EmAGO2 truncated recombinant protein, echinococcosis multilocularis indirect ELISA diagnostic kit based on truncated recombinant protein, and use method of echinococcosis multilocularis indirect ELISA diagnostic kit

Abstract

The invention discloses an EmAGO2 truncated recombinant protein, an echinococcosis multilocularis indirect ELISA (enzyme-linked immuno sorbent assay) diagnostic kit based on the truncated recombinant protein and a use method of the kit. The method comprises the following steps: designing a specific primer by selecting a sequence of 1-738bp at the N end of an Em-AGO2 gene, carrying out PCR (Polymerase Chain Reaction) amplification by taking reverse transcription echinococcosis multilocularis DNA (Deoxyribonucleic Acid) as a template, connecting an amplification product with a pCE2-TA / Blunt vector, and then transforming into a competent cell for culturing, so as to obtain a pCE2-T-EmAGO2 plasmid; respectively carrying out double enzyme digestion on the plasmid and an expression vector pET-28a (+), and connecting enzyme digestion fragments to obtain a recombinant protein expression plasmid pET-28a-EmAGO2; the preparation method comprises the following steps: taking EmAGO2 as a template, transforming the EmAGO2 into competent cells, selecting positive clones for induced expression, and carrying out ultrasonication treatment, separation and purification, concentration and desalination treatment on expressed thalli to obtain the EmAGO2 truncated recombinant protein. Furthermore, a mouse anti-EmAGO2 truncated recombinant protein and goat anti-rabbit IgG are respectively used as an antigen and an antibody to establish an indirect ELISA diagnostic kit for the echinococcosis multilocularis, and the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of the echinococcosis multilocularis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to an indirect ELISA diagnostic kit for alveolar echinococcosis based on a truncated recombinant protein EmAGO2 (1-246aa) The present application also relates to a method for using the indirect ELISA diagnostic kit. BACKGROUND

[0002] Alveolar echinococcosis, also known as alveolar hydatid disease (AE), is a serious zoonosis caused by the larva of Echinococcus multilocularis (Em) parasitizing in the liver of humans and animals. The disease is mainly distributed in the cold regions of the Northern Hemisphere. In China, it is mainly distributed in the northwest regions such as Xinjiang, Qinghai, Ningxia, Gansu and Tibet. Globally, an estimated 18,235 new AE cases are added each year. To date, AE remains a serious public health problem that needs to be addressed. Alveolar echinococcus exhibits tumor-like unlimited proliferation, infiltrative and invasive growth in the host body, hence the name "worm cancer" (Casulli et al., 2019). AE patients have a long incubation period, which can last for 10-15 years, during which the patients often have no obvious symptoms and are difficult to detect, which makes AE patients often miss the effective treatment opportunity. Therefore, it is particularly critical and urgent to develop a rapid, accurate, sensitive, specific and low-cost immunodiagnostic kit for early diagnosis of AE. SUMMARY

[0003] The present application provides a truncated recombinant protein EmAGO2 for early diagnosis of alveolar echinococcosis, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

[0004] The preparation method of the above-mentioned EmAGO2 truncated recombinant protein comprises the following steps:

[0005] (1) Construction of EmAGO2 truncated recombinant protein expression plasmid

[0006] The sequence of the N-terminal 1-738 bp of Em-AGO2 gene is selected, and specific primers are designed; total RNA of alveolar echinococcus is extracted, and cDNA reverse transcription is performed to obtain a reverse transcription product;

[0007] PCR amplification is performed on the reverse transcription product as a template, and the amplification product is connected to the pCE2-TA / Blunt vector and then transformed into the competent cells DH5α of Escherichia coli for overnight culture. The colonies are picked and cultured in LB culture solution to obtain a positive clone plasmid pCE2-T-EmAGO2 (1-738 bp);

[0008] The pCE2-T-EmAGO2 (1-738bp) plasmid and the expression vector pET-28a(+) are respectively subjected to double enzyme cutting with Hind III and Pst I restriction endonucleases, the enzyme cutting fragments are connected, and a recombinant protein expression plasmid pET-28a-EmAGO2 (1-738bp) is obtained.

