Indirect ELISA detection method and kit for Giardia duodenalis CP1 and CP2 genes

Abstract

This invention constructs pET-32a-CP1 and pET-32a-CP2 expression vectors based on the CP1 and CP2 protein genes of Giardia duodenalis, transforms them into Rosetta (DE3) competent cells, and induces expression using IPTG. After SDS-PAGE analysis of the recombinant protein expression, Ni-NTA affinity chromatography is used to purify the proteins. Two indirect ELISA methods are then established using these as coating antigens. The indirect ELISA methods based on the recombinant proteins CP1 and CP2 of this invention exhibit good specificity, sensitivity, and repeatability, with concordance rates of 98% and 94% with microscopic detection results, respectively.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to the expression vectors of Giardia lamblia CP1 and CP2 genes, the establishment of an indirect ELISA method, and a detection kit. Background Technology

[0002] Giardiasis is one of the most prevalent intestinal parasitic diseases worldwide, caused by Giardia, a species belonging to the phylum Sarcoptera, class Zoocybe, order Ditrichomoniata, family Hexaciridae, and genus Giardia. Symptoms primarily include poor growth, weight loss, and decreased productivity due to diarrhea. Extraintestinal complications include cognitive impairment, eye diseases, arthritis, allergies, hypokalemic myopathy, and even cancer. In 2004, Giardia lamblia, along with Cryptosporidium, was included in the WHO's List of Neglected Diseases. Currently, there is no vaccine for Giardia, but metronidazole is a relatively effective treatment.

[0003] The life cycle of Giardia lamblia is relatively simple, consisting of only two forms: cysts and trophozoites. Cysts detach with the host's feces. These cysts have a relatively thick wall, exhibiting moderate resistance to inactivation by various disinfectants (such as chlorine), and are environmentally stable, remaining infectious for several months in water or in cool, damp environments. The minimum infectious dose is considered to be as low as 10 cysts. When a host ingests Giardia lamblia cysts, the cysts exuviae in the duodenum to form trophozoites, which reproduce by longitudinal binary fission. If a trophozoite falls into the intestinal lumen and reaches the lower ileum or colon, it forms a cyst, which is then excreted in the feces, beginning a new infection cycle.

[0004] Giardia lamblia are transmitted via the fecal-oral route. Waterborne transmission is currently recognized as the most widespread route of transmission. Giardia lamblia cysts have been detected in various food products, such as fresh produce (leafy green vegetables, herbs, berries, onions, carrots, tomatoes, etc.), dairy products, meat, shellfish, and processed foods. Insects can also act as vectors under certain circumstances; Giardia lamblia cysts can survive for 3-4 days in some ticks and about 10 days in cockroaches.

[0005] Currently, the "gold standard" for diagnosing Giardia infection remains microscopic examination. However, this method is time-consuming, labor-intensive, subjective, requires extensive professional knowledge, has a low detection rate and low sensitivity, and is prone to false negatives when infection rates are low. Therefore, more and more immunological detection methods are being promoted, among which ELISA stands out due to its high specificity, ease of operation, and suitability for mass testing. Although several commercially available immunological diagnostic kits for Giardiasis are now on the market, several problems remain: First, most are imported products with high prices, making them difficult to distribute in the domestic market; second, when detecting antibodies, it is difficult to distinguish between current and past infections; third, when testing fecal samples, the antigen requirements are high, and it is difficult to distinguish cyst activity; fourth, various immunodiagnostic techniques have significant differences and insufficient stability, requiring further technical improvements and standardization in practical applications. In the event of an outbreak or epidemic, this detection method cannot meet the demand for testing large numbers of samples in a short period. In contrast, immunological detection methods have high specificity, high sensitivity, are simple to operate, and are easy to promote, demonstrating good application and development prospects. However, there is currently no immunological detection method specifically for Giardia duodenalis. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide expression vectors for Giardia lamblia CP1 and CP2 genes and to establish an indirect ELISA method, thereby opening up new avenues for the further development of colloidal gold test strips.

