Composite antigen for detecting haemonchus contortus infection and application thereof
阅读说明：本技术 一种检测捻转血矛线虫感染的复合抗原与其应用 (Composite antigen for detecting haemonchus contortus infection and application thereof ) 是由 严若峰 李祥瑞 徐立新 宋小凯 赖旭东 于 2021-08-02 设计创作，主要内容包括：本发明涉及一种检测捻转血矛线虫感染的复合抗原与其应用。一种复合型可用作该寄生虫早期感染诊断抗原,其氨基酸序列如SEQ ID NO.1所示。该抗原可以对感染捻转血矛线虫7天后的宿主血清中的抗体进行特异性识别,能在宿主排出虫卵2周前进行诊断。本发明利用该抗原建立了胶体金试纸条检测方法,具有敏感性好、特异性强、操作简便、用时短等优点,在牛、羊、鹿、骆驼等反刍动物捻转血矛线虫病的防控中具有较好的应用前景。(The invention relates to a composite antigen for detecting haemonchus contortus infection and application thereof. A compound antigen for diagnosing early infection of parasite has an amino acid sequence shown in SEQ ID NO. 1. The antigen can specifically recognize the antibody in the host serum 7 days after the infection of the haemonchus contortus, and can be diagnosed 2 weeks before the host expels eggs. The invention establishes a colloidal gold test strip detection method by using the antigen, has the advantages of good sensitivity, strong specificity, simple and convenient operation, short time and the like, and has better application prospect in the prevention and control of the haemonchus contortus disease of ruminants such as cattle, sheep, deer, camel and the like.)
1. The application of the haemonchus contortus composite antigen Hc-5C in preparing a haemonchus contortus infection diagnostic reagent is disclosed, wherein the amino acid sequence of the haemonchus contortus Hc-5C is shown as SEQ ID NO. 1.
2. The use of claim 1, wherein the complex antigen Hc-5C of haemonchus contortus is used in the preparation of a kit for molecular biological diagnosis or serological diagnosis of haemonchus contortus infection.
3. The use of claim 2, wherein the use of the complex antigen Hc-5C of haemonchus contortus for the preparation of a colloidal gold test strip for haemonchus contortus infection.
4. The use according to any one of claims 1 to 3, wherein the Haemonchus contortus complex antigen Hc-5C is prepared by: extracting total RNA of Haemonchus contortus, reverse transcribing to synthesize the first strand of cDNA, taking the cDNA as a template, performing RT-PCR amplification by using primers shown in SEQ ID NO.2-11, and performing overlap extension PCR on a PCR product to obtain Hc-5C gene; inserting the Hc-5C gene between BamHI and Xho I enzyme cutting sites of pET28a to obtain a recombinant expression plasmid pET28a/Hc-5C containing the Hc-5C gene; the plasmid is transformed into escherichia coli for induction expression, and the protein Hc-5C of the haemonchus contortus is obtained by separation and purification.
5. A colloidal gold test strip for detecting haemonchus contortus infection is characterized by comprising a gold-labeled pad prepared by using colloidal gold-labeled Hc-5C protein, a test strip with a detection line (T) coated by the Hc-5C protein and a quality control line (C) coated by an anti-Hc-5C protein antibody.
6. The colloidal gold test strip detection technology of claim 5, wherein the Haemonchus contortus complex antigen Hc-5C is prepared by the following method: extracting total RNA of Haemonchus contortus, reverse transcribing to synthesize the first strand of cDNA, taking the cDNA as a template, performing RT-PCR amplification by using primers shown in SEQ ID NO.2-11, and performing overlap extension PCR on a PCR product to obtain Hc-5C gene; inserting the Hc-5C gene between BamH I and Xho I enzyme cutting sites of pET28a to obtain recombinant expression plasmid pET28a-Hc-5C containing the Hc-5C gene; the plasmid is transformed into escherichia coli for induction expression, and the protein Hc-5C of the haemonchus contortus is obtained by separation and purification.
7. The colloidal gold test strip detection technology of claim 5, wherein the anti-Hc-5C antibody is a polyclonal antibody against Hc-5C.
8. The colloidal gold test strip detection technology of claim 7, wherein the anti-Hc-5C antibody is obtained by the following method: emulsifying the purified Hc-5C recombinant protein with Freund's complete adjuvant, immunizing rabbits, and emulsifying with Freund's incomplete adjuvant for 3 times to enhance immunization; collecting and purifying the hyperimmune serum.
