CN113354722B

CN113354722B - Application of echinococcus granulosus Severin protein in kit for detecting cystic echinococcosis and echinococcus granulosus infection

Info

Publication number: CN113354722B
Application number: CN202110644033.XA
Authority: CN
Inventors: 陈兆国; 吕亚洁; 龚海燕; 米荣升; 黄燕
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2022-12-09
Anticipated expiration: 2041-06-09
Also published as: CN113354722A

Abstract

The invention discloses application of a recombinant echinococcus granulosus Severin protein in a kit for detecting cystic echinococcosis and echinococcus granulosus infection; the coating antigen in the kit is recombinant echinococcus granulosus Severin protein. The echinococcus granulosus recombinant protein rEgSeverin is cloned and expressed for the first time, and immunoblotting shows that the protein has good reactogenicity; it is found that after mice are immunized by rEgSeverin, good humoral and cellular immune responses are induced, and the Th2 type cell factors are mainly induced. The invention establishes an indirect ELISA method for detecting the echinococcus granulosus antibody based on the rEgSeverin protein, prepares an ELISA kit, has higher specificity than a commercial ELISA kit, and provides an effective method for epidemiological investigation and diagnosis of the echinococcus granulosus infection and cystic echinococcosis of livestock.

Description

Application of echinococcus granulosus Severin protein in kit for detecting cystic echinococcosis and echinococcus granulosus infection

Technical Field

The invention belongs to the technical field of biology, and relates to application of echinococcus granulosus Severin protein in a kit for detecting cystic echinococcosis and echinococcosis infection, in particular to application of recombinant echinococcus granulosus Severin protein in an ELISA kit for detecting echinococcosis of dogs and livestock such as sheep.

Background

Echinococcus spp has a large number of types, and 5 main types of pathogens causing zoonosis include Echinococcus granulosus (Eg), echinococcus multilocularis (e.g., em), echinococcus furiosus (e.vogeli, ev), echinococcus canadensis (e.canadensis, ec), echinococcus oligosaccharensis (e.oligartha, eo) (MCMANUS et al, 2003). Echinococcus granulosus and Echinococcus multilocularis are common in China, and Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE) are respectively caused in animals such as humans, cows and sheep, wherein the CE caused by parasitizing Echinococcus granulosus of the middle-taenis larva in humans and animals is the most prevalent and serious in harm.

The echinococcus granulosus has a complex life history, including 5 developmental stages of ova, oncosphere, echinococcus, metacercaria and adults. Echinococcus granulosus must have two hosts (Thompson, 2017) to complete its life cycle, with the final host being a canine carnivore such as canine, wolf, etc.; the intermediate host is artiodactyl animals such as sheep, cattle, pigs, camels and the like, and the artillery can infect rodents, primates and people, particularly sheep; humans are considered as occasional or abnormal intermediate hosts, merely victims, not major transmission sources, and not involved in parasite cycles of life (JENKINS, 2001). Echinococcus granulosus adults develop in the small intestine of the terminal host, and the pregnancies or eggs of the adults are discharged outside the body, thereby polluting the surrounding environment. When the intermediate host eats the pregnant joint or the ovum by mistake, the ovum is incubated in the intestinal tract of the host to release the oncosphere, blood or lymph circulates to organs such as liver, lung and the like to develop the oncosphere, the oncosphere grows along with the development of the oncosphere sac of the oncosphere, and then the oncosphere develops into the original metacercaria, the stage is the cyst form, the cyst mainly comprises the cyst wall, cyst fluid, the original metacercaria and the like (slow dream, etc., 2018), is similar to the bladder-shaped structure and has infectivity. Continuously spreading and developing between two morphological stages of metacercaria and echinococcosis in the intermediate host. After echinococcus (cyst) is swallowed by the terminal host such as dog, every live prototheca included in the echinococcus (cyst) can develop into an adult, thereby completing the life cycle. From infection to development into adults and the elimination of ova and pregnancies takes about 56 days, most adults have a life span of about 5-6 m (Shixi' an, 2008).

In the intermediate host, echinococcus granulosus parasitism can cause echinococcosis in the liver, lung, abdominal cavity, brain, etc., with echinococcosis in the liver being the most common in clinical practice. Animals have no obvious symptoms in the early infection stage, and sometimes the bodies are found in necropsy. With the continuous spread of the disease, large-area damage of animal tissues and organs occurs, accompanied by cough, hair loss, inappetence, emaciation and long-time lying down, and finally complications are caused until the animal dies (stat, 2019). Echinococcus granulosus patients show nonspecific symptoms, and may take several months to several years to develop clinical symptoms, and generally during physical examination, cysts press body organ tissues or rupture to generate secretions, so that organs are atrophied and damaged, and the health of human bodies is seriously harmed. In the terminal host dog, pathogenic parasitism does not initially present symptoms, and long-term parasitism can cause dyspepsia, tissue atrophy and dysfunction, and the sick dog dies in severe cases.

