Indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein

Abstract

The invention discloses an indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein, comprising an ELISA plate coated with rabbit rotavirus VP8* protein; the amino acid sequence of the rabbit rotavirus VP8* protein is shown in SEQ ID NO.1. It also comprises a sample diluent, a concentrated washing solution, an enzyme conjugate working solution, etc. The indirect ELISA antibody detection kit is used in detecting rabbit rotavirus antibodies. The present invention uses the rabbit rotavirus VP8* protein as a coating antigen, which has high specificity for RVs antibodies, good safety, only specifically binds to RVs positive serum, does not cross-react with positive serum of other rabbit viruses, and has good antigenicity. The indirect ELISA antibody detection kit of the present invention has high specificity and sensitivity, can realize high-throughput detection of rabbit rotavirus antibodies, and provides technical support for the comprehensive prevention and control of rabbit rotavirus.

Description

Technical Field

[0001] The invention relates to an indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein, and belongs to the technical field of antibody detection. Background Art

[0002] Rotaviruses (RVs) are double-stranded RNA viruses of the genus Reovirus that can cause acute gastroenteritis in humans and animals. Rotavirus was first discovered in laboratory mice in 1974, and has since been found in mammals such as humans, pigs, cattle, and birds. Rabbit rotavirus mainly causes diarrhea in young rabbits aged 1 to 2 months, and rabbits of all stages can be infected. Rabbit rotavirus disease develops rapidly, has a high incidence rate, and can occur all year round.

[0003] The rotavirus genome consists of 11 discontinuous fragments, which can encode 6 structural proteins and 5 non-structural proteins. The main antigen VP4 of rotavirus can be cleaved into VP8* and VP5* under the action of trypsin. VP8* plays an important role in the process of the virus entering the host by changing from a dimer to a trimer. VP4 is an important target protein for the development of recombinant vaccines, but the complete VP4 has poor solubility. VP8* contains the main antigenic epitopes of the vp4 protein and can stimulate the body to produce neutralizing antibodies. At present, there have been many studies on subunit vaccines, antibody preparation, diagnostic methods, etc. for human, porcine, and bovine RVVP8*, and some progress has been made, but no research on rabbit rotavirus VP8* has been found. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides an indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein to achieve rapid detection of rabbit rotavirus antibodies.

[0005] The present invention is achieved through the following technical solutions:

[0006] An indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein, comprising an ELISA plate coated with rabbit rotavirus VP8* protein; the ELISA plate coated with rabbit rotavirus VP8* protein is prepared by using rabbit rotavirus VP8* protein as coating antigen and carbonate buffer as coating liquid. The amino acid sequence of the rabbit rotavirus VP8* protein is as follows, as shown in SEQ ID NO.1:

[0007] MASLIYRQLLNNSFTTNLSDEIEEIGSSSKSENVTINPGPFAQTGYAPVDWGPGETNDSTTIVPVLDGPYQPTNFNPPIEYWMLFAPSDKGVVAELTNNIDIWLAIILVEPSVPQESRTYT IFGQRSSLMVENTSQTKWKFIDFVKESQNGTYVPRDTLLSETKLQAAMKYGQRLFTFIGDTPNATPQERGYTTNNYSAINVTSLCDFYIVSRTPREVCRSYINHGLPPMQNTRNVVPVAL.

[0008] Furthermore, the rabbit rotavirus VP8* protein is prepared by the following method: using the RVZ3171 strain VP4 gene as a template, using specific primers for PCR amplification to obtain an amplified product (i.e., the expression gene of the rabbit rotavirus VP8* protein, the nucleotide sequence is shown in SEQ ID NO.2, 711 bp); transferring the amplified product into an engineered bacterium to obtain a recombinant engineered bacterium; culturing the recombinant engineered bacterium, collecting the bacteria, and purifying to obtain the rabbit rotavirus VP8* protein. The specific primers include an upstream primer and a downstream primer, and the nucleotide sequence is shown as follows:

[0009] Upstream primer P1: 5′-CCGCTCGAGATGCGCCAGTAGAC-3′, as shown in SEQ ID NO. 3;

[0010] Downstream primer P2: 5′-CGGAATTCTGATTGTGATGTCC-3′, as shown in SEQ ID NO.4;

[0011] The nucleotide sequence of the expression gene of rabbit rotavirus VP8* protein (direction 5′--3′, as shown in SEQ ID NO.2):

[0012] .