[0009] (2) Induced expression and purification of the EmAGO2 truncated recombinant protein

[0010] The recombinant protein expression plasmid pET-28a-EmAGO2 (1-738bp) is transformed into BL21 expression strain competent cells, positive clones are picked and transferred into LB liquid medium containing kanamycin, and after culture to the logarithmic growth phase, IPTG is added for induced expression, and after the induced expression is completed, the bacterial bodies are collected; the bacterial bodies are subjected to ultrasonic crushing treatment, separated and purified by a nickel ion affinity chromatography column, concentrated and desalted by an ultrafiltration centrifuge tube and a dialysis bag, and the EmAGO2 truncated recombinant protein is obtained.

[0011] As a preferred technical scheme of the EmAGO2 truncated recombinant protein preparation method, in step (1), the upstream primer sequence is shown in SEQ ID NO. 2, and the downstream primer sequence is shown in SEQ ID NO. 3.

[0012] Further, in step (1), the PCR amplification reaction is as follows:

[0013] The reaction system is 5x PrimeSTAR GXL buffer 10 μL, dNTP Mixture 4 μL, PrimeSTAR GXLDNA 1 μL, 2 μM upstream and downstream primers 2 μL each, template 4 μL, and Nuclease-Free Water up to 50 μL; the reaction condition is 95℃ pre-denaturation for 5 min; then 34 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 60 s are performed; finally, 72℃ extension for 5 min.

[0014] The application provides an indirect ELISA diagnostic kit for hydatid disease based on an EmAGO2 truncated recombinant protein, comprising: an enzyme-labeled plate coated with a mouse anti-EmAGO2 truncated recombinant protein antigen as claimed in claim 1, and a goat anti-rabbit IgG.

[0015] As a further preferred technical scheme of the indirect ELISA diagnostic kit, the kit further comprises a coating buffer, a blocking solution, a PBST diluent, a color developing solution and a termination solution.

[0016] A method for non-diagnostic purposes using the above indirect ELISA diagnostic kit, comprising the following steps:

[0017] (1) coating: EmAGO2 truncated recombinant protein is used as an antigen, and the enzyme-labeled plate is coated after dilution with carbonate coating buffer;

[0018] (2) blocking: the blocking solution is added to block the enzyme-labeled plate;

[0019] (3) sample addition: the serum sample to be detected is added for reaction;

[0020] (4) addition of enzyme-labeled secondary antibody: the goat anti-rabbit IgG is added for reaction;

[0021] (5) color development: color development is carried out in the dark after the addition of color developing solution;

[0022] (6) termination: the reaction is terminated after the addition of termination solution;

[0023] (7) reading: the OD450 value is measured by an enzyme-labeled instrument.

[0024] As a further preferred use method technical solution of the indirect ELISA diagnostic kit of the present application for non-diagnostic purposes, the step (1) coating operation is as follows: 1 μg / mL Em-AGO2 truncated recombinant protein is coated overnight at 4°C with 0.05M, pH 9.6 carbonate coating buffer.

[0025] Further, the step (2) blocking operation is as follows: 5% defatted milk powder is used for blocking at 37°C for 1h.

[0026] Further, in the steps (3) and (4), the serum sample to be detected and the goat anti-rabbit IgG are both diluted with the blocking solution and then added to the enzyme-labeled plate.

[0027] By using the above technical solution, the present application has the following beneficial effects:

[0028] The application selects the sequence of the first-738bp of the N terminal of Em-AGO2 gene to design specific primers, then reverses the RNA of hydatid cyst to obtain reverse transcription DNA, and carries out PCR amplification with the DNA as a template, connects the amplification product to pCE2-TA / Blunt vector, and then is transformed into E. coli competent cell DH5a to culture, selects the colony in LB culture solution to obtain pCE2-T-EmAGO2(1-738bp); the plasmid pCE2-T-EmAGO2(1-738bp) and the expression vector pET-28a(+) are respectively double enzyme cut with Hind III and Pst I restriction endonuclease, and the enzyme cutting fragments are connected to obtain the recombinant protein expression plasmid pET-28a-EmAGO2(1-738bp); the recombinant protein expression plasmid is transformed into BL21 expression strain competent cell, the positive clone is selected and transferred to LB liquid culture medium containing kanamycin, IPTG is added for induction expression after the logarithmic growth period, and the bacterial body is collected after the induction expression is completed; the bacterial body is ultrasonically broken, separated and purified by a nickel ion affinity chromatography column, concentrated and desalted by an ultrafiltration centrifuge tube and a dialysis bag, and the EmAGO2 truncated recombinant protein is obtained. Then, a hydatid cystosis indirect ELISA diagnostic kit is established by taking the mouse anti-EmAGO2 truncated recombinant protein and the goat anti-rabbit IgG as antigens and antibodies, the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of hydatid cystosis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 PCR amplification of the truncated gene (1～738bp) PCR amplification of the truncated gene and recombinant plasmid pET-28a-EmAGO2 (1～738bp) Double enzyme cutting electrophoresis result: wherein A is EmAGO2 (1～738bp) PCR amplification fragment of the truncated gene, B is pET-28a-EmAGO2 (1～738bp) Double enzyme cutting electrophoresis result

[0030] Figure 2 Induction expression and purification result of the EmAGO2 truncated recombinant protein

[0031] Figure 3 Column chart for selection and optimization of indirect ELISA coating liquid based on the EmAGO2 truncated recombinant protein

[0032] Figure 4 Column chart for selection and optimization of the concentration of indirect ELISA coating antigen based on the EmAGO2 truncated recombinant protein

[0033] Figure 5 Column chart for selection and optimization of the coating time of indirect ELISA based on the EmAGO2 truncated recombinant protein

[0034] Figure 6 Column chart for selection and optimization of indirect ELISA blocking solution based on EmAGO2 truncated recombinant protein;

[0035] Figure 7 Column chart for selection and optimization of serum dilution of indirect ELISA based on EmAGO2 truncated recombinant protein;

[0036] Figure 8 Column chart for selection and optimization of enzyme-labeled secondary antibody dilution of indirect ELISA based on EmAGO2 truncated recombinant protein;

[0037] Figure 9 Results of evaluation of the value of EmAGO2 truncated recombinant protein in diagnosis of mice infected with multilocular hydatid;

[0038] Figure 10 Results of positive detection rate of mouse serum at different infection stages detected by indirect ELISA based on EmAGO2 truncated recombinant protein. DETAILED DESCRIPTION

[0039] The present application will be described in detail below with specific embodiments and with reference to the accompanying drawings.

[0040] Example 1: Construction of a multilocular hydatid tapeworm EmAGO2 truncated recombinant protein expression plasmid

[0041] (1) Through analysis of the whole genome of multilocular hydatid tapeworm, it was found that multilocular hydatid tapeworm co-expressed four AGO proteins. In the Em-AGO protein family, the N-terminal sequence similarity between members is the lowest. In order to ensure the specificity of Em-AGO2 protein and avoid cross reaction with other members of the family, the present application selects the sequence of the N-terminal 1 to 738 bp of Em-AGO2 gene to prepare Em-AGO2 truncated recombinant protein. The amino acid sequence of the Em-AGO2 truncated recombinant protein is:

[0042] MLPSRPGRGTIGRKIVVEVNCWDFDVSDVSVLMYDITLTKLLSADGKEIKLKEKGIGKYVRSIAERKRGDVFHDGGRILFSLGPLDGKDGEVLNFSEKIADPLQNDDLTIEYVAERVGNISAREIREYLDNSRSKTSDLPQPAINMLDDLIKWVNRTSLPYLSKSAIFYDWPERDNTGGLFWIYRGYSLSFRPQWKCRLNIDMAHRAFFPAGNLADIIYAQYGDDMYSPSTWKHVKEDILSLHVEA (SEQ ID NO. 1).

[0043] Design specific primers based on the Em-AGO2 gene sequence:

[0044] The upstream primer sequence is 5'- AAGCTT ATGCTTCCTTCGCGTCCTGGTA-3'(SEQ ID NO.2); (The underlined part is the enzyme cleavage site: HindIII).

