阅读说明：本技术 犬腺病毒1型抗体elisa检测试剂盒及其应用 (Dog adenovirus type 1 antibody ELISA detection kit and application thereof ) 是由 朱言柱 闫喜军 廉士珍 于 2019-07-16 设计创作，主要内容包括：本发明公开了犬腺病毒1型抗体ELISA检测试剂盒及其应用。本发明利用pColdⅡ原核表达系统,制备具有良好反应原性的PB和Knob蛋白,对二者进行大量诱导表达,将复性后的两种蛋白分别作为包被抗原,与纯化的CAdV-1全病毒进行比对,最终确定Knob蛋白为最佳的ELISA包被抗原。在此基础上,本发明提供了一种高效、敏感性且特异性好的针对CAdV-1血清抗体的ELISA检测试剂盒,与SN法同时进行样品检测结果显示,本发明提供的ELISA检测试剂盒的敏感性为97.14％,特异性为90.00％,与SN符合率为93.57％,具有敏感度高,特异性好,重复性强等优点。(The invention discloses a canine adenovirus type 1 antibody ELISA detection kit and application thereof. The invention utilizes a pCold II prokaryotic expression system to prepare PB and Knob proteins with good reactogenicity, and carries out a large amount of induction expression on the PB and the Knob proteins, the two renatured proteins are respectively used as envelope antigens to be compared with purified CAdV-1 whole viruses, and finally the Knob protein is determined to be the best ELISA envelope antigen. On the basis, the invention provides an efficient ELISA detection kit with good sensitivity and specificity for the CAdV-1 serum antibody, and sample detection results are simultaneously carried out with an SN method, so that the sensitivity of the ELISA detection kit provided by the invention is 97.14%, the specificity is 90.00%, the rate of coincidence with SN is 93.57%, and the kit has the advantages of high sensitivity, good specificity, strong repeatability and the like.)

1. The dog adenovirus type 1 antibody ELISA detection kit comprises an ELISA plate coated with an antigen, an enzyme-labeled secondary antibody, a sample diluent, a washing solution, a confining liquid, a TMB developing solution, a stop solution, a standard CAdV-1 positive serum sample and a standard CAdV-1 negative serum sample, and is characterized in that the antigen is a Knob protein.

2. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the method for preparing the Knob protein comprises: and (3) expressing the coding gene of the Knob protein through a prokaryotic expression system under a low-temperature condition to obtain the recombinant Knob protein.

3. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 2, comprising: (1) amplifying a Knob full-length gene by using the extracted CAdV-1DNA as a template and primers shown in SEQ ID NO.1 and SEQ ID NO. 2; (2) and connecting the amplified Knob full-length gene to a prokaryotic expression vector, and performing induced expression and purification in escherichia coli to obtain the recombinant protein.

4. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the enzyme-labeled secondary antibody is HRP-labeled Goat anti-DogIgG.

5. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the blocking solution is BSA.

6. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 5, wherein the blocking solution is 3% BSA.

7. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the concentration of the Knob protein-coated ELISA plate is 2.5. mu.g/mL.

8. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the washing solution is PBST.

9. The ELISA detection kit for canine adenovirus type 1 antibody according to claim 1, wherein the stop solution is HCL solution with a concentration of 1M.

10. Use of the canine adenovirus type 1 antibody ELISA detection kit of any one of claims 1-9 in the preparation of a reagent for detecting canine adenovirus type 1 antibodies.

Technical Field

The invention relates to a canine adenovirus type 1 antibody detection kit, in particular to a canine adenovirus type 1 antibody ELISA detection kit and a preparation method thereof, and further relates to application of the canine adenovirus type 1 antibody ELISA detection kit in detection of canine adenovirus type 1 antibodies, belonging to the field of canine adenovirus type 1 antibody ELISA detection kits.

Background

Canine adenovirus type 1 (CAdV-1) is a double-stranded DNA virus. CAdV-1 causes mainly hepatitis, nephritis (dogs) and encephalitis (bears and foxes), is highly pathogenic and has a broad spectrum of infections, and can cause fatal infections in wild carnivorous mammals (wolves, brown bears, black bears, otters) in addition to infections in dogs and foxes (Jeong-Won et al, 2014; Lepczyk, 2015). CAdV-1 infection has been reported around the world and has attracted considerable attention. The monitoring of the CAdV-1 virus requires a detection method with strong specificity, high sensitivity and accuracy. The traditional detection method of the CAdV-1, such as separation and identification of virus, SN, HA-HI and the like, is time-consuming and labor-consuming, and HAs larger error of results; although the detection sensitivity of PCR and qPCR is high, the PCR and qPCR are very likely to cause virus nucleic acid pollution in the environment during the operation (Tasker et al, 2003).