[0013] Furthermore, the indirect ELISA antibody detection kit also includes the following components: sample diluent, concentrated washing solution, enzyme conjugate working solution, color developing solution, stop solution, positive serum control and negative serum control.

[0014] Furthermore, the sample diluent is a phosphate buffer (0.01 M, pH 7.4) containing 0.05% Tween-20.

[0015] Furthermore, the concentrated washing solution is phosphate buffered saline (0.1 M, pH 7.4) (PBST) containing 0.5% Tween-20.

[0016] Furthermore, the enzyme conjugate working solution is HRP-goat anti-rabbit IgG.

[0017] Furthermore, the color developing solution is TMB-hydrogen peroxide urea solution.

[0018] Furthermore, the stop solution is 0.31% hydrofluoric acid solution.

[0019] The indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein is used in detecting rabbit rotavirus antibodies.

[0020] A method for detecting rabbit rotavirus antibodies using an indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein comprises the following steps:

[0021] (1) Dilute the sample to be tested 1:100 with sample diluent and add 100 μL / well to the ELISA plate coated with rabbit rotavirus VP8* protein. Set up negative serum control and positive serum control at the same time and incubate at 37°C for 45 min.

[0022] (2) Discard the liquid in the reaction wells, add 350 μL of washing solution to each well, wash 3 to 5 times, with 1 min interval between each wash, and pat dry;

[0023] (3) Add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 45 min;

[0024] (4) Discard the liquid in the reaction wells, add 350 μL of washing solution to each well, wash 3 to 5 times, with 1 min interval between each wash, and pat dry;

[0025] (5) Add 100 μL of color development solution and incubate at 37°C in the dark for 15 min;

[0026] (6) Add 50 μL of stop solution and measure the absorbance OD value of each well at 650 nm using an ELISA reader. Calculate and determine the result. If the OD value of the sample to be tested is 650nm OD value and negative control 650nm The ratio of values ​​(P / N) is ≥ 2.1, and the OD of the sample to be tested is 650nm If the value is greater than 0.247, it is considered positive.

[0027] The indirect ELISA antibody detection kit of the present invention uses the synthetic rabbit rotavirus VP8* protein as the coating antigen, has high specificity for RVs antibodies, good safety, does not contain irrelevant foreign proteins, specifically binds only to RVs positive serum, does not cross-react with positive serum of other rabbit viruses, and has good antigenicity, so that the detection kit of the present invention has high specificity and sensitivity.

[0028] The indirect ELISA detection kit based on rabbit rotavirus VP8* protein of the present invention can evaluate the RVs antibody level, is suitable for the detection of batch samples, is simple and fast to operate, and greatly improves the speed of rabbit rotavirus serological diagnosis. The present invention uses genetic engineering technology to express the VP8* protein of the current epidemic strain of rabbit rotavirus as a coating antigen, establishes an antibody detection ELISA method, can realize high-throughput detection of rabbit rotavirus antibodies, and provides technical support for the comprehensive prevention and control of rabbit rotavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of the test results of serum at different time points after rabbit rotavirus vaccine immunization. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0031] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0032] Example 1 Preparation of rabbit rotavirus VP8* protein

[0033] Here are the steps:

[0034] (1) Using the VP4 gene of RVZ3171 strain as a template, specific primers were used for PCR amplification to obtain the recombinant plasmid pMD-18T-VP8*.

[0035] The specific primers include an upstream primer and a downstream primer, and the nucleotide sequence is as follows:

[0036] Upstream primer P1: 5′-CCGCTCGAGATGCGCCAGTAGAC-3′, as shown in SEQ ID NO. 3;

[0037] Downstream primer P2: 5′-CGGAATTCTGATTGTGATGTCC-3′, as shown in SEQ ID NO.4.