[0007] In one aspect, the present invention provides an indirect ELISA detection kit for the Giardia lamblia CP1 or CP2 gene, the kit comprising a recombinant protein CP1 polyclonal antibody or a recombinant protein CP2 polyclonal antibody as a primary antibody.

[0008] Furthermore, the titers of the recombinant protein CP1 polyclonal antibody and the recombinant protein CP2 polyclonal antibody were detected using an indirect ELISA method, and the titers were above 128,000.

[0009] Furthermore, the recombinant protein CP1 polyclonal antibody or recombinant protein CP2 polyclonal antibody is prepared by immunizing New Zealand white rabbits with the recombinant protein of the CP1 or CP2 gene as an antigen according to the immunization program, and then collecting blood from the carotid artery after the titer is qualified by ELISA.

[0010] Furthermore, the recombinant protein of the CP1 or CP2 gene is prepared by the following method:

[0011] The CP1 or CP2 specific primers were used to amplify the total genome extracted from Giardia trophozoites as a template to obtain the CP1 and CP2 genes. These genes were then ligated into the cloning vector pMD18-T to construct the recombinant plasmids pMD18-T-CP1 and pMD18-T-CP2, respectively.

[0012] The constructed recombinant plasmids pMD18-T-CP1 and pMD18-T-CP2 were transformed into competent DH5α cells for amplification culture. The pMD18-T-CP1 and pMD18-T-CP2 cloning vectors with correct sequencing results were subjected to a double enzyme digestion reaction with the pET-32a(+) vector. The digested CP1 and CP2 genes were ligated with the digested pET-32a(+) vector to prepare the pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids.

[0013] The above-mentioned pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids were transformed into Rosetta(DE3) competent cells, and the recombinant protein expression was induced by IPTG. The recombinant proteins of CP1 or CP2 were then purified.

[0014] Furthermore, the sequences of the specific primers for the CP1 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2; and the sequences of the specific primers for the CP2 gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0015] In a second aspect, the present invention provides an indirect ELISA detection method for the Giardia lamblia CP1 or CP2 gene, the detection method comprising:

[0016] (1) Preparation: Lyse the sample to be tested, centrifuge, transfer the supernatant to a new sterile centrifuge tube, add 10 μL of protease inhibitor to obtain the lysate;

[0017] (2) Antigen coating: Add 100 μL of the above lysis buffer to each well and coat overnight at 4°C;

[0018] (3) Washing: Discard the liquid in the well, wash with PBST, shake and pat dry on absorbent paper;

[0019] (4) Blocking: Add 200 μL of blocking solution to each well and block at 37 °C for 1 h. Then repeat step (3).

[0020] (5) Primary antibody incubation: The above-mentioned recombinant protein CP1 polyclonal antibody or recombinant protein CP2 polyclonal antibody was used as the primary antibody, and the serum was diluted with the primary antibody dilution buffer; 100 μL of serum of each dilution factor was added to each well of the ELISA plate; incubated at 37 ℃, and then step (3) was repeated.

[0021] (6) Secondary antibody incubation: horseradish peroxidase-labeled goat anti-rabbit IgG was diluted in secondary antibody dilution buffer, 100 mL was added to each well, and incubated at room temperature. Then step (3) was repeated.

[0022] (7) Reaction: Add 100 μL of TMB substrate solution to each well for colorimetric reaction and incubate at 37 °C for 15 min;

[0023] (8) OD value determination: Use 50 μL of ELISA stop reaction solution for each well, and read the OD450 value with a microplate reader within 30 min. If the P value (OD value of the sample to be seen: OD value of the negative control) > 2.1, it is considered positive.

[0024] Furthermore, in step (2), the antigen coating amount is 0.25 ug / mL.

[0025] Furthermore, when using recombinant protein CP1 monoclonal antibody as primary antibody, the primary antibody dilution factor in step (5) is 1600 and the primary antibody incubation time is 1 h; the secondary antibody dilution factor in step (6) is 20000 and the secondary antibody incubation time is 1 h.