[0045] The downstream primer sequence is 5'- CTGCAG AGCCTCAACATGGAGTGACAGT-3' (SEQ ID NO.3) (the underlined part indicates the restriction enzyme site: PstI). Primers were synthesized by Xi'an Qingke Biotechnology Co., Ltd.

[0046] (2) Total RNA was extracted from Echinococcus multilocularis larvae, and cDNA was reverse transcribed using the RevertAid cDNA first strand synthesis kit. The synthesized product was stored at -20℃ for later use.

[0047] (3) PCR amplification was performed using the product synthesized in (2) as a template. The reaction system consisted of: 10 μL of 5×PrimeSTAR GXL buffer, 4 μL of dNTP Mixture, 1 μL of PrimeSTAR GXL DNA, 2 μL each of forward and reverse primers (2 μM), 4 μL of template, and Nuclease-Free Water to a final volume of 50 μL. The reaction conditions were set as follows: pre-denaturation at 95℃ for 5 min; followed by 34 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s); and finally extension at 72℃ for 5 min. After PCR products were detected by 1% agarose gel electrophoresis, PCR products with matching bands were purified and recovered using an agarose gel purification kit. The recovered products were ligated into the pCE2-TA / Blunt vector and transformed into competent E. coli DH5α cells. The cells were cultured overnight at 37°C. Independently distributed colonies were picked and cultured in LB medium at 37°C. Positive clones identified by PCR were sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing and named pCE2-T-EmAGO2 (1-738bp).

[0048] (4) pCE2-T-EmAGO2 (1-738bp) plasmid and expression vector pET-28a (+) were double-digested with Hind III and Pst I restriction enzymes respectively, and the digested fragments were recovered. The target fragment and the double-digested fragment of pET-28a (+) were ligated, and the recombinant plasmid vector was named pET-28a-EmAGO2 (1-738bp). The recombinant plasmid vector was transformed into E. coli DH5α competent cells, and the transformation product was plated on LB solid medium containing kanamycin and incubated at 37°C overnight. Single colonies were selected from the plate and subjected to PCR identification. Positive colonies were inoculated into LB liquid medium containing kanamycin and incubated at 37°C with shaking. After plasmid extraction, double digestion identification was performed to ensure successful construction of the recombinant plasmid.

[0049] (5) Experimental results

[0050] The results of nucleic acid electrophoresis detection showed that the applicant successfully obtained an EmAGO2 gene truncated fragment with a length of 738bp. The double-digested recombinant expression plasmid pET-28a-EmAGO2 (1-738bp) was consistent with the expected results, indicating that the EmAGO2 (1-738bp) recombinant expression plasmid was successfully constructed (as shown in Figure 1 ).

[0051] Example 2: Induction expression and purification of EmAGO2 truncated recombinant protein

[0052] (1) Induction expression of EmAGO2 truncated recombinant protein

[0053] The successfully constructed EmAGO2 (1-738bp) recombinant expression plasmid was transformed into BL21 expression strain competent cells. Positive clones identified by PCR were selected and inoculated into LB liquid medium containing kanamycin and incubated at 37°C with shaking until the logarithmic growth phase. IPTG was then added for induction expression, and key parameters such as IPTG concentration, induction temperature and induction time were systematically optimized. After induction, the bacterial cells were collected and subjected to ultrasonic disruption. The supernatant and precipitate were collected by centrifugation at 10000 r / min at 4°C for 15 min. The expression of recombinant protein was detected by SDS-PAGE electrophoresis to determine the optimal induction expression conditions.

[0054] (2) Purification and identification of EmAGO2 truncated recombinant protein

[0055] Under the optimal conditions, the EmAGO2 truncated recombinant protein was induced to express in large quantities. After the induction, the bacterial cells were collected and treated by ultrasonic disruption. The nickel ion affinity chromatography column was used for separation and purification, and the target protein was eluted by imidazole elution buffer with different concentration gradients. After collecting the eluate, the purity was verified by SDS-PAGE electrophoresis analysis. The purified EmAGO2 truncated recombinant protein was concentrated and desalted by ultrafiltration centrifuge tube and dialysis bag, so as to meet the purity and concentration requirements of subsequent experiments. Finally, the purified protein was divided and stored in a refrigerator at -80°C for standby.