[0038] (2) The recombinant plasmid pMD-18T-VP8* and the expression vector pET-28a(+) were double-digested with EcoRⅠ and XhoⅠ, respectively. The vector fragment and the target fragment were recovered and ligated. The ligation product was transformed into Escherichia coli Trans5α competent cells using the thermal transformation method. After double enzyme digestion and identification, the positive recombinant plasmid was obtained.

[0039] (3) The positive recombinant plasmid was double-digested with EcoRⅠ and XhoⅠ, the target fragment was recovered, connected at 16°C for 12 hours, transformed into BL21(DE3) competent cells, and the plasmid was extracted. After double-digestion identification, the positive recombinant plasmid pET-28a(+)-Z3171-VP8* was obtained.

[0040] (4) The recombinant bacteria containing the positive recombinant plasmid pET-28a(+)-Z3171-VP8* were cultured at 37°C. When the OD600 value reached 0.6, IPTG was added to a final concentration of 1.0 mmol / L and induced for expression at 30°C for 4 h. The bacteria were collected, disrupted by ultrasonic wave, centrifuged, and the supernatant was purified by Ni2+ column affinity chromatography to obtain rabbit rotavirus VP8* protein.

[0041] Example 2 Preparation of ELISA Plates Coated with Rabbit Rotavirus VP8* Protein

[0042] The rabbit rotavirus VP8* protein (prepared in Example 1) was diluted to a concentration of 6.25 μg / mL using a 0.05 M carbonate buffer solution at pH 9.6; the protein solution was added to an ELISA reaction plate at a dose of 100 μL / well, incubated at 37° C. for 2 hours, and then coated at 4° C. for 12 hours; the coating solution was discarded (patted dry), and blocked with pigskin gelatin at 37° C. for 2 hours; the plate was washed with a phosphate buffer solution (0.01 M, pH 7.4, containing 0.05% Tween-20), dried, and stored in a packaging bag containing a desiccant for later use.

[0043] The coating solution is prepared by taking 1.5 g of sodium carbonate, 2.9 g of sodium bicarbonate, and 0.2 g of sodium azide, adding double distilled water to a volume of 1000 ml, and adjusting the pH to 9.6.

[0044] Example 3 Preparation of other components in ELISA antibody detection kit

[0045] The sample diluent is a phosphate buffer (0.01M, pH 7.4) containing 0.05% Tween-20. The preparation method is as follows: take 0.2 g of KH2PO4, 2.9 g of NaHPO4·12H2O, 8 g of NaCl, and 0.5 mL of Tween-20, mix them, and add double distilled water to make up to 1000 mL.

[0046] The concentrated washing solution (10×) is a phosphate buffer (0.1 M, pH 7.4) containing 0.5% Tween-20. The preparation method is as follows: take 2 g of KH2PO4, 29 g of NaHPO4·12H2O, 80 g of NaCl, and 5 mL of Tween-20, mix them, and add double distilled water to make up to 1000 mL.

[0047] The color developing solution is TMB-hydrogen peroxide urea solution, and the preparation method is as follows:

[0048] Take 200 mg of tetramethylbenzidine (TMB), dissolve it in 100 mL of anhydrous ethanol, and dilute to 1000 mL with double distilled water to obtain solution A;

[0049] Take 21 g of citric acid (C6H8O7·H2O), 28.2 g of anhydrous disodium hydrogen phosphate (Na2HPO4), 6.4 mL of 0.75% urea hydrogen peroxide, dilute to 1000 mL with double distilled water, adjust the pH to 5.0, and obtain solution B;

[0050] Mix equal volumes of solution A and solution B to obtain the color developing solution.

[0051] The stop solution is 0.31% hydrofluoric acid solution, and the preparation method is as follows: take 0.31 ml of hydrofluoric acid and dilute to 100 ml with double distilled water.

[0052] Positive serum control: The RVs positive serum obtained by screening was diluted 1:100 with sample diluent (OD 650nm ≥1.0)(referring to OD after 100-fold dilution 650nm ≥1.0), add penicillin-streptomycin at a final concentration of 1000 U / mL, and sterile filter as a positive control.