9. The colloidal gold test strip detection technology of claim 5, wherein the gold pad is obtained by the following method: labeling the purified Hc-5C recombinant protein with colloidal gold; soaking and drying the gold label pad; cutting into proper size; is adhered to the test strip bottom plate.
Technical Field
The invention belongs to the technical field of veterinary immunology and molecular biology, and relates to a serological diagnosis method of animal parasitic diseases.
Background
Haemonchus contortus is a common alimentary canal parasite of ruminants such as cattle, sheep, deer, camels and the like, parasitizes in the fourth stomach of a host and takes the blood of the host as nutrition. The symptoms of anemia, emaciation, edema, diarrhea and the like appear after the animal is infected, the young animals, particularly lambs, can die in large batches, the death rate of adult animals is low, but the growth is slow after the infection, the feed reward is reduced, and huge economic loss is caused and is often not discovered. With the vigorous development of herbivores such as cattle and sheep and related industries in China, the harm of the haemonchus contortus and the economic loss caused by the haemonchus contortus are increasingly obvious and serious, and the method has positive significance for early diagnosis of the parasite infection condition of animals.
At present, a commercial vaccine is used for preventing the haemonchus contortus, but the commercial vaccine has limited application due to high production cost. The prevention and control of the disease still mainly treats benzimidazole, levamisole, ivermectin and other medicaments, but irregular administration accelerates the generation and spread of drug-resistant strains of the haemonchus contortus. The research and development of new vaccines and new drugs in a short period are difficult to have breakthrough progress, the disease is quickly and accurately diagnosed, proper drugs are selected for early treatment, and the phenomenon that the imagoes generate eggs to pollute grasslands and colony houses in the later stage of infection is avoided, so that the method plays an important role in the prevention and control of the disease.
The diagnosis of haemonchus contortus disease is mainly based on the evaluation of clinical symptoms and the results of fecal examinations. Diagnosis based on symptoms is convenient and simple, but requires a skilled technician, and clinical symptoms usually become apparent only when the infection is severe, and is not suitable for mild infections. The fecal test method is currently most widely used, but requires that eggs be detected in the feces about 3 weeks after infection, and is difficult to diagnose for early infection. The necropsy method can be used for accurate diagnosis, but animals need to be killed, so that the cost is high and the popularization is difficult. The establishment of new diagnostic techniques and methods suitable for early infection and mild infection in animals is urgent and necessary.
Disclosure of Invention
The invention aims to provide a composite diagnostic antigen Hc-5C of haemonchus contortus and application thereof, aiming at the defects of the prior art.
It is still another object of the present invention to provide a technique for diagnosing early infection of Haemonchus contortus.
The purpose of the invention can be realized by the following technical scheme:
the application of the composite diagnosis antigen Hc-5C of the haemonchus contortus in preparing a reagent for diagnosing haemonchus contortus infection is disclosed, wherein the amino acid sequence of the composite diagnosis antigen Hc-5C of the haemonchus contortus is shown in SEQ ID NO. 1.
As a preferable selection of the invention, the application of the composite diagnosis antigen Hc-5C of the haemonchus contortus in preparing a molecular biological diagnosis or serological diagnosis kit for haemonchus contortus infection.
As a further optimization of the invention, the application of the composite diagnosis antigen Hc-5C of the haemonchus contortus in the preparation of a colloidal gold test strip detection technology for haemonchus contortus infection is provided.
Preferably, the composite diagnostic antigen Hc-5C of Haemonchus contortus is prepared by the following method: extracting total RNA of haemonchus contortus, and performing reverse transcription to synthesize a first strand of cDNA; taking the cDNA as a template, respectively carrying out RT-PCR amplification by using primers shown by SEQ ID NO.2/SEQ ID NO.3, SEQ ID NO.4/SEQ ID NO.5, SEQ ID NO.6/SEQ ID NO.7, SEQ ID NO.8/SEQ ID NO.9 and SEQ ID NO.10/SEQ ID NO.11, and carrying out overlap extension PCR on a PCR product to obtain an Hc-5C gene; inserting the Hc-5C gene between BamH I and Xho I enzyme cutting sites of pET28a to obtain recombinant expression plasmid pET28a-Hc-5C containing the Hc-5C gene; the plasmid is transformed into escherichia coli for induction expression, and the protein Hc-5C of the haemonchus contortus is obtained by separation and purification.
A colloidal gold test strip detection technology for detecting haemonchus contortus infection comprises a gold-labeled pad prepared by using colloidal gold-labeled Hc-5C protein, a test strip with a detection line (T) coated by the Hc-5C protein and a quality control line (C) coated by an anti-Hc-5C protein antibody.