[0056] After the Em-AGO2 truncated recombinant protein was separated by SDS-PAGE, it was transferred to a PVDF membrane by a semi-dry transfer method (Western blot). 5% (w / v) skimmed milk powder blocking solution was used for blocking at room temperature for 1 hour. After the blocking solution was discarded, the positive serum infected with Echinococcus multilocularis was added at a dilution ratio of 1:200, and incubated at 4°C overnight. The membrane strip was washed with PBST buffer for 3 times (5 minutes each time), and a HRP-labeled goat anti-mouse IgG secondary antibody was added at a dilution ratio of 1:10,000, and incubated at room temperature for 1 hour. The membrane strip was washed with PBST buffer for 3 times (5 minutes each time) again. The prepared ECL chemiluminescence working solution was uniformly covered on the surface of the PVDF membrane, and after about 1 minute of dark reaction, it was immediately placed in a chemiluminescence imaging system for exposure and signal collection.

[0057] (3) Experimental results

[0058] The SDS-PAGE detection results showed that the EmAGO2 truncated recombinant protein was mainly expressed in the supernatant, with a molecular weight of about 27 kDa, which was consistent with the expected size. When the imidazole concentration was 300 mM, the elution effect was the best (as shown in Figure 2 ). The Western blot results showed that the serum of mice infected with Echinococcus multilocularis could specifically recognize the EmAGO2 truncated recombinant protein, and a clear specific reaction band appeared at about 27 kDa, while there was no reaction with the negative serum, indicating that the EmAGO2 truncated recombinant protein had good immunogenicity Figure 3 .

[0059] Example 3: Establishment and optimization of indirect ELISA detection method based on EmAGO2 truncated recombinant protein

[0060] An indirect ELISA diagnostic method for detecting mouse Echinococcosis multilocularis was established and optimized based on the EmAGO2 truncated recombinant protein of Echinococcus multilocularis.

[0061] (1) Collection and preservation of specimens (standard and control)

[0062] Serum: Mouse whole blood samples were placed at 37°C for 1 hour, then placed in a centrifuge at 2500g for 15 minutes, and the supernatant was taken for detection.

[0063] (2) Operation steps

[0064] 1) The coating antigen EmAGO2 truncated recombinant protein was diluted to an appropriate concentration with coating buffer and added to a 96-well enzyme-labeled plate at 100 μL / well, coated at 4°C overnight, and blank control wells were set up;

[0065] 2) Blocking: The coating solution was discarded, the 96-well plate was washed with PBST 3 times, 5 min each time, 7.5% skimmed milk powder was added at 200 μL per well, and then incubated at 37°C for 1 h;

[0066] 3) Add primary antibody: Discard the blocking solution, wash the plate as in step 2), dilute the serum to be tested, add 100 μL per well to the 96-well enzyme-labeled plate, mix well, and incubate at 37°C for 1 h;

[0067] 4) Add enzyme-labeled secondary antibody: Discard the primary antibody, wash the plate as in step 2), dilute the goat anti-rabbit IgG as the secondary antibody, add 100 μL per well, and incubate at 37°C for 1 h;

[0068] 5) Color development: Discard the secondary antibody, wash the plate as in step 2), add 50 μL of TMB color developing solution to each well, and develop color at 37°C for 10 min;

[0069] 6) Stop the reaction: Add 2 mol / L sulfuric acid at 50 μL per well, read the OD value at 450 nm, and record the test data.

[0070] (3) Optimize the conditions for the reaction by using chessboard titration method:

[0071] Select PBS (pH 7.4), 0.05M CBS (pH 9.6), and 0.05M Tris-HCl (pH 8.0) as coating buffers, dilute the purified EmAGO2 truncated recombinant protein to four concentration gradients of 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, add 100 μL per well to the 96-well enzyme-labeled plate, set 2 repeats for each sample, and perform 4°C overnight coating and 37°C 1 h incubation; select 2% BSA, 2% skimmed milk powder, 5% BSA, and 5% skimmed milk powder as blocking solutions; dilute the serum to be tested at gradients of 1:100, 1:200, 1:400, and 1:800; dilute the enzyme-labeled secondary antibody at gradients of 1:2500, 1:5000, and 1:10000. Measure the OD450 value of each well according to the above operation steps, calculate the average P / N value, and select the condition combination with the maximum P / N value as the optimal reaction system.