[0026] Furthermore, when using recombinant protein CP2 monoclonal antibody as primary antibody, the primary antibody dilution factor in step (5) is 3200 and the primary antibody incubation time is 1.5 h; the secondary antibody dilution factor in step (6) is 20000 and the secondary antibody incubation time is 1 h.

[0027] In a third aspect, the present invention also provides a method for detecting Giardia lamblia, the method comprising:

[0028] The OD450 of CP1 and CP2 protein-negative serum samples was detected using the indirect ELISA method described above. The mean (AV) and standard deviation (SD) within each group were calculated. The AV+3×SD value was calculated as the positive cutoff value, and the AV+2×SD value was calculated as the negative cutoff value. Values ​​between the two were considered suspicious and required retesting.

[0029] Furthermore, the number of negative serum samples is more than 20.

[0030] Technical effect

[0031] The CP1 and CP2 proteins selected in this invention belong to the CatB-like proteases of the CA family in Giardia cysteine ​​proteases (CPs). During the trophozoite stage, CatB-like proteins are the most highly expressed secreted proteins. CP1 protein is mainly involved in decapsulation and is located in the endoplasmic reticulum. CP2 protein is considered to be the most highly expressed protein in both the trophozoite and cyst stages, and is one of the main proteins involved in cyst wall formation (CWP). It has multiple functions, not only participating in decapsulation and cyst formation, but also playing important roles in immune invasion and apoptosis induction. The properties of CP1 and CP2 proteins are relatively stable and suitable for detection using the online software ExPASy.

[0032] Inclusion body proteins obtained using the *E. coli* expression system underwent denaturation, renaturation, purification, dialysis, and concentration with 8M urea to become soluble proteins with the required concentration and purity, a relatively complex process. A reasonable immunoassay procedure was established to prepare polyclonal antibodies, and the combined results of Western blot and immunofluorescence localization demonstrated that the antibodies exhibited good reactivity. An indirect ELISA method was established using the purified recombinant protein and polyclonal antibody, showing good sensitivity, specificity, and reproducibility. Using the established ELISA method to detect clinically confirmed samples, the sensitivity for CP1 protein was 95.7% and the concordance rate was 98%, while the sensitivity for CP2 protein was 87.0% and the concordance rate was 94%. Preliminary comparisons suggest that the indirect ELISA detection of CP1 protein is better than that of CP2 protein, providing a theoretical basis for future rapid detection methods and opening up new avenues for the further development of colloidal gold test strips. Attached Figure Description

[0033] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products of the CP1 and CP2 genes in an embodiment of the present invention;

[0034] Figure 2 The results of double enzyme digestion identification of recombinant plasmids pET-32a-CP1 and pETt-32a-CP2 in embodiments of the present invention are shown.

[0035] Figure 3 The Western blot verification results of the CP1 and CP2 recombinant proteins according to embodiments of the present invention are shown.

[0036] Figure 4 The results of solubility analysis of recombinant proteins CP1 and CP2 from embodiments of the present invention are shown.

[0037] Figure 5 The purification results of recombinant proteins CP1 and CP2 from embodiments of the present invention are shown.

[0038] Figure 6 The results of ELISA titer assays for polyclonal antibodies according to embodiments of the present invention are shown.

[0039] Figure 7 The Western blot validation results of the polyclonal antibody according to an embodiment of the present invention are shown;

[0040] Figure 8 The immunofluorescence localization of CP1 and CP2 proteins in Giardia trophozoites is shown in an embodiment of the present invention.

[0041] Figure 9 The results of sensitivity tests on CP1 and CP2 proteins according to embodiments of the present invention are shown. Detailed Implementation

[0042] The following describes several embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0043] Example

[0044] Insect species: The Giardia lamblia species used in this embodiment is type A WB strain, which was donated by Professor Zhang Xichen of Jilin University.

