificial sequence <400> 2 ccgcagcagt tcatcaacaa cctccaagtt gcgttcctga aagttgacaa cgctgttgct 60 tcttacgacc cggaccagaa accgatagtt gacaaaaacg accgtgacaa tcgtcaggcg 120 ttcgaaggta tctcgcagct gcgtgaagaa tactctaaca aagctatcaa aaacccgacc 180 aaaaaaaacc agtacttctc tgacttcatc gaaaaatcta acgacctgat aaacaaagac 240 aacctgatag acgttgaatc ttctaccgaa tctttccgta aattcggtga ccagcgttac 300 cgtatcttca cctcttgggt ttctcaccag aacgacccgt ctaaaatcaa cacccgttct 360 atccgtaact tcatggaaca caccatccag ccgccgatac cggacgacaa agaaaaagct 420 gaatttctga aatctgctaa acagtctttc gctggtatca tcatcggtaa ccagatacgt 480 accgaccaga aattcatggg tgttttcgac gaatctctga aagaacgtca ggaagctgaa 540 aaaaacggtg gtccgaccgg tggtgactgg ctcgacatct tcctgagctt catcttcgac 600 aagaaacagt cttctgacgt taaagaagct atcaaccagg aaccggttcc gcacgttcag 660 ccggacatcg ctacctctac cacccacatc cagggtctgc cgccggaatc tcgtgacctg 720 ctggacgaac gtggtaactt ctctaaattc accctgggtg acatggaaat gctggacgtt 780 gaaggtgttg ctgacatgga cccgaactac aaattcaacc agctgctgat acacaacaac 840 gctctgtctt ctgttctgat gggttctcac gacggtatcg aaccggaaaa agtttctctg 900 ctgtacgctg gtaacggtgg tttcggtgac aaacacgact ggaacgctac cgttggttac 960 aaagaccagc agggtaacaa cgttgctacc atcatcaacg ttcacatgaa aaacggttct 1020 ggtctggtta tcgctggtgg tgaaaaaggt atcaacaacc cgtctttcta cctgtacaaa 1080 gaagaccagc tgaccggttc tcagcgtgct ctgtctcagg aagaaatcct gaacaaaatc 1140 gacttcatgg aatttctggc tcagaacaac gctaaactgg acaacctgtc tgaaaaagaa 1200 aaagaaaaat tccgtaacga aatcaaagac ttccagaaag actctaaacc gtacctggac 1260 gctctgggta acgaccgtat caccttcgtt tctaaaaaag acccgaaaca ctctgctctg 1320 ataaccgaat ttaacaaagg tgacctgtct tacaccctga aagactacgg taaaaaagct 1380 gacaaagctc tggaccgtga aaaaaacgtt accctgcagg gttctctgaa acacgacggt 1440 gttatgttcg ttaactactc taacttcaaa tacaccaacg cttctaaatc tccgaacaaa 1500 ggtgttggcg ttactaacgg tgtaagccac ctggaggcgg gcttctctaa agttgcggtt 1560 ttcaacctgc cgaacctgaa caacctggct atcacctctg ttgttcgtcg tgacctggaa 1620 gacaaactga tagctaaagg tctgccgccg caggaagcta acaaactggt taaaggtttc 1680 ctgtcttcta acaaagaact ggttggtaaa gctctgaact tcaacaaagc tgttgctgaa 1740 gctaaaaaca ccggtaacta cgacgaagtt aaacgtgctc agaaagacct ggaaaaatct 1800 ctgaaaaaac gtgaacgtct ggaaaaagac gttgctaaaa acctggaatc taaatctggt 1860 aacaaaaaca aaatggaagc taaatctcag gctaactctc agaaagacga aatcttcgct 1920 ctgataaaca aagaagctaa ccgtgacgct cgtgctatcg cttacaccca gaacctgaaa 1980 ggtatcaaac gtgaactgtc tgacaaactg gaaaacatca acaaagacct gaaagacttc 2040 tct 2043 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <400> 3 cgcggatcca tgccgcagca gttcatcaac aacctc 36 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <400> 4 ccgctcgagt taagagaagt ctttcaggtc tttgtt 36

Claims

1. A Helicobacter pylori CagA antibody detection strip, characterized in that, The test strip uses the fusion protein as the detection antigen for Helicobacter pylori CagA antibody; the amino acid sequence of the fusion protein is shown in SEQ ID NO.1; The test strip includes a chromatography membrane; one end of the chromatography membrane is connected to an adsorption pad; the adsorption pad carries the immunolabeled fusion protein and the immunolabeled first antibody; a detection line and a control line are sequentially arranged on the chromatography membrane along the liquid chromatography direction; The detection line is coated with the aforementioned fusion protein; the quality control line is coated with a second antibody; the second antibody can specifically bind to the first antibody. The immunolabeling is colloidal gold labeling, colloidal carbon labeling, colored latex labeling, or fluorescent latex labeling.

2. The Helicobacter pylori CagA antibody test strip as described in claim 1, characterized in that, The loading capacity of the fusion protein on the colloidal gold adsorption pad is 0.1~1 μg / cm³. 2 .

3. The Helicobacter pylori CagA antibody test strip as described in claim 1, characterized in that, The loading capacity of the first antibody on the colloidal gold adsorption pad was 0.1~5 μg / cm³. 2 .

4. The Helicobacter pylori CagA antibody test strip as described in claim 1, characterized in that, The detection line is formed by coating the detection line location with a fusion protein solution of concentration of 0.5~1.5 mg / mL at a liquid volume of 0.5~1.5 μL / cm.

5. The Helicobacter pylori CagA antibody test strip as described in claim 1, characterized in that, The control line is formed by coating the control line location with a second antibody solution of concentration of 1~2 mg / mL at a liquid volume of 1~2 μL / cm.

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