Preferably, the composite diagnostic antigen Hc-5C of Haemonchus contortus is prepared by the following method: extracting total RNA of haemonchus contortus, and performing reverse transcription to synthesize a first strand of cDNA; taking the cDNA as a template, carrying out RT-PCR amplification by using primers shown in SEQ ID NO.2-11, and carrying out overlap extension PCR on a PCR product to obtain a Hc-5C gene; inserting the Hc-5C gene between BamH I and Xho I enzyme cutting sites of pET28a to obtain recombinant expression plasmid pET28a-Hc-5C containing the Hc-5C gene; the plasmid is transformed into escherichia coli for induction expression, and the protein Hc-5C of the haemonchus contortus is obtained by separation and purification.
Preferably, the anti-Hc-5C antibody is a polyclonal antibody against Hc-5C.
Preferably, the anti-Hc-5C antibody is obtained by: emulsifying the purified Hc-5C recombinant protein with Freund's complete adjuvant, immunizing experimental rabbits, and emulsifying with Freund's incomplete adjuvant for 3 times to enhance immunization; collecting and purifying the hyperimmune serum.
As a preferred aspect of the present invention, the gold-labeled pad is obtained by the following method: labeling the purified Hc-5C recombinant protein with colloidal gold; soaking and drying the gold label pad; cutting into proper size; is adhered to the test strip bottom plate.
The present invention builds on the following findings:
previous researches of the applicant find that 5 proteins including a cold shock binding domain protein (CS), a hepatoma cell-associated antigen 59(HCA59), a methyltransferase 12 type protein (Mt12), NADH, ubiquinone oxidoreductase domain protein (NDUDC) and Ras domain protein (Ras) of Haemonchus contortus have certain diagnostic potential, and indirect ELISA methods established based on the proteins have certain sensitivity and stability, but the specificity of the indirect ELISA methods needs to be further improved.
In order to further improve the diagnostic capability of the 5 proteins, a peptide segment with strong specificity is screened from each protein and is sequentially named as Hc-CSf, Hc-HCA59f, Hc-Mt12f, Hc-NDUDCf and Hc-Rasf, and the Hc-5C composite antigen is formed according to the sequence. The diagnostic potential of the composite antigen is analyzed and a double-antigen sandwich colloidal gold immunochromatographic test strip detection method is established. The sensitivity and specificity of the method are analyzed, and the detection method is applied to the detection of clinical samples.
The colloidal gold test strip detection technology is a fast, convenient and safe serological detection method, and the method is not reported to be used for detecting haemonchus contortus infection. Hc-5C is used as a compound antigen and is obtained by fusion expression of 5 specific peptide fragments screened from 5 proteins with early diagnosis potential, and the immune function, the immunodiagnosis potential and the like of the Hc-5C are not reported.
The invention has the following advantages and effects:
1. the specificity of the ELISA technology for detecting the haemonchus contortus antibody reported by the current data is not strong, the invention forms a composite antigen by screening the specific epitope of 5 antigens, and the colloidal gold test strip detection technology established based on the composite antigen has high specificity.
2. The fecal examination can be diagnosed about 21 days after the animal is infected, and the method can make accurate diagnosis 7 days after the infection and has the advantage of early diagnosis.
3. The colloidal gold test strip detection technology established based on the antigen has the advantages of simple and convenient operation, short required time, no need of a detection instrument and the like.
Drawings
FIG. 1 analysis of B cell epitope (A) and T cell epitope (B) of Hc-5C complex protein
FIG. 2 agarose gel electrophoresis of PCR amplification products of fragments Hc-CSf, Hc-HCA59f, Hc-Mt12f, Hc-NDUDCf, Hc-Rasf, etc
M: DNA standard molecular weight; 1-5: respectively are PCR amplification products of Hc-CSf, Hc-HCA59f, Hc-Mt12f, Hc-NDUDCf and Hc-Rasf
FIG. 3 agarose gel electrophoresis of fusion amplification products
M is DNA standard molecular weight; 1-4 are PCR amplification products of gene A, gene B, gene C and Hc-5C respectively
FIG. 4 double restriction enzyme identification of recombinant plasmid pET-28a/Hc-5C
M is DNA standard molecular weight; 1, recombinant plasmid pET-28a/Hc-5C BamH I and Xho I double digestion product
FIG. 5 expression of recombinant Hc-5C
M is protein standard molecular weight; 1-2, sequentially inducing no-load thalli for 0h and 6h to obtain cleavage products; 3-9, sequentially inducing 0, 1, 2, 3, 4, 5 and 6h of cracking products by the recombinant thallus; 10-11, respectively preparing supernatant of the cracking product after the recombinant thallus is induced for 6h after ultrasonic treatment, and precipitating the cracking product after the recombinant thallus is induced for 6h after ultrasonic treatment
FIG. 6 purification of recombinant Hc-5C
M is protein standard molecular weight; unpurified inclusion body protein; 2, the purified recombinant Hc-5C protein.