CE and AE are both common diseases of neglected animals and humans, where CE is globally distributed, especially common in countries and regions dominated by animal husbandry, mainly in the northern hemisphere, most parts of asia in the east, europe and north america, and parts of africa (MORO et al, 2009), where china is also one of the most widespread countries for CE distribution, and most parts of the west, inner mongolia in the north, and black dragon river in the northeast are high incidence areas (ZHANG et al, 2009). It is a local disease in Xinjiang, qinghai, ningxia and Gansu, western region of Sichuan province and autonomous region of Tibet (Jiang Xian, 2002). Due to wide distribution and high regional prevalence rate, the overall disease burden is high. According to incomplete statistics, the population threatened by CE in China is about 5000 million, the number of sick people is about 50-60 million, the infection rate of domestic dogs is 7-71%, the infection rate of sheep is 3.3-90%, and the direct economic loss of animal husbandry caused by animal infection is up to billions of yuan each year (JIANG, 2002 WANG et al, 2008). AE is more lethal than CE, especially in asia. Xinjiang, gansu, qinghai, ningxia, tibet, sichuan, inner Mongolia and Heilongjiang are also the main distribution areas of AE.

At present, methods such as imaging, clinical diagnosis and immunology are available for the diagnosis of CE. The imaging diagnosis method can diagnose invisible carriers, but the wounds are large, and the diagnosis is assisted by means of etiology. Surgical removal is the only possible radical treatment method for clinically treating echinococcosis, liver transplantation is the ultimate treatment, but the advanced prognosis effect is poor, and the overall radical treatment rate is low (BRUNETTI et al, 2010). The immunological diagnosis method has the advantages of rapidness, low price, sensitivity, specificity and the like, and is limited by sensitivity and specificity, so that the immunological diagnosis often plays an auxiliary role, is beneficial to supporting or confirming diagnosis of echinococcosis (HIGUITA et al, 2016), and is widely used in clinical diagnosis and epidemiological research of the echinococcosis. The major immunological diagnostic methods include enzyme linked immunosorbent assay (ELISA), indirect Hemagglutination Assay (IHAT), latex Agglutination assay (LAT), and immunofluorescence antibody assay (IFAT), among which ELISA is the most common. In recent years, molecular biological methods such as Polymerase Chain Reaction (PCR), random Amplified Polymorphic DNA (RAPD), loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA) and the like have been used, but expensive instruments and equipment are required, and the sensitivity, specificity and repeatability are greatly different, and are still under continuous optimization and development.

Screening and production of highly specific and sensitive antigens is a prerequisite for the establishment of CE serological diagnostic methods (KALANTARI et al, 2010). Echinococcus granulosus has a very complex antigenic component, and different developmental stages of the parasite can generate different immune responses, and the immunogenicity of candidate molecules of different antigens is also significantly different, and the immune mechanisms are also different (SBIHI et al, 1996 sun and zhao, 2011. The echinococcosis diagnosis antigen is selected, and the diagnosis of which animal is used needs to be judged while analyzing the development stage of the parasite in the body. Currently, the most common serodiagnostic antigen for CE is encapsulated fluid (HCF) (GONZALEZ-SAPIENZA et al, 2000), which is complex in composition and reactive to antigen. Studies have shown that cyst fluid antigens have high sensitivity (ZHANG et al, 2003), but low specificity and are capable of cross-reacting with echinococcus multilocularis, taenia suis, nematodes or blood fluke infected sera (LIGHT WLERS et al, 1996). In addition, the preparation of native HCF is difficult and expensive, the purification process is not standardized, large-scale purification production is not possible, and the requirements for current-stage diagnostics cannot be met (jinn et al, 2013 iraq, 2016. To this end, many scholars propose to isolate individual antigenic molecules in HCF, of which one of the most used for immunodiagnostic purposes is antigen B (AgB), which was determined in ELISA tests using sheep serum and found to be highly specific but less sensitive to cyst fluid (kittel berger et al, 2002), suggesting that AgB is not a suitable antigen for the detection of echinococcus granulosus infection in ruminants. Since the discovery of the Eg95 protein, which was mainly focused on immunoprotection evaluation, it was found that lambs immunized against Eg95 produced specific IgG antibodies, that immunized ewes transmitted protection to offspring, and that sheep vaccinated with Eg95 had shown superior immunoprotection (WOOLLARD et al, 2000). Meanwhile, eg95 recombinant protein was also used in sheep serum detection, but the diagnostic sensitivity was low (5.2%) (kittel berger et al, 2002) and there was severe cross-reaction with cercaria tenuis (MCMANUS et al, 2012). Crude extracts of protozoan larvae and adults may be suitable sources of antigens for serological testing in dogs and other canines, but ELISA methods using these antigens vary widely from 40% to 90% (GASSER et al, 1992) and have varying levels of cross-reactivity. In contrast, the use of recombinant proteins for serum detection has good specificity (CHEN et al, 2014), and the selection of highly reactive recombinant proteins as candidate antigen molecules is also the focus of current CE diagnostic studies.

Myogenin (Severin) is an important actin-binding protein (ABPs) belonging to the gelsolin superfamily. Currently, tens of ABPs have been discovered, of which the gelsolin superfamily is the most widely studied class of proteins and is conserved in both mammalian and non-mammalian organisms (lyzhang super and songxin, 2012). Large-scale gene sequencing work has identified gelsolin homologs of many parasites (ZHOU et al, 2009, cortez-herrerra et al, 2001.