ELISA is used as a traditional serological diagnosis method with the widest application, has strong operability, high specificity and sensitivity and low price and cost, and is an ideal diagnosis method for monitoring the immune effect of the CAdV-1 vaccine and infecting animals. At present, most of CAdV-1 antibody ELISA detection methods are in the laboratory development stage (Jianelili et al, 2008; Kudzuvine et al, 2010; Walker et al, 2016), but no commercial CAdV-1 antibody ELISA detection kit is provided in the market.

Disclosure of Invention

One of the purposes of the invention is to provide an optimal envelope antigen of the ELISA detection kit for the canine adenovirus type 1 antibody;

The other purpose of the invention is to provide a dog adenovirus type 1 antibody ELISA detection kit;

The third purpose of the invention is to provide a method for preparing the ELISA detection kit for the canine adenovirus type 1 antibody.

The above object of the present invention is achieved by the following technical solutions:

The invention firstly determines the best coating antigen of the kit for ELISA detection of the Knob protein as the canine adenovirus type 1 antibody.

The preparation method of the Knob protein comprises the following steps: and (3) expressing the coding gene of the Knob protein through a prokaryotic expression system under a low-temperature condition to obtain the recombinant Knob protein. More preferably, the method for preparing the recombinant Knob protein comprises the following steps: (1) amplifying a Knob full-length gene by using the extracted CAdV-1DNA as a template and primers shown in SEQ ID NO.1 and SEQ ID NO. 2; (2) and connecting the amplified Knob full-length gene to a prokaryotic expression vector, and performing induced expression and purification in escherichia coli to obtain the recombinant protein.

The invention utilizes a pColdII prokaryotic expression system to obtain a soluble expressed recombinant CAdV-1 structural protein Penton Base (PB) and Knob, obtains a purified CAdV-1F1301 strain culture product by a CsCl density gradient centrifugation method, uses three antigens to respectively react with CAdV-1 negative serum and positive serum, and determines the optimal ELISA envelope antigen according to the OD450 value and the P/N value; the results show that the Knob protein is the best antigen for ELISA.

Penton is composed of a substrate (Base) and a fibril (Fiber) and is one of the main structural proteins of CAdV-1, the top of the Penton substrate comprises two important bulge structures, wherein one bulge is a hyper-variable Loop structure, and the other bulge comprises a structural domain interacting with host cell integrin, and when a virus invades a host cell, the interaction is generated after the virus is combined with the host cell integrin protein, and virus particles are internalized into the cell. The Knob protein is the distal structure of trimer Fiber and is spherical. The Knob protein is the main receptor binding protein of CAdV-1, and in the process of CAdV-1 infection of a host, the Knob is combined with a host cell surface specific receptor to anchor virus particles on the cell surface, so that an environmental basis is provided for further infection of the host cell. The Knob protein has hemagglutination property, can specifically agglutinate red blood cells, and is a main antigen protein for distinguishing CAdV-1 from CAdV-2. Both Penton Base (PB) and Knob proteins have CAdV-1 major type-specific epitopes, both of which can be used as diagnostic antigens for CAdV-1 (Cao et al, 2012; Simao et al, 2016).

On the basis of determining that the Knob protein is used as the optimal envelope antigen of the ELISA detection kit for the canine adenovirus type 1 antibody, the invention provides the ELISA detection kit for the canine adenovirus type 1 antibody, which comprises the following components in percentage by weight: the kit comprises an ELISA plate for coating the Knob protein, an enzyme-labeled secondary antibody, a diluent, a washing solution, a confining solution, a developing solution, a stopping solution, a standard CAdV-1 positive serum sample or a standard CAdV-1 negative serum sample.

The enzyme-labeled secondary antibody is HRP-labeled Goat anti-Dog IgG; the blocking solution is preferably BSA, and most preferably 3% BSA; the wash solution is PBST; the sample diluent is a washing solution containing 5% of skimmed milk powder; the stop solution is a HCL solution with the concentration of 1M; the color development liquid is TMB color development liquid.