FIG. 7 WB results of 7 rHc-5C and goat positive sera at different infection periods
M is standard protein molecular weight; 1-10 binding of goat positive sera as primary antibody to recombinant Hc-5C protein, pre-infection (day 0) and post-infection 7, 14, 21, 28, 35, 42, 49, 56 and 61 days, respectively.
FIG. 8 shows a structure of a colloidal gold test strip
FIG. 9 test strip specificity analysis
1-2: positive and negative serum for haemonchus contortus infection, respectively; 3-4 positive serums of trichina, toxoplasma and fasciola gigantica respectively
FIG. 10 test strip sensitivity assay
1-7, the dilution times of the positive serum are 1:5, 1:15, 1:25, 1:35, 1:45, 1:55 and 1:65 in sequence
Detailed Description
Base material:
1. haemonchus contortus strain: the laboratory separates, identifies and preserves.
2. Experimental animals: 4 local goats, 5-6 months old, purchased from a certain farm in sentence-capacity city, Jiangsu province and raised in animal houses of Nanjing agriculture university.
PCR primers: the sequences of the upstream and downstream primers for amplifying the 5 gene fragments are shown in Table 1.
4. Tool enzyme and reagent: the restriction endonucleases BamH I, Xho I, DNA Marker (DL2000Plus, DL5000Plus) andII 1st Strand cDNA Synthesis Kit reverse transcription kits were purchased from Nanjing Novozam Biotech Ltd;reagent was purchased from ThermoFisher Biotech; the plasmid small quantity extraction kit and the agarose gel recovery kit are American E.Z.N.A.TMCompany, BCA protein quantitative analysis kit is a product of the United states Thermo company; HisTrapTMFF protein affinity chromatographic column is purchased from GE company, DAB color development kit, and HRP-labeled rabbit anti-goat IgG is purchased from Shanghai Bin Yuntian Biotech company; the 20nm colloidal gold solution was purchased from Nanjing Xiancheng nanomaterial science and technology Limited; the pH test paper is purchased from Shanghai Sanaisi reagent Co., Ltd; colloidal gold consumables such as Sartorius CN140 NC membrane, gold label pad and sample pad are purchased from Shanghainey Biotech Co., Ltd; octanoic acid and ammonium sulfate were purchased from Shanghai Allantin Biotechnology Ltd.
5. Clinical test samples: 54 goats were randomly selected from different sheep farms in Jiangsu province, and serum, feces and abomasum samples were collected.
6. The main apparatus comprises: PCR amplification instrument (TaKaRa company), desk type refrigerated centrifuge (Eppendorf), electric pressure steam sterilizer (Shanghai Shen' an medical instrument factory), ultrasonic crusher (Ningbo Xinzhi scientific research instrument research institute), gel imaging system, protein electrophoresis system, semi-dry transfer system, enzyme labeling instrument (Bio-Red), XYZ large platform three-dimensional film-drawing gold spraying instrument HM3260 (Shanghai gold-labeled organism), and microcomputer automatic cutting machine ZQ2002 (Shanghai gold-labeled organism).
EXAMPLE 1 preparation of composite antigen Hc-5C for diagnosis
1.1 screening of specific peptide fragment and epitope analysis of Hc-5C composite protein
The amino acid sequences of Haemonchus contortus Hc-CS (GenBank accession number CDJ84294.1), Hc-HCA59(CDJ80864.1), Hc-Mt12(CDJ87424.1), Hc-NDUDC (CDJ88764.1) and Hc-Ras (CDJ86500.1) were subjected to Blast alignment in the NCBI database, respectively. Fragments (about 25-60 amino acids in length) of 5 proteins were screened for amino acid differences from M.circinelloides, using "M.circinelloides" shared among the results and infected with cattle and sheep as a standard. And comparing the selected fragments with an NCBI database, and filtering the result by taking 'Nematode' and 'Goat' as key words respectively to confirm that the selected fragments are different from other nematodes infecting the cattle and sheep and have no similar fragments with Goat protein. Finally, 5 peptide fragments respectively containing 9-58, 10-61, 137-189, 8-40 and 28-76 amino acid residues of the original protein are selected, potential B cell epitopes and potential T cell epitopes of each target fragment are analyzed by using a DNAStar Protean program, and each peptide fragment respectively contains 3, 5, 4 and 2B cell epitopes and 2, 3, 2 and 1T cell epitopes. The 5 peptide fragments are fused to obtain the Hc-5C composite protein, the sequence is shown as SEQ ID NO.1, the Hc-5C composite protein totally encodes 251 amino acids, the relative molecular mass (M.W.) is 28.54kDa, the theoretical isoelectric point (pI) is 5.97, the coincidence rate of the similar parts of the Hc-5C composite protein and other nematode proteins is lower than 25%, the similarity is 36-88%, and a large amount of B cell epitope and T cell epitope are contained (see figure 1).