At present, there are 7 protein members of the gelsolin superfamily, and myoglobin, an important member of the gelsolin superfamily, has Ca ²⁺ Dependent filamentous actin (F-actin) cleavage Activity (KHAI)TLINA et al, 2013), control actin organization or filamentous actin remodeling. Gelsolin and villin (villin) proteins with molecular weights of about 80-90 kDa, and myoglobin (severin) proteins with molecular weights of about 40kDa were isolated from vertebrates and invertebrates, respectively. YIN et al (1990) studies have shown that the main structure of severin is organized in a simpler manner than gelsolin. Two actin binding sites of Severin have functions of cleavage and nucleation, while these activities in gelsolin are divided into three actin binding sites. The Severin protein has various biological activities and functions, and plays an important role in the processes of cell secretion, cell signal transduction regulation, cell immunity, tumorigenesis and the like. Its activity is affected by Ca ²⁺ The concentration, intracellular pH, and membrane phospholipids are regulated only in Ca ²⁺ The filamentous actin cleavage activity could only be detected at concentrations above 6 μmol/L, whereas phosphophosphatidylinositol could inhibit the cleavage activity of myogenin (lispoin and songxin, 2012). Interestingly, each member of the gelsolin superfamily contains 3 or 6 gelsolin-like domains (SILACCI et al, 2004), and thus highly similar functions and structures exist between members.

Echinococcus granulosus chopping protein (EgSeverin) is encoded by 366aa, has a molecular weight of approximately 40.5kDa, and is a cytoskeletal regulatory protein. Studies have shown that the three-dimensional structure of Echinococcus multilocularis severin (EmSeverin) is 33.97% identical to that of human gelsolin protein. This protein is an important antigenic molecule of Echinococcus and is presumed to be closely involved in evading the host immune response (Wangfen et al, 2019). CHEN and the like (2013) prove that Clonorchis sinensis myoglobin (CsSeverin) can cause obvious apoptosis inhibition effect in a human HCC cell line and is possibly a target for preventing apoptosis; the tissue-specific distribution of CsSeverin at muscle sites, and it is presumed that CsSeverin may be involved in regulating the locomotion of the worm itself (gaily et al, 1990), a component of clonorchis sinensis secretion/excretion products (ESPs), which release large amounts of ESP when the clonorchis sinensis is persistently parasitic on the host, is likely to participate in the interaction between the host and the parasite.

The laboratory analyzes echinococcus granulosus metacercaria soluble protein by adopting an immunoproteomics and mass spectrometry technology to obtain a batch of diagnostic antigen candidate molecules; performing system analysis on the structure and function of candidate antigen molecules by using bioinformatics software, screening EgSeverin which can possibly become specific diagnostic antigen molecules as a research object, preparing a recombinant protein antibody, and analyzing the immunoreactivity and the immune effect of the antigen molecules; an indirect ELISA detection method for echinococcus granulosus and echinococcus ovirinus infection is established, is used for the primary detection of field samples, and provides technical support for the prevention and control of cystic echinococcosis.

Disclosure of Invention

The invention aims to provide application of echinococcus granulosus Severin protein in a kit for detecting cystic echinococcosis and echinococcosis infection, and particularly relates to application of recombinant echinococcus granulosus Severin protein in an ELISA kit for detecting echinococcosis granulosus infection of dogs and cystic echinococcosis of livestock such as sheep.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention relates to application of a recombinant echinococcus granulosus Severin protein in preparation of a specific antibody and/or a kit for detecting echinococcus granulosus infection and cystic echinococcosis.

As an embodiment of the invention, the recombinant echinococcus granulosus Severin protein is a protein shown in SEQ ID NO.1, or is a protein with the homology being ≧ 90% with the amino acid sequence shown in SEQ ID NO.1 and having immunogenicity. Preferably, the homology is ≧ 95%.

As an embodiment of the invention, the coding gene sequence of the recombinant echinococcus granulosus Severin protein is shown in SEQ ID No. 2.

As one embodiment of the invention, the recombinant echinococcus granulosus Severin protein is a recombinant protein obtained by taking echinococcus granulosus protozoon cDNA as a template and amplifying to obtain egseverin gene segment, connecting and transforming the egseverin gene segment with a vector to obtain a recombinant plasmid, and transforming escherichia coli to induce and express the recombinant plasmid; the egseverin gene segment is shown as SEQ ID NO. 2.

As an embodiment of the present invention, the vector is pET-28a. Coli BL21 (DE 3).

As one embodiment of the invention, the sequences of the primer pairs used for amplification are shown as SEQ ID NO.3 and SEQ ID NO.4.

As an embodiment of the invention, the kit is an indirect ELISA detection kit or an immunoblot detection kit.

As an embodiment of the present invention, the sample to be tested is serum of domestic animals (canine, ovine, etc.); through further development, the kit can be used for detecting dog excrement.

In a second aspect, the invention also relates to an ELISA kit for detecting livestock cystic echinococcosis and echinococcus granulosus infection, and the coating antigen in the kit is recombinant echinococcus granulosus Severin protein.

As an embodiment of the invention, the kit also comprises the conventional components of an ELISA detection kit, wherein the conventional components comprise antigen coating liquid, an ELISA plate, confining liquid, an enzyme-labeled secondary antibody and a substrate.

The antigen coating solution is 0.015mol/L carbonate buffer solution with pH 9.6.

The confining liquid is 5% skimmed milk powder confining liquid.

The enzyme-labeled secondary antibody is an HRP-labeled rabbit anti-sheep IgG antibody (for detecting sheep cystic echinococcosis) or an HRP-labeled rabbit anti-dog IgG antibody (for detecting echinococcus granulosus infection).

The substrate was 200mL of phosphate-citrate buffer (pH 5.0) containing 10mg of TMB and 2mL of DMSO, and 30% hydrogen peroxide (1.5. Mu.L/mL) was added just before use.