[0053] Negative serum control: RVs negative serum obtained by screening (OD 650nm ≤0.22), penicillin-streptomycin was added at a final concentration of 1000 U / mL, and sterile filtered as a negative control.

[0054] Example 4 ELISA antibody detection kit

[0055] It is composed of the following components:

[0056] ELISA strips (96 wells): 5 pieces;

[0057] Sample diluent: 400mL;

[0058] Concentrated washing solution: 400 mL (diluted 1:10 before use);

[0059] Enzyme conjugate working solution: goat anti-rabbit enzyme-labeled secondary antibody (KPL, USA), 100 mL;

[0060] Color developing solution: 200mL;

[0061] Stop solution: 100mL;

[0062] Positive serum control (+): 2 mL;

[0063] Negative serum control (-): 2 mL.

[0064] Example 5 Determination of indirect ELISA reaction conditions

[0065] The optimal working concentrations of polypeptide antigen and serum were determined by square array test.

[0066] The rabbit rotavirus VP8* protein was serially diluted with coating solution and coated on ELISA reaction plate, with the dose controlled at 100 μL / well; the positive serum and negative serum against RVs were serially diluted with sample diluent at 1:50, 1:100, 1:200, and 1:400, respectively; indirect ELISA determination was performed; color development solution was added for color development, and the reaction was terminated with stop solution; the OD value at a light wavelength of 650 nm was measured, and the results are shown in Table 1.

[0067] Table 1 Determination of optimal working concentrations of expressed antigens and serum (OD 650nm value)

[0068]

[0069]

[0070] Take positive serum OD 650nm 1.0, negative serum OD 650nm 0.22, and positive serum OD 650nm / Negative serum OD 650nm That is, the antigen concentration and serum dilution with the largest P / N value are the optimal working concentrations. The results show that the optimal serum dilution is 1:100, and the optimal protein coating concentration is 6.25μg / mL, or 0.625μg / well.

[0071] The 30 collected rabbit sera without RVs antibodies were subjected to indirect ELISA under the optimal working conditions to determine the absorbance range of rabbit sera when there was no RVs infection. The results were: Therefore, the OD of the sample to be tested is determined 650nm Value and negative OD 650nm The ratio of values ​​(P / N) is ≥ 2.1, and the OD of the sample to be tested is 650nm Value>0.247 was judged as positive.

[0072] Example 6 Method of using the ELISA antibody detection kit

[0073] Here are the steps:

[0074] (1) Dilute the sample to be tested 1:100 with sample diluent and add 100 μL / well to the ELISA plate coated with rabbit rotavirus VP8* protein. Set up negative serum control and positive serum control at the same time and incubate at 37°C for 45 min.

[0075] (2) Discard the liquid in the reaction wells, add 350 μL of washing solution to each well, wash 3 to 5 times, with 1 min interval between each wash, and pat dry;

[0076] (3) Add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 45 min;

[0077] (4) Discard the liquid in the reaction wells, add 350 μL of washing solution to each well, wash 3 to 5 times, with 1 min interval between each wash, and pat dry;

[0078] (5) Add 100 μL of color development solution and incubate at 37°C in the dark for 15 min;

[0079] (6) Add 50 μL of stop solution and measure the absorbance OD value of each well at 650 nm using an ELISA reader. Calculate and determine the result. If the OD value of the sample to be tested is 650nm OD value and negative control 650nm The ratio of values ​​(P / N) is ≥ 2.1, and the OD of the sample to be tested is 650nm If the value is greater than 0.247, it is considered positive.

[0080] Example 7 Specificity Test

[0081] The ELISA antibody detection kit established in Example 4 was used to detect rabbit rotavirus-positive serum, SPF rabbit serum, rabbit coronavirus-positive serum, rabbit hemorrhagic disease virus (type 1)-positive serum, and rabbit astrovirus-positive serum, respectively. Four replicates were performed for each sample to perform cross-reactivity assays. The test results showed that the rabbit rotavirus-positive serum was positive, and the rest were negative, indicating that the detection kit had no cross-reaction with rabbit coronavirus, rabbit hemorrhagic disease virus (type 1), rabbit astrovirus, etc.