[0072] (4) Experimental Results

[0073] The optimal detection conditions were determined as follows: 1 μg / mL Em-AGO2 truncated recombinant protein was coated overnight at 4°C with 0.05 M (pH 9.6) carbonate coating buffer (CBS); blocked with 5% skim milk powder at 37°C for 1 h; 100 μL of the test serum (diluted 1:200 with blocking buffer) was reacted at 37°C for 1 h; 100 μL of enzyme-labeled secondary antibody (diluted 1:10,000 with blocking buffer) was reacted at 37°C for 1 h; substrate was added and the mixture was incubated in the dark for 10 min; OD was measured after adding 50 μL of stop solution. 450 Value (e.g.) Figures 3-8 ).

[0074] Example 4: Specificity and sensitivity analysis of the ELISA detection method

[0075] Forty-eight serum samples from healthy control mice were tested under optimal ELISA conditions (Table 1), and the average value was calculated based on the results. The value was 0.4086, and the standard deviation (SD) was 0.0406, according to the cut-off... The critical value was determined to be 0.5304.

[0076] Table 1. OD values ​​of 48 serum samples from healthy control mice 450 value

[0077]

[0078] Using the optimal detection conditions in Example 3, 24 positive serum samples from mice infected with Echinococcus multilocularis for 3 months were tested. The results showed that their OD450 nm values ​​were all greater than 0.5304 (Table 2). Figure 9 A); ROC analysis showed that the Em-AGO2 truncated recombinant antigen had 100% specificity and sensitivity in diagnosing mouse multilocular echinococcosis infection, with an AUC of 1.000 (AUC = 1.000). Figure 9 B). Further indirect ELISA validation experiments showed that the OD450 values ​​of serum samples positive for parasites such as *Echinococcus granulosus*, *Echinococcus cerebralis*, *Fasciola hepatica*, *Toxoplasma gondii*, *Haemaphysalis contortus*, and *Taenia scabra* were all below the set threshold. Figure 9 C) indicates that the detection method has good specificity and no obvious cross-reactivity.

[0079] Table 2. OD values ​​of four positive serum samples from mice infected with Echinococcus multilocularis 3 months prior. 450 value

[0080]

[0081]

[0082] Example 5: Evaluation of the value of EmAGO2 truncated recombinant protein in early diagnosis of multilocular hydatidosis

[0083] To explore the early diagnosis potential of EmAGO2 in the mouse model of multilocular hydatidosis, the present application used the optimized indirect ELISA detection method to detect 32 serum samples of mice infected with multilocular hydatid at different stages (7d-20d).

[0084] The results showed that the positive detection rate of EmAGO2 truncated recombinant protein was 71.88% at 7d of infection; the detection rate was 84.38% at 10d of infection; the detection rate reached 100% at 15d and above (such as Figure 10 ), which indicated that EmAGO2 truncated recombinant protein had potential application value in the early serological diagnosis of mouse multilocular hydatidosis.

Claims

1. A truncated recombinant protein of EmAGO2 for early diagnosis of multilocular echinococcosis, characterized in that, The amino acid sequence of the truncated recombinant protein is shown as SEQ ID NO.

1.