As an embodiment of the present invention, the conventional composition further comprises a color development reaction stop solution, a washing solution and a diluting solution.

The chromogenic reaction termination solution is 2mol/L H ₂ SO ₄ 。

The washing solution was 0.01mol/L PBS (pH 7.2) containing 0.05 Tween-20.

The diluent is 0.015mol/L carbonate buffer solution with pH9.6.

As one embodiment of the invention, the kit is an indirect ELISA kit for detecting echinococcus granulosus infection of dogs or an indirect ELISA kit for detecting echinococcus granulosus infection of sheep.

In the specific ELISA detection of the canine serum sample, the optimal coating concentration is 4.0 mu g/mL, the optimal serum dilution is 1. Under the above conditions, serum OD _450nm At least 0.297, the sample is judged to be positive and OD _450nm The water tank is less than 0.297 and is judged as negative.

In the specific ELISA detection of the goat serum sample, the optimal coating concentration is 1.0 mu g/mL, the optimal serum dilution is 1. Under the above conditions, serum OD _450nm A value of not less than 0.479 is positive, OD _450nm Values less than 0.479 were negative.

Compared with the prior art, the invention has the following beneficial effects:

1) The echinococcus granulosus recombinant protein rEgSeverin is cloned and expressed for the first time, and immunoblotting shows that the protein has good reactogenicity; it is found that after rEgSeverin is used in immunizing mouse, excellent humoral and cellular immune response is induced, and Th2 type cell factor is mainly induced.

2) The invention establishes an indirect ELISA detection method for echinococcus canicola infection based on rEgSeverin protein, and the specificity of the indirect ELISA detection method is higher than that of a commercial ELISA kit.

3) The invention establishes an indirect ELISA detection method for the echinococcus granulosus infection of sheep based on the rEgSeverin protein, and has higher sensitivity and specificity.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic of PCR amplification of the egseverin gene; wherein, M: DL2000 DNA molecular mass standard; 1. 2: egseverin gene amplification product;

FIG. 2 is a schematic diagram of the double digestion identification of recombinant plasmids; wherein, M: DL5000 DNA molecular weight standard; 1: double enzyme digestion products of the recombinant plasmid pET-28a-egseverin; 2: the recombinant plasmid pET-28a-egseverin is not subjected to enzyme digestion; 3: PCR product of egseverin gene;

FIG. 3 is a schematic diagram showing the induced expression of pET-28a-egseverin transformed bacteria; wherein, M: low molecular weight protein quality standard; 1: pET-28a transforming bacteria before induction; 2: transforming the induced pET-28a into bacteria; 3: pET-28a-egseverin transformed bacteria before induction; 4: induced pET-28a-egseverin transforming bacteria; 5: cracking supernatant of the induced pET-28a-egseverin converting strain;

FIG. 4 is a schematic SDS-PAGE analysis of EgSeverin purified protein; wherein, M: low molecular mass protein standards; 1. 6: cracking supernatant of the induced pET-28a-egseverin transformed bacteria; 2 to 5: 120. 150, 200, 250mM imidazole eluted product;

FIG. 5 is a schematic diagram of Western blot analysis of rEgSeverin recombinant protein; wherein, M: low molecular weight protein quality standard; 1: echinococcus granulosus infected sheep serum; 2: healthy sheep serum; 3: echinococcus granulosus infected dog serum; 4: healthy dog serum; 5: murine anti-rEgSeverin IgG serum; 6: serum of healthy mice;

FIG. 6 is a schematic diagram of the serum titer of immunized mice;

FIG. 7 is a schematic diagram showing the changes in serum IgG antibodies of immunized mice;

FIG. 8 is a schematic diagram showing the detection of the variation levels of IL-2, IL-12 and IFN-gamma in the serum of an immunized mouse;

FIG. 9 is a schematic diagram showing the detection of the variation levels of IL-4, IL-6 and IL-10 in the serum of the immunized mouse;

FIG. 10 is a schematic cross-reactivity diagram of an indirect ELISA method;

FIG. 11 is a schematic diagram of the sensitivity and specificity analysis of indirect ELISA; wherein, A: the sensitivity and specificity of rEgSeverin-iELISA are analyzed, and the critical value is 0.297; b: the detection kit has sensitivity and specificity analysis, and the critical value is 0.202.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the invention.

Example 1: amplification of EgSeverin coding gene and construction of recombinant plasmid