1.2 Synthesis of primers
5 pairs of specific primers are designed according to the fragments screened by 5 proteins including Hc-CS, Hc-HCA59, Hc-Mt12, Hc-NDUDC, Hc-Ras and the like, and 20bp overlapping parts are ensured to exist at the junctions, and the sequences are shown in Table 1.
TABLE 1 PCR primer sequences
Note: all primer wavy lines indicate restriction sites and the shaded portions indicate overlapping portions.
1.3 extraction of Haemonchus contortus Total RNA
The method for extracting the total RNA of the imagoes is carried out according to the Inviotgen TRIzol instruction book, and the specific steps are as follows:
(1) 20 haemonchus contortus were picked and placed in a homogenizer treated with DEPC water, and nitrogen was added to grind the worms. Adding 1mL of TRIzol reagent after the polypide is sufficiently ground, and sufficiently grinding on ice (the grinding time on ice is more than 30 min);
(2) transferring the ground polypide into a centrifuge tube without RNA enzyme, standing for 5min or centrifuging at 4 ℃ at 12000rpm to take supernatant;
(3) transferring the supernatant to a new centrifuge tube, adding 200uL of chloroform pre-cooled at 4 ℃, shaking for 30s for emulsification, and standing for 5 min;
(4) centrifuging at 12000rpm for 15min at 4 deg.C, and carefully transferring the upper water phase to a new centrifuge tube; adding cooled isopropanol, shaking upside down for 1min, and standing for 10 min;
(5) centrifuging at 12000rpm for 10min at 4 deg.C, and discarding supernatant;
(6) resuspending the pellet with 75% ethanol in 1ml of EPC water, centrifuging at 12000rpm at 4 ℃ for 10min, and discarding the supernatant;
(7) repeating the step (6) once, airing the centrifuge tube under a fan to volatilize residual ethanol, and dissolving RNA by DEPC water at 30uL55 ℃;
(8) after the purity and concentration of RNA are determined, reverse transcription or storage at-80 ℃ is carried out immediately.
1.3 Synthesis of cDNA
And (4) synthesizing cDNA chains of the qualified total RNA of the worm bodies extracted in the previous step. The first strand of cDNA is synthesized by using a reverse transcription kit by using total RNA of Haemonchus contortus as a template.
1.4 RT-PCR amplification
Using cDNA as a template, and adopting the following reaction system to perform RT-PCR: cDNA template 2.0. mu.L, 10 XPCR Buffer 5. mu.L, MgCl25 μ L (25mM), 2 μ L of upstream primer (10pM), 2 μ L of downstream primer (10pM), 0.5 of LA Taq enzyme (5U/. mu.L)mu.L, adding sterilized ultrapure water to 50 mu.L, fully mixing, performing pre-denaturation at 95 ℃ for 1min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, performing 30 cycles, and extension at 72 ℃ for 10min on a PCR instrument. The 5 gene fragments were successfully amplified and named Hc-CSf, Hc-HCA59f, Hc-Mt12f, Hc-NDUDCf and Hc-Rasf respectively, with sizes of 169bp, 182bp, 188bp, 128bp and 179bp, as shown in FIG. 2.
1.5 overlap extension PCR to obtain the Gene of the Complex protein Hc-5C
Overlap extension PCR was performed using Hc-CSf, Hc-HCA59f, Hc-Mt12f, Hc-NDUDCf and Hc-Rasf gene fragments as templates, in the order of Hc-CSf and Hc-HCA59f (the new product is fusion gene A), gene A and Hc-Mt12f (the new product is fusion gene B), gene B and Hc-NDUDCf (the new product is fusion gene C), and gene C and Hc-Rasf (the new product is Hc-5C coding gene), with fusion genes A, B, C and Hc-544C being 331bp, 436bp, 766bp, respectively, as shown in FIG. 3.