[0045] Main reagents: tryptone, agar powder, yeast extract, ampicillin, IPTG, sodium phosphate, SDS, DMSO, EDTA, Tris, DEPC, ethidium bromide, bromophenol blue, DTT, Coomassie Brilliant Blue R-250, Tricine, Triton X-100, BamHI restriction endonuclease, XhoI restriction endonuclease, DNA Marker, color pre-stained protein maker, EZNA stool DNAkit kit, gel extraction kit, plasmid mini-extraction kit, 10% PAGE gel preparation kit, BCA kit, etc.

[0046] Major instruments and equipment: PCR amplification instrument purchased from Applied Biosystems; BDY-2 constant voltage and constant current electrophoresis apparatus purchased from Shanghai Botong Experimental Instrument Factory; DYB-1 electrophoresis apparatus purchased from Shanghai Botong Experimental Instrument Factory; constant voltage and constant current electrophoresis apparatus purchased from Beijing Liuyi Instrument Factory; ultrasonic cell disruptor purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; NanoDrop 2000 purchased from Thermo Fisher Scientific; ice maker purchased from Beijing Changfeng Instrument Co., Ltd.; constant temperature water bath purchased from Beijing Changfeng Instrument Co., Ltd.; Thermo-BIRT4i low temperature high speed centrifuge purchased from Shanghai Thermo Fisher Scientific Technology Co., Ltd.; horizontal shaker purchased from Shanghai Huake Experimental Equipment Co., Ltd.; OLYMPUS-BL53 biological microscope purchased from Zhengzhou Nanbei Instrument Equipment Co., Ltd.; AB204-N electronic balance purchased from Mettler-Toledo Group.

[0047] Example 1: Construction of expression vector

[0048] Referring to the CP1 (Gene ID: GL50803_10217) and CP2 (Gene ID: GL50803_14019) sequences in Giardia DB (https: / / giardiadb.org / ), specific primers for adding restriction enzyme sites to the CP1 and CP2 genes were designed using Permer Peimer 5.0 software. The sequences are shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The total genome extracted from Giardia tadpoles was used as a template for amplification, and the PCR products were sent to Beijing Nuosai Biotechnology Co., Ltd. for sequencing.

[0049] Table 1. Primer sequences for PCR amplification

[0050]

[0051] PCR amplification of the CP1 and CP2 genes, followed by agarose gel electrophoresis analysis, revealed gene lengths of 922 bp and 915 bp, respectively, consistent with expectations. The results are as follows: Figure 1 As shown, M: DNA marker D2000; 1: CP1 gene PCR product; 2: CP2 gene PCR product. The sequencing results of the PCR products are compared with... Giardia The CP1 (Gene ID: GL50803_10217) and CP2 (Gene ID: GL50803_14019) gene sequences in DB are identical.

[0052] The target gene was ligated to the cloning vector pMD18-T. The ligation system was prepared according to the following ratio: 5 μL Solution, 0.5 μL PMD18-T vector, and 4.5 μL target gene. The mixture was shaken and mixed at room temperature, then centrifuged briefly at low speed. The PCR instrument was set to 16°C overnight. The constructed recombinant plasmids pMD18-T-CP1 and pMD18-T-CP2 were transformed into competent DH5α cells for amplification culture. For pMD18-T-CP1 and pMD18-T-CP2 cloning vectors with correct sequencing results, a double enzyme digestion reaction was performed with the pET-32a(+) vector. The digested CP1 and CP2 genes were ligated into the digested pET-32a(+) vector using a ligation system prepared with 10 × 1 μL Ligation Buffer, 3 μL Digested pET-32a(+), 5 μL Digested PCR product, and 1 μL T4 DNA Ligase. The mixture was vortexed at room temperature, briefly centrifuged at low speed, and incubated overnight at 16°C using a PCR instrument. The ligated pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids were then double-digested for verification and sequenced.