Primers were designed and restriction sites were introduced based on the Echinococcus granulosus Severin gene sequence (protein accession No.: EUB57054.1 SEQ ID NO.1; gene accession No.: genBank: APAU02000095.1SEQ ID NO. 2) in GenBank, where EgSeverin F1 is: 5' -CGGGATCCATGGCGGGTCTTGTGAAAGC-3' (underlined BamHI cleavage site) SEQ ID No.3, R1 is: 5' -ATTGCGGCCGCCTACTCATCCCAAATGCCC-3' (underlined Not I cleavage site) SEQ ID NO.4. The Echinococcus granulosus prototheca is used for extracting total RNA of the Echinococcus granulosus prototheca, the total RNA is reversely transcribed into cDNA, the cDNA is used as a template, and synthesized upstream and downstream primers are used for amplifying to obtain egseverin (figure 1), the size of the egseverin is about 1101bp, and the size of the egseverin accords with the expected size. The target fragment was recovered using a DNA recovery kit, and the eg14-3-3 ζ gene fragment and pET-28a (+) vector recovered from the gel were digested with SalI and Not I, respectively, in a thermostatic water bath at 37 ℃ for 2 hours. The concentrations were determined separately using a spectrophotometer, according to the concentration of the fragment of interest: the pET-28a (+) vector concentration was 3 to 4 in a ratio of 1, and was ligated overnight in a microthermal apparatus at 16 ℃ using T4 ligase. Transforming 10. Mu.L of the ligation product into E.coli DH 5. Alpha. Competent cells, and standing on ice for 30min; thermally exciting for 90s in a water bath kettle at 42 ℃, and then exciting for 3min; adding 800 μ L of non-resistant LB into the mixed solution, and shaking at 200r/min for 45min. The supernatant was discarded by centrifugation, and 100. Mu.L of nonreactive LB was added for resuspension. The bacterial liquid is smeared (containing kanamycin) and placed in a constant temperature incubator at 37 ℃ for overnight. Picking single colony from each plate, transferring to LB liquid culture medium containing kanamycin resistance, shaking at 200r/min for 6h, taking 3 mu L to carry out PCR identification on bacteria liquid, and carrying out PCR identification on the bacteria liquid and a target stripSamples of consistent size were sent for sequencing. And (3) aligning sequences, inoculating the completely correct bacterial liquid according to the proportion of 1. The recombinant plasmid is extracted by adopting an endotoxin-free plasmid large-scale extraction kit according to the instruction method, and is correctly identified by BamH I and Not I double enzyme digestion (figure 2), and the nucleotide sequence of the registered egseverin gene (accession number: APAU 02000095.1) is determined and shown to have 100 percent of homology with the nucleotide sequence of the registered egseverin gene (accession number: APAU 02000095.1).

Example 2: expression and purification of recombinant proteins

The successfully constructed recombinant plasmid pET-28a-egseverin is transformed into E.coli BL21 (DE 3) to express recombinant protein. Under IPTG induction, the recombinant plasmid transformant successfully expresses recombinant EgSeverin (recombinant EgSeverin, rEgSeverin), and the recombinant protein is soluble (FIG. 3). After affinity purification with a Ni-NTA-His column, a high purity protein was obtained with a relative molecular weight of about 45kDa (FIG. 4), consistent with the expected size. The rEgSeverin concentration was 1.071mg/mL as determined by BCA.

Example 3: antigenic analysis of recombinant proteins

The antigenicity of the recombinant protein rEgSeverin was analyzed by Western blot, and the results showed that the recombinant protein rEgSeverin recognized echinococcus granulosus-infected goat serum, echinococcus granulosus-infected dog serum, and murine anti-rEgSeverin IgG serum, with a distinct single band at about 45kDa (FIG. 5).

Example 4: preparation of mouse anti-rEgSeverin serum and titer determination

Mice were randomly divided into 3 groups of 10/group, into rEgSeverin immune group, PBS immune group and blank control group. The primary immunization was completely emulsified with Freund's complete adjuvant, followed by the booster immunization with Freund's incomplete adjuvant using subcutaneous multi-site injection. Immunizations were performed every two weeks for a total of 3 times. 1w after 3 rd immunization, the mice are anesthetized by ether, blood is collected, and the mice are kept stand for more than 2 hours in a refrigerator at 4 ℃. Separating serum, and detecting the titer of the immune mouse serum by an indirect ELISA method. The results show that OD of experimental wells at dilution 1 _450nm And negative serum OD _450nm Is greater than 2.1 (fig. 6).

Example 5: detection of serum antibody IgG levels

Sera were immunized at different time periods and the results showed that from 2w (2 w after primary immunization) to 6w (2 w after tertiary immunization), the levels of serum antibodies IgG in the rsegseverin immunized group were all very significantly elevated compared to the control group and before immunization (p < 0.0001) (fig. 7).

Example 6: cytokine detection

And (3) detecting the content changes of IL-2, IL-4, IL-6, IL-10, IL-12 (p 70) and IFN-gamma in the serum of the immune group, the blank and the PBS control group by using a cytokine detection kit. As a result, the rEgSeverin immune group with the level of IL-2 has no significant difference compared with the control group (p > 0.05). The increase of IL-12 (p 70) level in rEgSeverin immune group is very different from that in control group (p < 0.0001). The increase in IFN- γ levels in the rsegseverin immunized group was significantly different (p < 0.05) compared to the control group, with statistical significance for the difference (fig. 8). The rEgSeverin immune group showed a marginal increase in IL-6 variation level (p < 0.0001) compared to the control group. For IL-4, IL-10, rEgSeverin immunized group was significantly higher and very significantly different (p < 0.0001) compared to control group (FIG. 9).

Example 7: establishment of echinococcus canicola indirect ELISA detection method and kit assembly

7.1 determination of optimal antigen coating concentration and serum dilution

The optimal antigen coating concentration and serum dilution were determined using a square matrix method. The results show that the optimal coating concentration of rEgSeverin-iELISA was 4.0. Mu.g/mL, and the optimal serum dilution was 1.

TABLE 1 determination of optimal antigen coating concentration and serum dilution for rEgSeverin-iELISA

7.2 determination of optimal action time of serum

Adding 100 μ L/well of serum to be tested, incubating at 37 deg.C for 1h, 1.5h and 2h, and determining OD _450nm The condition that the P/N value is maximum is the optimum condition. The results showed that the optimal serum duration of action of rEgSeverin-iELISA was 2h at 37 ℃ (Table 2).