1.6 construction of prokaryotic expression vectors
Electrophoresis was performed on 1% agarose gel containing 25. mu.l of the Hc-5C PCR product, the agarose gel was cut under an ultraviolet lamp, and the target fragment was recovered and purified by using a gel recovery kit from Dalibao Bio Inc., according to the instructions. Purified Hc-5C and pET28a vectors are subjected to double enzyme digestion by BamH I and Xho I, Hc-5C and pET28a fragments are recovered and are connected according to a proper proportion, a connection product is transformed into competent Escherichia coli BL21, plasmids are extracted and subjected to double enzyme digestion identification by BamH I and Xho I, and the result is shown in a figure 4. The positive clone is subjected to sequencing analysis, and the coded amino acid sequence is shown as SEQ ID NO. 1.
1.7 purification of expression products
1.7.1 preparation of Inclusion body proteins
pET28a-Hc-5C expression bacteria cultured in 1L LB medium were induced with IPTG at a concentration of 1mM, collected by centrifugation at 8000r/min for 5min, resuspended in PBS (40 ml), sonicated at 600W for 1s at intervals of 3s, and disrupted for 30 min. Centrifuging the crushed suspension at 10000r/min for 20min, taking the precipitate, and removing the supernatant to obtain the inclusion body. Adding 20ml of Elution Buffer into the inclusion body, fully sucking and suspending by using a large-size syringe, dissolving the inclusion body overnight at 4 ℃, centrifuging for 20min at 10000r/min after most of the inclusion body is dissolved, removing the precipitate, taking the supernatant, filtering by using a 0.22 mu m filter membrane, and finishing the preparation of the sample (see figure 5).
1.7.2 affinity chromatography purification of recombinant proteins
Protein samples were passed slowly through His Tag affinity chromatography columns (1 ml. times.3 in series) stored in 20% ethanol at 4 ℃ at a flow rate of 0.5ml/min, and the columns were washed using a Binding Buffer of 5 column volumes. The column was washed with Binding Buffer at a flow rate of 2ml/min using 5-10 bed volumes. The desired protein was eluted using an Elution Buffer at a flow rate of 0.5ml/min (see FIG. 6).
Example 2 Western blot analysis of Complex protein Hc-5C
2.1 preparation of serum from goat infected Haemonchus contortus at different stages
4 local goats, 4-6 months old, purchased from a farm in sentence-capacitor city, Jiangsu province and raised in animal houses of Nanjing agriculture university. After anthelmintic infestation, and confirmation of absence of parasite infection by fecal examination, Haemonchus contortus third-stage larvae were artificially infected (L3, 5000 larvae per nematode). Blood was collected and serum was isolated before infection (day 0, negative control) and 7, 14, 21, 28, 35, 42, 49, 56, 61 days post infection, respectively.
2.2 Western blot analysis of recombinant proteins
And (3) carrying out Western blot analysis on the Hc-5C recombinant protein by using the serum of the goat infected at different periods as a primary antibody and using the rabbit anti-goat HRP-IgG as a secondary antibody. As a result (FIG. 7), it was found that Hc-5C reacted with serum from 7 to 61 days after infection, but goat serum not infected with Haemonchus contortus did not react with the recombinant protein. It was shown that Hc-5C could be used for diagnosis of early and long-term infection of Haemonchus contortus (Table 2).
TABLE 2 results of the interaction between goat positive serum and recombinant protein Hc-5C at different infection stages
Example 3 establishment of colloidal gold test strip detection technology based on recombinant Hc-5C protein
3.1 Assembly of colloidal gold immunochromatographic test strip
(1) Soaking the gold label pad in colloidal gold labeled antigen solution for 10min, oven drying at 37 deg.C for 30min, repeating for 3 times, cutting into appropriate size, and drying at 4 deg.C for storage.
(2) Preparing a T line and a C line on an NC film by using a film scribing instrument according to 1 mu L/cm, drying the T line and the C line in a 37 ℃ oven for 30min, and cutting the T line and the C line to a proper size by using a slitter.
(3) The serum pad, the NC membrane and the absorbent paper are sequentially stuck on the bottom plate.
(4) And (3) installing and pressing the cut materials according to the sequence of a figure 8, and drying and storing the materials at 4 ℃ for later use.