[0053] The theoretical sizes of the enzyme digestion products of pET-32a-CP1 were 922 bp and 5900 bp, respectively; the theoretical sizes of the enzyme digestion products of pET-32a-CP2 were 915 bp and 5900 bp, respectively. The truncated enzyme digestion products were consistent with the theoretical values, proving that the recombinant plasmids pET-32a-CP1 and pET-32a-CP2 were successfully constructed. The results are as follows: Figure 2 As shown, M: DNA marker D10000; 1: Double digestion product of recombinant plasmid pET-32a-CP1; 2: Double digestion product of recombinant plasmid Pet-32a-CP2.

[0054] Example 2: Expression, purification, and identification of recombinant proteins

[0055] The correctly sequenced pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids were transformed into Rosetta(DE3) competent cells. The bacterial culture was inoculated into 1.5 mL LB medium containing 100 μg / mL ampicillin and incubated overnight at 37°C with shaking at 220 rpm. The next day, the cells were inoculated at a 1:100 ratio into 20 mL LB medium containing 100 μg / mL ampicillin and incubated at 37°C with shaking at 220 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to the remaining culture to a final concentration of 1 mM, and the culture was incubated at 37°C with shaking at 220 rpm for 4 h to induce fusion protein expression. The culture was then removed, centrifuged at 10000 rpm for 2 min at room temperature, the supernatant was discarded, and the bacterial pellet was resuspended in PBS. After sonication, the supernatant and pellet were resuspended separately in loading buffer. 10% SDS-PAGE analysis was performed, and Coomassie brilliant blue staining was used to analyze the expression pattern of recombinant proteins. Inclusion body proteins were washed three times with inclusion body washing buffer, dissolved in 8M urea, and then dialyzed into buffer solution to change the precipitation pattern of inclusion bodies. The recombinant proteins were then purified by Ni-NTA affinity chromatography, and the concentration of recombinant proteins was determined by the BCA method. Image J analysis was used to analyze protein purity.

[0056] Western blot analysis of the recombinant proteins showed that the molecular weights of recombinant proteins CP1 and CP2 were approximately 48 kDa. The expression levels of the recombinant proteins were significantly higher in the group with the inducer than in the group without the inducer, demonstrating successful induction of recombinant protein expression. The results are as follows: Figure 3 As shown, M: pre-stained marker; 1: uninduced expression product of CP1 bacterial culture; 2: expression product of CP1 bacterial culture after induction; 3: uninduced expression product of CP2 bacterial culture; 4: expression product of CP2 bacterial culture after induction.

[0057] Solubility analysis of the recombinant proteins showed that recombinant CP1 and CP2 proteins mainly existed in the form of inclusion bodies and were almost absent from the supernatant. Figure 4 As shown, M: pre-stained marker; 1: uninduced bacterial culture expression product; 2: induced bacterial culture expression product; 3: supernatant; 4: precipitate.

[0058] The purification results of the recombinant protein were identified by SDS-PAGE gel electrophoresis. The final purified protein was as follows: Figure 5 As shown (M: color pre-stained marker; 1: purified CP1 recombinant protein; 2: purified CP2 recombinant protein), the concentrations of CP1 and CP2 recombinant proteins were determined to be 0.52 mg / mL and 0.48 mg / mL, respectively, by BCA method; the purities of CP1 and CP2 recombinant proteins were analyzed by ImageJ software and found to be 91.27% and 93.33%, respectively.

[0059] Example 3: Preparation and Identification of Polyclonal Antibodies

[0060] The recombinant protein obtained in Example 2 was used as an antigen to immunize New Zealand white rabbits according to the immunization program. After the titer was qualified by ELISA, blood was collected from the carotid artery to prepare polyclonal antibodies, which were then verified by Western blot and immunofluorescence localization.

[0061] The titers of recombinant CP1 and CP2 polyclonal antibodies were both above 128,000 using indirect ELISA. The results are as follows: Figure 6 As shown.