The invention further optimizes the preparation conditions of the ELISA detection kit for the canine adenovirus type 1 antibody, and the optimal coating concentration of the Knob protein is determined to be 2.5 mug/mL through the discovery of an optimized test result; when sealing is carried out by adopting sealing liquid, the most suitable sealing condition is 37 ℃ for 2 h; the optimal dilution of serum is 1:100, respectively; the optimal dilution of the enzyme-labeled secondary antibody is 1: 8000; the optimal action conditions of the serum and the secondary antibody are respectively 1h at 37 ℃ and RT1 h; the final cut-off value for positive and negative samples was determined to be 0.376.

For reference, the invention provides a method for detecting canine adenovirus type 1 antibody in a serum sample by using the canine adenovirus type 1 antibody ELISA detection kit, which comprises the following steps:

(1) Diluting the protein antigen of Knob to 5 mu g/mL by using 50mM buffer solution with pH9.6, coating an enzyme label plate, performing reaction at 100 mu L/hole for 2h at 37 ℃; washing with washing solution for 3 times, 100 μ L/well/time; adding confining liquid, acting at 100 μ L/hole for 2 hr at 37 deg.C, and washing the plate with washing liquid for 3 times;

(2) Adding serum (1:100 dilution) to be detected, setting positive and negative control in the same enzyme label plate at 100 μ L/hole, and acting at 37 deg.C for 0.5 h; washing the plate for 5 times;

(3) Adding HRP-labeled Goat anti-Dog IgG (1:5000 dilution), 100 mu L/hole, and RT1 h; washing the plate for 5 times;

(4) Adding TMB color development solution, standing for 10min in dark at a concentration of 100 μ L/well, adding 1M HCL at a concentration of 100 μ L/well, placing the ELISA plate in an ELISA reader, reading OD value at 450nm, and calculating P/N value according to OD value of positive and negative serum.

(5) The OD450 value of the serum to be detected is greater than 0.376 and is determined as positive, and the OD450 value of the serum to be detected is less than 0.376 and is determined as negative.

According to the invention, 12 serum samples are selected for repeated experiments, and the results show that the difference coefficient between ELISA plates and the difference coefficient in ELISA plates are within 10%. The specificity test result shows that the method does not have cross reaction with CDV, CPV and CPIV positive serum, and has good specificity. Sensitivity experiment results show that the highest dilution factor of positive samples detectable by ELISA is 1:3200, which has a sensitivity well above 1:256 of SN. The ELISA method and the SN method established by the invention are used for simultaneously detecting samples, and the result shows that the ELISA sensitivity is 97.14 percent, the specificity is 90.00 percent, and the rate of coincidence with the SN is 93.57 percent; the comprehensive results show that the ELISA detection kit for the canine adenovirus type 1 antibody provided by the invention has the advantages of high sensitivity, good specificity, strong repeatability and the like, and has a certain application prospect.

Detailed description of the invention

The invention utilizes a pCold II low-temperature prokaryotic expression system to induce the expression of the target protein under the condition of 15 ℃, compared with the condition of 37 ℃, the low-temperature condition is favorable for the folding of a protein high-grade structure, the soluble expression efficiency is improved, the soluble PB and Knob fusion protein is successfully expressed, and the invention provides the envelope antigen for the establishment of a CAdV-1 antibody ELISA method. In the test, after the enzyme label plate is coated with the purified CAdV-1 complete virus antigen and reacts with CAdV-1 negative and positive serum samples, the OD value of the positive sample is 2.48, and the positive sample has a good reaction effect, but the OD value of the negative sample is 0.48, which is twice higher than that of the positive sample when the negative sample is coated with the Knob protein, and the positive sample is easy to miss detection in clinical application. In the traditional ELISA method using the CAdV-1 whole virus as the coating antigen, virus particles are mostly obtained in an ultracentrifugation mode, the concentration and the purity of the virus are low, and the sensitivity and the specificity of the ELISA detection method are influenced to a certain extent. In the virus purification process, because a centrifugal tube special for the ultracentrifuge cannot be sealed, virus particles are easy to escape, and the environment is polluted. Compared with the traditional method, the protein obtained by prokaryotic expression not only has higher concentration and purity, but also can not cause environmental pollution (Lebarbenchon et al, 2012). PB is one of main structural proteins of CAdV-1, the purified recombinant PB protein coats the ELISA plate and reacts with negative and positive serum samples of CAdV-1, the OD value of the positive sample is 2.14, the good reaction effect is achieved, but the OD value of the negative sample is too high and is 0.72, the P/N value is 2.97, and the overall specificity is low. Compared with the Knob protein, the PB protein has a much more complex structure, is wholly in the shape of a pentameric tulip, and the insufficient exposure of specific antigen epitopes can be the reason for poor reaction with CAdV-1 negative and positive serum (Fuschiotti et al, 2006; Galinier et al, 2002).