3.2 colloidal gold-labeled Hc-5C antigen
(1) 10mL of the colloidal gold solution was added to a 50mL centrifuge tube using 0.1% K2CO3Adjusting the pH value of the solution to 8.0;
(2) adding 200ul recombinant Hc-5C (0.5mg/mL) and mixing, standing at room temperature for 20min, and standing at 4 deg.C for 30 min;
(3) adding 10% BSA to a final concentration of 1%, mixing, and standing at room temperature for 15 min;
(4) adding 5% PEG20000 to a final concentration of 0.5%, mixing, and standing at room temperature for 15 min;
(5) adding 10% NaCI solution to the final concentration of 1%, and mixing;
(6) centrifuging at 4 deg.C and 1500rpm for 20min, and collecting supernatant;
(7) centrifuging at 4 deg.C and 12000rpm for 60min, and removing supernatant;
(8) the pellet was resuspended in 1/5 original volume of 0.01mol/L PB solution (containing 2.5% sucrose and 1% BSA) and stored at 4 ℃ protected from light.
3.3 detection line (T line) coating
(1) Adjusting the concentration of the recombinant Hc-5C protein to 0.774mg/mL, 0.500mg/mL, 0.250mg/mL and 0.125mg/mL by using 0.01mol/L PBS (pH7.4), respectively dividing a membrane to be used as a T line, and drying in an incubator at 37 ℃ for 30 min;
(2) assembling the gold label pad, the absorbent paper, the serum pad, the bottom plate and the NC membrane;
(3) 50 mu L of goat positive serum infected with Haemonchus contortus diluted according to the ratio of 1:25 is dripped into the serum pad by each test strip, C, T lines are observed after 10min, and the optimal T line antigen coating concentration is determined to be 0.774 mg/mL.
3.4 coating of quality control line (C line)
(1) Preparation of rabbit anti-recombinant Hc-5C polyclonal antibody
The anti-Hc-5C polyclonal antibody is obtained by 4 times of immunization of 8-week-old female New Zealand white rabbits. Firstly, exempting from: completely emulsifying 500ug of rHc-5C with equal volume of Freund's complete adjuvant, and performing subcutaneous multipoint injection immunization; and (2) avoiding: two weeks apart, 500ug rHc-5C was completely emulsified with equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple sites for immunization; three and four times of immunization: all the components are separated by 1 week; on the 10 th day after the four-immunization, blood is collected from the artery in the ear, serum is separated, the serum is diluted in a multiple proportion, and the titer of the anti-rHc-5C polyclonal antibody is detected by rHc-5C indirect ELISA; after the titer reaches the standard, 7% chloral hydrate is injected into ear margin vein to anaesthetize white rabbit, blood is collected from abdominal middle artery, serum is separated, and the serum is subpackaged and stored at-70 ℃.
(2) Purification of polyclonal antibody by octanoic acid-ammonium sulfate method
Melting serum at 4 ℃, adding 0.06mol/L acetate buffer solution (pH4.0) into the serum according to the ratio of 1: 2-1: 4, and adjusting the pH of the solution to 4.5-4.8 by using 0.1mol/L NaOH or 0.1mol HCI; n-octanoic acid (25. mu.L/mL) was added dropwise slowly over 30min at room temperature, followed by standing at 4 ℃ for 2 h; centrifuging at 4 deg.C and 5000rpm for 30min, collecting supernatant, and filtering with 0.22 μm filter; adding 0.01mol/L PBS (pH7.4) buffer solution with the volume of 1/10 of the original solution, and then adjusting the pH to 7.4 by using 0.1mol/L NaOH; in ice bath, slowly adding an isovolumetric saturated ammonium sulfate solution dropwise within 30min, stirring, and standing overnight at 4 ℃; centrifuging at 4 deg.C and 12000rpm for 30min the next day, and removing supernatant; the pellet was resuspended in 1/10 volumes of 0.01mol/L PBS (pH7.4) buffer and transferred to dialysis bags and dialyzed against 20 volumes of 0.01mol/L PBS (pH7.4) buffer at 4 ℃ for 3 times, 6h each; after concentration with PEG8000, the mixture was filtered through a 0.22 μm filter, and then stored at-20 ℃ after being dispensed, and the protein concentration was measured using BCA protein concentration kit.
(3) Film-scribing and coating C line
Streaking and coating with polyclonal antibody dilution (2mg/mL) and oven drying at 37 deg.C for 30 min.
3.5 specific assay
And dripping goat positive serum infected with Haemonchus contortus, trichina positive serum, toxoplasma gondii positive serum and fasciola gigantica positive serum diluted by 1:25 into different test strip serum pads respectively, observing C, T lines after 10min, and judging the specificity condition of the test strips. The results (FIG. 9) show that there were no bands in T-line and bands in C-line of positive serum from Trichinella spiralis, Toxoplasma gondii and fasciola gigantica. Indicating that the method has good specificity.