TABLE 2 optimal serum action time for rEgSeverin-iELISA

7.3 determination of the optimal working concentration of enzyme-labeled Secondary antibody

The experimental results show that the optimal working concentration of the enzyme-labeled secondary antibody of the rEgSeverin-iELISA is 1.

TABLE 3 optimal enzyme-labeled secondary antibody working concentration of rEgSeverin-iELISA

7.4 determination of optimal working time of enzyme-labeled Secondary antibody

The results showed that the optimal enzyme-labeled secondary antibody of rEgSeverin-iELISA had an action time of 1h at 37 ℃ (Table 4).

TABLE 4 optimal enzyme-labeled secondary antibody action time of rEgSeverin-iELISA

7.5 determination of the cut-off value

The cutoff value was determined by testing 28 canine negative serum samples. The results showed that the mean value of rEgSeverin-iELISA was 0.174, the standard deviation was 0.041, cut-off

0.297, OD in serum detected _450nm Not less than 0.297, the product is theoretically positive, OD _450nm The peptide below 0.297 was judged negative.

7.6 repeatability test

The results of the repeated experiments showed that the highest value of the intra-batch coefficient of variation of rEgSeverin-iELISA was 8.30% (less than 10%), and the highest value of the inter-batch coefficient of variation of different batches was 9.41%, less than 10% (Table 5). The established rEgSeverin-iELISA method has higher repeatability.

TABLE 5 rEgSeverin repeatability test

7.7 Cross-reaction test results

The cross reaction test is respectively carried out with the blood serum of mice infected by Echinococcus multilocularis, sparganosis, toxoplasma, cryptosporidium, babesia, trichinella and schistosoma. The results showed that rEgSeverin-iELISA detected OD _450nm Values all serum samples were below the cut-off value except those infected with echinococcus multilocularis (fig. 10). The rEgSeverin-iELISA can be used for simultaneously detecting echinococcus granulosus and echinococcus multilocularis canine serum.

7.8 sensitivity and specificity assays

From 24 known canine positive sera, 22 positive sera were detected in total; 45 negatives were detected in 46 canine negative sera with a sensitivity of 91.67% (22/24) and a specificity of 97.82% (45/46). A Canine echinococcosis Kit (Canine Hydatidosis IgG ELISA Kit, available from Rissenberg Biotech, inc. of Shanghai) was used to detect 24 positive samples in 24 known Canine positive sera; since 41 negative clones were detected in 46 canine negative sera, the sensitivity was 100% (24/24) and the specificity was 89.13% (41/46) (FIG. 11). The result shows that the sensitivity of rEgSeverin-iELISA is higher than that of an ELISA kit, and the indirect ELISA method is an effective and reliable detection tool for the echinococcus canicola.

7.9 kit Assembly

Antigen coating solution (0.015 mol/L carbonate buffer solution with pH of 9.6), 5% skimmed milk powder blocking solution, diluent (0.015 mol/L carbonate buffer solution with pH of 9.6), 1HRP-labeled rabbit anti-canine IgG antibody at 000 dilutions, positive serum, negative serum, TMB-H ₂ O ₂ Substrate developing solution [ phosphoric acid-citric acid buffer solution containing TMB and DMSO (pH 5.0), and 30% hydrogen peroxide (1.5. Mu.L/mL)]2mol/L sulfuric acid stop solution, washing solution (0.01 mol/L pH 7.2PBS containing 0.05 Tween-20) and an instruction for use, and assembling into a kit.

Example 8: establishment of indirect ELISA detection method for echinococcus granulosus of sheep and assembly of kit

8.1 determination of optimal antigen coating concentration and serum dilution

The same samples were made in bursts and the optimal antigen coating concentration and serum dilution were determined. The results show that the optimal coating concentration for rEgSeverin-iELISA was 1.0. Mu.g/mL, and the optimal serum dilution was 1.

TABLE 6 determination of optimal antigen coating concentration and serum dilution of rEgSeverin

8.2 determination of optimal time to Effect of serum

The results of the experiments showed that the optimal serum duration of action of rEgSeverin-iELISA was 2h at 37 ℃ (Table 7).

TABLE 7 optimal serum action time for rEgSeverin-iELISA

8.3 determination of the optimal working concentration of enzyme-labeled Secondary antibodies

The experimental results show that the optimal working concentration of enzyme-labeled secondary antibody of rEgSeverin-iELISA is 1.

TABLE 8 working concentration of the optimal enzyme-labeled secondary antibody for rEgSeverin-iELISA

8.4 determination of optimal working time of enzyme-labeled secondary antibody

The optimal enzyme-labeled secondary antibody of rEgSeverin-iELISA has the action time of 1h at 37 ℃ (Table 9).

TABLE 9 optimal enzyme-labeled secondary antibody action time of rEgSeverin-iELISA

8.5 determination of the threshold value

The threshold was calculated using 40 sheep negative serum samples and the results showed that the mean value of rEgSeverin-iELISA was 0.337, the standard deviation was 0.0472, cut-off

0.479 when detecting serum OD _450nm A value of not less than 0.479 is positive, OD _450nm Values less than 0.479 are negative.

8.6 repeatability test

The results of the repeatability tests show that the variation coefficients of the rEgSeverin-iELISA in batches are 3.51-8.3% and less than 10%, and the variation coefficients of the rEgSeverin-iELISA in batches are 4.91-12.94% and less than 15%, which indicates that the method of the rEgSeverin-iELISA has good repeatability (Table 10).