[0062] The purified recombinant proteins CP1 and CP2 were validated by Western blot using polyclonal antibodies as primary antibodies. The results are as follows: Figure 7 As shown, this demonstrates that recombinant proteins CP1 and CP2 both possess good reactivity.

[0063] Fluorescence microscopy revealed that CP1 and CP2 proteins were mainly located in the cytoplasm of Giardia lamblia trophozoites, consistent with previous studies. No fluorescence signal was observed in the negative control group, demonstrating that the recombinant protein's reactivity was consistent with the original protein. Results are as follows: Figure 8 As shown, the scale bar is 75 μm and the value is 40×.

[0064] Example 4: Establishment of an Indirect ELISA Method

[0065] The polyclonal antibodies prepared in Example 3 were used to detect recombinant proteins CP1 and CP2, and the optimal antigen coating amount, blocking solution, blocking time, primary antibody dilution factor, primary antibody incubation time, secondary antibody dilution factor, and secondary antibody incubation time were determined. The optimized indirect ELISA method was used for detection, and the OD450 of 20 negative serum samples was recorded. The mean (AV) and standard deviation (SD) within each group were calculated. AV+3×SD and AV+2×SD values ​​were calculated. According to statistical principles, the former is the positive cutoff value, and the latter is the negative cutoff value. Values ​​between the two are considered suspicious and require retesting.

[0066] As described above, the optimized reaction conditions are as follows:

[0067] The antigen coating amount of recombinant protein CP1 was 0.25 ug / mL. The blocking buffer was 5% skim milk powder, the blocking time was 1 h, the primary antibody was diluted 1600 times, the primary antibody incubation time was 1 h, the secondary antibody was diluted 20000 times, and the secondary antibody incubation time was 1 h.

[0068] The antigen coating amount of recombinant protein CP2 was 0.25 ug / mL. The blocking buffer was 5% skim milk powder, and the blocking time was 1 h. The primary antibody was diluted 3200 times and incubated for 1.5 h. The secondary antibody was diluted 20000 times and incubated for 1 h.

[0069] Indirect ELISA detection using anti-CP1 protein polyclonal antibodies yielded the following OD450 values ​​for 20 Giardia negative proteins: 0.171, 0.266, 0.181, 0.198, 0.163, 0.174, 0.147, 0.126, 0.188, 0.141, 0.139, 0.176, 0.130, 0.125, 0.142, and 0.173. Indirect ELISA detection was performed using polyclonal antibodies against the anti-CP2 protein. The OD450 values ​​of 20 Giardia lamblia-negative proteins were: 0.181, 0.166, 0.281, 0.178, 0.173, 0.204, 0.147, 0.146, 0.168, 0.111, 0.169, 0.186, 0.133, 0.165, 0.122, and 0.143. The cut-off values ​​were determined using the cut-off formula: for recombinant protein CP1, an OD450 > 0.267 was considered positive, and an OD450 < 0.233 was considered negative; for recombinant protein CP2, an OD450 > 0.281 was considered positive, and an OD450 < 0.243 was considered negative. Values ​​between these values ​​were considered suspicious and required retesting.

[0070] Example 5: Evaluation of Detection Methods

[0071] Sensitivity test

[0072] Total protein was extracted from Giardia trophozoites, and protein concentration was determined using the BCA method. The protein was diluted with PBS to concentrations of 1 mg / mL, 500 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, and 0 ng / mL. Different concentrations of Giardia trophozoite total protein were used to replace recombinant protein for antigen coating. The antigen was detected under optimized reaction conditions, and the results were interpreted using an ELISA cutoff value of 2.4.

[0073] The results are as follows Figure 9 As shown, the lowest detectable antigen concentration of anti-CP1 serum is 5 μg / mL, and that of anti-CP2 serum is 100 μg / mL, indicating that anti-CP1 serum has higher sensitivity than anti-CP2 serum.