The indirect ELISA detection method established by the recombinant Knob protein and aiming at the CAdV-1 has high stability, the in-plate difference coefficient is 0.36-8.07%, and the inter-plate difference coefficient is 0.43-5.40%, and both are less than 10%. The sensitivity test is carried out on serum samples diluted in multiple proportions, and the result shows that the highest dilution of CAdV-1 positive samples which can be detected by ELISA is 1:3200, which has a sensitivity well above 1:256 of SN. The serum has no cross reaction with positive serum of three common canine diseases of CDV, CPV and CPIV, and has strong specificity. The sensitivity (Se) and specificity (Sp) of the ELISA detection method and the SN detection method of the CAdV-1 antibody are 97.14 percent and 90 percent respectively, and the coincidence rate is 93.57 percent. The result shows that the ELISA detection method established by the test has high sensitivity, good specificity and strong repeatability, can replace a neutralization test to carry out large-scale clinical diagnosis, and has certain economic value.

The invention utilizes a pCold II prokaryotic expression system to prepare PB and Knob proteins with good reactogenicity, and carries out a large amount of induction expression on the PB and the Knob proteins, and the two renatured proteins are respectively used as envelope antigens to be compared with purified CAdV-1 complete viruses to screen out the optimal ELISA envelope antigen. Through serial optimization, an indirect ELISA detection method of a serum antibody is established, an efficient ELISA detection kit which has good sensitivity and specificity and aims at the CAdV-1 serum antibody is provided, and the parallel relation between the kit and a neutralization test detection method is evaluated.

Drawings

FIG. 1 gene amplification and recombinant plasmid double restriction enzyme identification; (A) PB and Knob genes are amplified by PCR, and 1, DNA molecular mass standard; 2. a PB gene; 3. a Knob gene; 4. negative control (B) double enzyme digestion identification of recombinant plasmid, 1, DNA molecular mass standard; 2. pCold Π -PB plasmid; 3. pCold Π -Knob plasmid.

FIG. 2 expression and purification of recombinant proteins; 1: protein molecular mass standard; 2: purifying the protein; 3: a whole-bacterium lysate; 4: the upper clear of thallus schizolysis; 5: non-induced control

FIG. 3 antigen coating concentration versus serum dilution.

FIG. 4 confining liquid optimization curve.

Figure 5 secondary antibody optimization curves.

FIG. 6 is a closed time optimization curve.

Figure 7 serum incubation condition optimization curve.

Figure 8 secondary antibody incubation conditions.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Primary biomaterials and reagents

70 parts of CAdV-1 standard positive serum, 32 parts of CAdV-1 inactivated vaccine immune silver fox positive serum prepared in the laboratory, 20 parts of CAdV-1 inactivated vaccine immune Chinese rural dog positive serum and 18 parts of CAdV-2 live vaccine immune silver fox positive serum. Obtaining 10 parts of CAdV standard negative serum by SN screening, wherein all the serum is derived from healthy silver foxes. CDV, CPV and CPIV positive sera were all preserved. The CAdV-1F1301 strain, the CAdV-2 vaccine strain and the MDCK cell are all preserved in the laboratory. DH5 alpha, BL21(DE3) competent cells and pColdII prokaryotic expression vector, purchased from TaKaRa company.

Fast pfu high-fidelity DNA polymerase and T₄DNA ligase was purchased from lnvitrogen;His-Bind Purification Kit was purchased from Thermo Fisher; goat Anti-Mouse IgG (HRP), Goat Anti-Dog IgG (HRP) and Anti-6X His tags from abcam; CAdV-1 standard positive sera were stored by the laboratory.