TABLE 10 rEgSeverin repeatability test

8.7 Cross-reactivity test results

Uses rEgSeverin protein as antigen coating, and makes them implement cross reaction with sheep serum infected with Echinococcus multilocularis, haemonchus contortus and schistosome respectivelyIn response to the assay, the assay results showed that all serum samples except those infected with Echinococcus multilocularis goat serum samples were tested for OD by rEgSeverin-iELISA ₄₅₀ The values were all below the critical value (table 11). The rEgSeverin-iELISA can simultaneously detect echinococcus granulosus and echinococcus multilocularis infected goat serum, and does not generate cross reaction on other parasite serum samples.

TABLE 11 Cross-reactivity test

8.8 sensitivity and specificity of the detection method

31 parts of sheep positive serum are detected by rEgSeverin-iELISA, 22 positive serum are detected in total, and the sensitivity is 70.97% (22/31); 70 sheep negative sera were tested and found to be 61 negative, so the specificity was 87.14% (61/70). Detecting 31 parts of sheep positive serum by using a commercial detection kit rapid detection kit for the sheep echinococcosis antibody (purchased from Shenzhen Lushi source biotechnology Limited), detecting 25 parts of positive serum together, wherein the sensitivity is 80.65% (25/31); 70 sheep-negative sera were tested and 66 negatives were detected, so the specificity was 94.29% (66/70) (Table 12).

TABLE 12 assay sensitivity and specificity analysis

8.9 field sample test results

Respectively detecting 540 parts of sheep serum samples by using an established rEgSeverin-iELISA and a commercial detection kit for rapid detection of the sheep echinococcosis antibody (purchased from Shenzhen poetry source biotechnology Limited), wherein 132 parts of the rEgSeverin-iELISA are positive in detection, and the positive rate is 24.44% (132/540); 146 parts of the positive test sample are detected by the kit, and the positive rate is 27.04% (146/540).

8.10 kit Assembly

Coating antigen solution (A)0.015mol/L carbonate buffer solution with pH 9.6), 5% skimmed milk powder blocking solution, diluent (0.015 mol/L carbonate buffer solution with pH 9.6), HRP-labeled rabbit anti-sheep IgG antibody diluted 1 ₂ O ₂ Substrate developing solution [ phosphoric acid-citric acid buffer solution containing TMB and DMSO (pH 5.0), and 30% hydrogen peroxide (1.5. Mu.L/mL)]2mol/L sulfuric acid stop solution, washing solution (0.01 mol/L pH 7.2PBS containing 0.05 Tween-20) and an instruction for use, and assembling into a kit.

In conclusion, the echinococcus granulosus recombinant protein rEgSeverin is cloned and expressed for the first time, and immunoblotting shows that the protein has good reactogenicity; it is found that after mice are immunized by rEgSeverin, good humoral and cellular immune responses are induced, and the Th2 type cell factors are mainly induced. The invention establishes an indirect ELISA detection method for echinococcus canicola based on rEgSeverin protein, and the specificity of the indirect ELISA detection method is higher than that of a commercial ELISA kit. The indirect ELISA detection method for the echinococcus granulosus of the sheep is established based on the rEgSeverin protein, and the sensitivity and the specificity are high.

The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers for animal health and epidemiology)

<120> application of echinococcus granulosus Severin protein in kit for detecting cystic echinococcosis and echinococcus granulosus infection

<130> DD14383

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 366

<212> PRT

<213> Echinococcus granulosus (Echinococcus grandis)