[0074] Specificity test

[0075] The established indirect ELISA method was used to detect five intestinal pathogens, including Bacillus cystis, Cryptosporidium, Coccidia, Escherichia coli, and Salmonella. The ELISA cutoff value in Example 4 was used to determine the results. The results were negative regardless of whether anti-CP1 serum or anti-CP2 serum was used as the primary antibody, indicating that both have good specificity.

[0076] Repeatability test

[0077] The established indirect ELISA method was used for intra-assay and inter-assay replication to test its repeatability. For intra-assay assays, 10 positive samples were selected, with 3 control groups for each sample, and the mean and coefficient of variation were calculated. For inter-assay assays, two batches of protein coating assays were performed, with 10 positive samples tested in each batch, and the mean and coefficient of variation were calculated.

[0078] Regardless of whether anti-CP1 serum or anti-CP2 serum was used as the primary antibody, the coefficient of variation was less than 10%, indicating that the method had good reproducibility. The results are shown in Tables 2 and 3.

[0079] Table 2. Results of repeatability tests for CP1 protein

[0080]

[0081] Table 3. Results of repeatability tests for CP2 protein

[0082]

[0083] Clinical sample testing

[0084] The established indirect ELISA method was used to test 50 stool samples (23 positive and 27 negative), and the results are shown in Table 4. The ELISA method established using anti-CP1 serum is more sensitive, has a higher concordance rate, and is more suitable for detection.

[0085] Table 4. Detection results of clinical samples

[0086]

[0087] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Use of reagents for the preparation of an indirect ELISA test kit for the detection of Giardia duodenalis CP1 or CP2 proteins, characterized in that, The kit comprises a recombinant protein CP1 polyclonal antibody or a recombinant protein CP2 polyclonal antibody as a primary antibody reagent; The titer of the recombinant protein CP1 polyclonal antibody or the recombinant protein CP2 polyclonal antibody is above 128,000; The recombinant protein CP1 or CP2 is prepared by the following method: The CP1 or CP2 gene is obtained by amplification using specific primers with total genomic DNA extracted from the Giardia trophozoite as a template, wherein the primer sequence of the CP1 gene is SEQ ID NO. 1: 5'-GGATCCATGGCTTTATCTTTGCTCTTGG-3' and SEQ ID NO. 2: 5'-CTCGAGCTAGTCAAGATATACAGCAT-3', and the primer sequence of the CP2 gene is SEQ ID NO. 3: 5'-GGATCCATGAAGCTCTTTCTCCT-3' and SEQ ID NO. 4: 5'-CTCGAGTTACTCATCGAAGAAGCCCG-3'; The amplified CP1 or CP2 gene is linked to a cloning vector pMD18-T to construct a recombinant plasmid pMD18-T-CP1 or pMD18-T-CP2; The constructed recombinant plasmid pMD18-T-CP1 and pMD18-T-CP2 are transformed into competent cells DH5α for expansion culture, and the pMD18-T-CP1 and pMD18-T-CP2 cloning vectors with correct sequencing results are subjected to BamHI / XhoI double enzyme digestion, and the digested CP1 and CP2 genes are linked to the digested pET-32a(+) vector to prepare pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids; The pET-32a-CP1 and pET-32a-CP2 recombinant expression plasmids are transformed into Rosetta(DE3) competent cells, and after expression induced by 1mM IPTG at 37℃ for 4 hours, the bacterial bodies are collected, ultrasonically broken, and the inclusion bodies are separated, denatured by 8M urea, renatured by gradient dialysis, and purified by Ni-NTA affinity chromatography to obtain CP1 or CP2 recombinant proteins with a purity of ≥91% and a concentration of ≥0.48 mg / mL.

2. Use according to claim 1, characterized in that, The recombinant protein CP1 polyclonal antibody or the recombinant protein CP2 polyclonal antibody is prepared by immunizing New Zealand white rabbits according to an immunization procedure with the recombinant protein of the CP1 or CP2 gene as an antigen, and is obtained after ELISA detection of a qualified titer.

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