2. A method for the preparation of EmAGO2 truncated recombinant protein for early diagnosis of multilocular echinococcosis according to claim 1, characterized in that, The method comprises the following steps: (1) Construction of EmAGO2 truncated recombinant protein expression plasmid The sequence of the N-terminal 1-738 bp of Em-AGO2 gene is selected, and specific primers are designed; Total RNA of multilocular hydatid is extracted, and cDNA reverse transcription is performed to obtain a reverse transcription product; PCR amplification is performed on the reverse transcription product as a template, and the amplification product is connected to the pCE2-TA / Blunt vector and then transformed into E. coli competent cells DH5a for overnight culture, and the colonies are picked and cultured in LB culture solution to obtain a positive clone plasmid pCE2-T-EmAGO2(1-738 bp); The pCE2-T-EmAGO2(1-738 bp) plasmid and the expression vector pET-28a(+) are double-digested respectively, the digested fragments are connected, and a recombinant protein expression plasmid pET-28a-EmAGO2(1-738 bp) is obtained; (2) Induced expression and purification of EmAGO2 truncated recombinant protein The recombinant protein expression plasmid pET-28a-EmAGO2(1-738 bp) is transformed into BL21 expression strain competent cells, positive clones are picked and transferred into LB liquid medium containing kanamycin, and after culture to the logarithmic growth phase, IPTG is added for induced expression, the induced expression bacteria are subjected to ultrasonic crushing treatment, nickel ion affinity chromatography column separation and purification, concentration and desalting treatment, and the EmAGO2 truncated recombinant protein is obtained.

3. A method for the preparation of EmAGO2 truncated recombinant protein for early diagnosis of multilocular echinococcosis according to claim 2, characterized in that, In the specific primers in step (1), the sequence of the upstream primer is shown as SEQ ID NO. 2; and the sequence of the downstream primer is shown as SEQ ID NO.

3.

4. A method for preparing EmAGO2 truncated recombinant protein for early diagnosis of multilocular echinococcosis according to claim 3, characterized in that, The PCR amplification reaction in step (1) is as follows: The reaction system is 5×PrimeSTAR GXL buffer 10 μL, dNTP Mixture 4 μL, PrimeSTAR GXL DNA 1 μL, 2 μM of each of the upstream and downstream primers 2 μL, template 4 μL, and Nuclease-Free Water up to 50 μL; and the reaction conditions are pre-denaturation at 95 ℃ for 5 min, followed by 34 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 60 s, and finally extension at 72 ℃ for 5 min.

5. An indirect ELISA diagnostic kit for cystic echinococcosis based on EmAGO2 truncated recombinant protein, characterized by, It comprises: An enzyme-labeled plate coated with the mouse anti-EmAGO2 truncated recombinant protein antigen of claim 1, and goat anti-rabbit IgG.

6. The EmAGO2 truncated recombinant protein based indirect ELISA diagnostic kit for cystic echinococcosis according to claim 5, wherein, The kit further comprises coating buffer, blocking solution, PBST diluent, color developing solution and termination solution.

7. A method for non-diagnostic purposes using a cystic echinococcosis indirect ELISA diagnostic kit based on EmAGO2 truncated recombinant protein according to claim 6, characterized in that, The method comprises the following steps: (1) Coating: EmAGO2 truncated recombinant protein is used as an antigen, and after dilution with carbonate coating buffer, an enzyme-labeled plate is coated; (2) Blocking: Blocking solution is added to block the enzyme-labeled plate; (3) Sample addition: The serum sample to be detected is added for reaction; (4) Addition of enzyme-labeled secondary antibody: Goat anti-rabbit IgG is added for reaction; (5) Color development: Color developing solution is added for color development in the dark; (6) Termination: Termination solution is added to terminate the reaction; (7) Reading: The OD450 value is determined by an enzyme-labeled instrument.

8. A method for non-diagnostic purposes using a cystic echinococcosis indirect ELISA diagnostic kit based on EmAGO2 truncated recombinant protein according to claim 7, characterized in that, The step (1) coating operation is: 1 μg / mL Em-AGO2 truncated recombinant protein is coated with 0.05 M, pH 9.6 carbonate coating buffer at 4°C overnight.

9. A method for non-diagnostic purposes using a cystic echinococcosis indirect ELISA diagnostic kit based on EmAGO2 truncated recombinant protein according to claim 8, characterized in that, The step (2) blocking operation is: 5% mass fraction of skimmed milk powder is used for blocking at 37°C for 1 h.

10. A method for non-diagnostic purposes using a cystic echinococcosis indirect ELISA diagnostic kit based on EmAGO2 truncated recombinant protein according to any one of claims 7 to 9, characterized in that, In the step (3) and step (4), the serum sample to be detected and goat anti-rabbit IgG are diluted by the blocking solution and then added to the enzyme-labeled plate.

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