<400> 1

Met Ala Gly Leu Val Lys Ala Lys Asp Tyr Asp Trp Lys Asp Ser Asn

1 5 10 15

Met Glu Leu Phe Gly Ser Ser Lys Asp Arg Gln Val Lys Lys Glu Ser

20 25 30

Ala Met Thr Glu Lys Cys Trp Glu Pro Val Gly Arg Ala Thr Ser Pro

35 40 45

Phe Leu Met Val Trp Arg Val Asn Gln Phe Thr Leu Glu Pro Val Pro

50 55 60

Ser Asp Glu Ile Gly Asn Phe Tyr Asn Gly Asp Ser Tyr Val Ile Cys

65 70 75 80

Lys Ala Thr Arg Ser Pro Gly Gly Asp Lys Leu Leu Tyr Asn Val His

85 90 95

Phe Trp Ile Gly Lys His Ser Thr Ala Asp Glu Tyr Gly Thr Ala Ala

100 105 110

Tyr Lys Thr Val Glu Leu Asp Thr Phe Leu Asp Asp Ala Ala Val Gln

115 120 125

His Arg Glu Val Glu Gly Tyr Glu Ser Gln Leu Phe Lys Ser Tyr Phe

130 135 140

Asp Lys Leu Val Ile Leu Lys Gly Gly Tyr Ala Ser Gly Phe Arg His

145 150 155 160

Val Lys Pro Asp Glu Tyr Arg Pro Arg Leu Leu Arg Phe Cys Lys Glu

165 170 175

Gly Lys Thr Thr Tyr Met Arg Gln Val Ala Phe Ser Lys Gln Ser Val

180 185 190

His Ser Gly Asp Val Phe Ile Leu Asp Leu Gly Ser Arg Ala Tyr Gln

195 200 205

Phe Asn Gly Ser Lys Cys Ser Ala Phe Glu Lys Ser Ser Ala Ala Ala

210 215 220

Phe Leu Gln Asp Leu Glu Ser Lys Arg Asn Gly Arg Cys Asn Thr Ser

225 230 235 240

Val Leu Asp Glu Ala Asp Thr Pro Gln Asp His Glu Phe Trp Thr Ala

245 250 255

Leu Pro Asp Val Pro Val Lys Glu Leu Glu Pro Pro Lys Glu Val Ile

260 265 270

Lys Ser Leu Tyr Lys Leu Ser Asp Ser Ser Gly Lys Leu Glu Leu Thr

275 280 285

Ile Val Ser Glu Gly Ser Ala Ser Lys His Asp Ile Lys Pro Asp Asp

290 295 300

Val Tyr Ile Ile Leu Thr Lys Glu Gly Leu Phe Val Tyr Ile Gly Lys

305 310 315 320

Asp Cys Ser Val Leu Glu Lys Arg Asn Ala Leu Ser Asn Ala His Lys

325 330 335

Phe Leu Gln Thr Cys Pro Asn Pro Phe Leu Pro Ile Thr Val Val Thr

340 345 350

Asp Glu Gln Ala Glu Ser Phe Leu Lys Gly Ile Trp Asp Glu

355 360 365

<210> 2

<211> 1101

<212> DNA

<213> Echinococcus granulosus (Echinococcus grandis)

<400> 2

atggcgggtc ttgtgaaagc taaggactat gactggaaag actcaaacat ggagcttttc 60

ggctcctcaa aggataggca ggtaaaaaag gagtccgcga tgacggagaa gtgttgggag 120

cctgttggca gagcaacgtc gcccttcctc atggtctggc gtgtcaatca atttaccctc 180

gagccggtgc ccagtgatga aattgggaat ttctacaatg gcgattccta cgtcatctgc 240

aaggcaacga gaagccctgg tggtgacaag ctgctctaca atgtccattt ctggattggc 300

aagcacagca cagctgatga atacggtacc gctgcctaca agactgtcga attggacacc 360

ttcctcgatg atgctgccgt ccaacatcgc gaggtcgaag gctacgagtc gcaactcttc 420

aagagctact ttgacaagct tgtcatccta aagggtggct acgcctccgg tttccgccat 480

gtaaagccag acgagtacag gccacgtttg ttgcgcttct gcaaggaagg taaaaccacc 540

tatatgcgcc aggtggcctt cagcaagcaa tccgtccact ctggtgacgt cttcattctg 600

gacctgggca gccgggccta tcagtttaac ggctccaagt gctctgcttt tgagaagagc 660

tcggctgcag cttttctgca ggatttggag agcaagcgta atggacgctg caatacttcc 720

gtcttggatg aagccgacac accacaggat cacgaattct ggactgcgct acccgatgta 780

cctgtgaaag agctagaacc tcccaaagaa gttatcaagt cgctctacaa gttgtctgac 840

tctagtggaa aattggagtt gacgattgtg agcgagggat cggcttccaa gcatgacatt 900

aagcccgacg atgtctacat aatcctcacc aaggagggtc tcttcgtcta cataggcaag 960

gattgctctg ttttggagaa gagaaacgct ctttctaatg cccacaaatt cttacagacc 1020

tgccccaacc ctttcttgcc catcaccgtt gtgactgatg agcaggcaga atcattccta 1080

aagggcattt gggatgagta g 1101

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatccat ggcgggtctt gtgaaagc 28

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

attgcggccg cctactcatc ccaaatgccc 30

Claims

1. An application of a recombinant echinococcus granulosus Severin protein in preparation of a specific antibody and/or a kit for detecting cystic echinococcosis and echinococcus granulosus infection, wherein the recombinant echinococcus granulosus Severin protein is a protein shown as SEQ ID No. 1.

2. The use according to claim 1, wherein the recombinant echinococcus granulosus Severin protein has a coding gene sequence represented by SEQ ID No. 2.

3. The use of claim 1, wherein the recombinant Echinococcus granulosus Severin protein is obtained by amplification using Echinococcus granulosus prototheca cDNA as a templateegseverinA gene fragment of a gene which is capable of expressing,egseverinconnecting and transforming the gene fragment and a vector to obtain a recombinant plasmid, and transforming escherichia coli into recombinant protein obtained by induced expression by using the recombinant plasmid; the above-mentionedegseverinThe gene segment is shown in SEQ ID NO. 2.

4. The use of claim 3, wherein the sequences of the primer pairs used for amplification are shown as SEQ ID No.3 and SEQ ID No.4.

5. The use of claim 1, wherein the kit is an indirect ELISA detection kit or an immunoblot detection kit.

6. An indirect ELISA kit for detecting cystic echinococcosis and echinococcus granulosus infection, wherein an envelope antigen in the kit is recombinant echinococcus granulosus Severin protein, and the recombinant echinococcus granulosus Severin protein is a protein shown as SEQ ID No. 1.

7. The indirect ELISA kit for detecting echinococcosis granulosus infection and cystic echinococcosis according to claim 6, wherein the kit further comprises a conventional composition of ELISA detection kit, and the conventional composition comprises antigen coating solution, an ELISA plate, confining solution, an enzyme-labeled secondary antibody and a substrate.

8. The indirect ELISA kit of claim 7 where the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-sheep IgG antibody or HRP-labeled rabbit anti-canine IgG antibody.

9. The ELISA kit for detecting echinococcosis granulosa and echinococcosis granulosa infection of claim 6 wherein the standard composition further comprises a color reaction stop solution, a washing solution and a dilution solution.