3.6 sensitivity assay
Diluting goat positive serum artificially infected with Haemonchus contortus with PBS according to the ratio of 1:5, 1:15, 1:25, 1:35, 1:45, 1:55 and 1:65, respectively dripping 50 μ L of the diluted goat positive serum on different test strips, observing C, T lines, and judging the sensitivity of the test strips. The results (fig. 10) show that the test strip can still distinguish the positive serum and the negative serum of the haemonchus contortus after the serum is diluted by 1:35, and has better sensitivity.
EXAMPLE 4 clinical application of the diagnostic method
Serum samples and abomasum samples were collected from 19 goats, respectively. The samples were separately tested by adult counting and colloidal gold test strips, and the two diagnostic methods were compared. The results (table 3) show that 14 positive samples were detected by the colloidal gold test strip detection technique, the detection rate was 73.4% (14/19), and the coincidence rate of the method and the autopsy result was 84.2% ((14+ 2)/19).
TABLE 3 comparison of Hc-5C-based colloidal gold assay techniques with the results of a dissection
Serum and fecal samples were collected from 35 goats, respectively. The samples were subjected to egg counting and colloidal gold test strip detection. The results (table 4) show that 35 positive sera were detected by the colloidal gold test strip detection technique, the detection rate was 100% (35/35), and the detection rate was higher than that of the fecal examination method. The coincidence rate of the colloidal gold test strip detection technology and the fecal detection result is 85.7% ((30+ 0)/35).
TABLE 4 comparison of results with stool based on Hc-5C colloidal gold detection technique
Sequence listing
<110> Nanjing university of agriculture
<120> composite antigen for detecting haemonchus contortus infection and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 251
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Ser Met Ile Trp Ala Gln Arg Glu Ser Phe Leu Tyr Val Thr Ile
1 5 10 15
Glu Val Asp Asp Val Thr Ile Asp Asp Leu Ser Ala Asp Asp Asn Lys
20 25 30
Met His Phe Lys Gly Thr Ser His Thr Glu Val Tyr Glu Thr Thr Leu
35 40 45
Glu Phe Phe Ala Glu Glu Phe Met Asn Glu Ile Arg Glu Cys Phe Asn
50 55 60
Val Tyr Ser Gln Asp Gly Ile Val His Ser Ala Pro Gln Leu Arg Cys
65 70 75 80
Ile Leu Arg Ser Leu Gly Tyr Ser Pro Thr Ala Ala Lys Thr Ala Ala
85 90 95
Tyr Phe Ser Lys Ile Lys Gln Pro Met Asn Phe Glu Leu Met Glu Trp
100 105 110
Asp Thr His His Arg Met Phe Leu Tyr Ser Cys Asp Tyr Ser Ala Val
115 120 125
Ala Val Lys Val Leu Lys Glu His Glu Lys Tyr Asp Gly Ser Arg Ile
130 135 140
Ser Gly Phe Val Trp Asp Ile Thr Lys Asp Ala Pro Glu Ala Ala Pro
145 150 155 160
Ala Lys Glu Val Asp Met Gly Val Asp Lys Leu Ile Lys Phe Gly Arg
165 170 175
Ile Ile Lys Gln Ile Gly Gly Val Arg Ala Ala Leu Lys Lys Arg Tyr
180 185 190
Leu Met Asp Val Thr Arg Ala Lys Leu Met Arg Pro Leu Pro Asp Phe
195 200 205
Val Ser Lys Pro Gln Ile His Pro Thr Thr Val Ala Glu Gln Gln Val
210 215 220
Tyr Ser Gln Val Ala Val Pro Tyr Pro Ala Val Gln Ala His Val Glu
225 230 235 240
Pro Ala Val Ser Thr Gln Asp Ala Lys Glu Asn
245 250
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgggatccat gatttgggca caacgag 27
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaattctt cagcgaagaa ctcca 25
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttcttcgctg aagaattcat gaatgagatc cgtgaatgtt 40
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgagctcga agttcattgg ctgct 25
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccaatgaact tcgagctcat ggagtgggac acacatcaca ga 42
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgtcgactt cttttgccgg agcag 25
<210> 8
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccggcaaaag aagtcgacat gggtgtcgac aagttaataa agtt 44
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ataagcttag cacgcgtgac atcc 24
<210> 10
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtcacgcgtg ctaagcttat gcgacccttg ccagat 36
<210> 11
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccctcgagtt aattttcctt agcatcttgt g 31