[0082] Example 6 Sensitivity Test

[0083] The rabbit rotavirus positive serum and negative serum were diluted 1:50 to 1:6400 respectively, and the other conditions were tested by ELISA according to the optimal reaction conditions. The results are shown in Table 2. The results show that when the rabbit rotavirus positive serum was diluted to 1:3200, its OD 650nm It is still higher than the critical value of 0.247, which proves that the ELISA detection kit of the present invention has high sensitivity.

[0084] Table 2 Results of sensitivity test

[0085]

[0086] Example 7 Repeatability Test

[0087] The ELISA antibody detection kit established in Example 4 was used to detect 5 RVs positive sera and 5 negative sera respectively. Each sample was tested 5 times, and the coefficient of variation CV% (CV=SD / X×100%, SD: standard deviation, X: arithmetic mean) was determined. The results showed that the maximum coefficient of variation was 3.8% and the minimum was 1.24%. The coefficients of variation of the 10 sera were small, with good repeatability.

[0088] Example 8 Clinical application of ELISA antibody detection kit

[0089] The samples for clinical application test were rabbit serum at 3d, 7d, 14d, 21d, 35d and 42d after immunization with rabbit rotavirus vaccine. The ELISA antibody detection kit established in Example 4 was used for detection. The results were as follows: Figure 1 As shown. Figure 1 It can be seen that the test results of serum at different time points after immunization with rabbit rotavirus vaccine are consistent with the theoretical increase and decrease of antibodies of rabbit rotavirus inactivated vaccine. The ELISA antibody detection kit established by the present invention is suitable for clinical application.

[0090] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. An indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein, characterized in that: An ELISA plate coated with a rabbit rotavirus VP8* protein; the amino acid sequence of the rabbit rotavirus VP8* protein is shown in SEQ ID NO.1; The indirect ELISA antibody detection kit also includes the following components: sample diluent, concentrated washing solution, enzyme conjugate working solution, color developing solution, stop solution, positive serum control and negative serum control; The enzyme conjugate working solution is HRP-goat anti-rabbit IgG; The sample diluent is phosphate buffer containing 0.05% Tween-20, 0.01 M, pH 7.4; The concentrated washing solution is phosphate buffer containing 0.5% Tween-20, 0.1 M, pH 7.4; The color developing solution is TMB-hydrogen peroxide urea solution; The stop solution is 0.31% hydrofluoric acid solution.

2. The indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein according to claim 1, characterized in that: The rabbit rotavirus VP8* protein is prepared by the following method: using the VP4 gene of the RV Z3171 strain as a template, using specific primers to perform PCR amplification to obtain an amplified product; transferring the amplified product into engineering bacteria to obtain recombinant engineering bacteria; culturing the recombinant engineering bacteria, collecting the bacteria, and purifying to obtain the rabbit rotavirus VP8* protein.

3. The indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein according to claim 2, characterized in that: The specific primers include an upstream primer and a downstream primer, and the nucleotide sequence is as follows: Upstream primer P1: 5′-CCGCTCGAG ATGCGCCAGTAGAC-3′, as shown in SEQ ID NO. 3; Downstream primer P2: 5′-CGGAATTCTGATTGTGATGTCC-3′, as shown in SEQ ID NO.

4.

4. The indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein according to any one of claims 1 to 3, characterized in that: It is composed of the following components: ELISA plates coated with rabbit rotavirus VP8* protein: 5; Sample diluent: 400 mL; Concentrated washing solution: 400 mL; Enzyme conjugate working solution: goat anti-rabbit enzyme-labeled secondary antibody, 100 mL; Color development solution: 200 mL; Stop solution: 100 mL; Positive serum control: 2 mL; Negative serum control: 2 mL.

5. Use of the indirect ELISA antibody detection kit based on rabbit rotavirus VP8* protein according to any one of claims 1 to 4 in the preparation of a product for detecting rabbit rotavirus antibodies.

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