Establishment method of indirect ELISA (enzyme-Linked immuno sorbent assay) for detecting IBR (infectious bronchitis Virus) based on gL protein
阅读说明：本技术 一种基于gL蛋白检测IBR的间接ELISA的建立方法 (Establishment method of indirect ELISA (enzyme-Linked immuno sorbent assay) for detecting IBR (infectious bronchitis Virus) based on gL protein ) 是由 吴同垒 史秋梅 张志强 柳翠翠 付祥 白和平 周诗淼 刘勃兴 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种基于gL蛋白检测IBR的间接ELISA的建立方法,属于生物工程技术领域。本发明公开的一种基于gL蛋白检测IBR的间接ELISA的建立方法,对传染性鼻气管炎病毒gL蛋白进行原核表达和纯化,作为包被抗原,建立了牛传染性鼻气管炎的间接ELISA检测方法。本发明方法具有良好的敏感性、特异性、重复性等,可以用于大规模临床样品检测,为牛传染病鼻气管炎疫苗免疫后的抗体水平检测和检疫防控提供了重要的技术手段。(The invention discloses a method for establishing indirect ELISA (enzyme-Linked immuno sorbent assay) for detecting IBR (immunoglobulin B receptor) based on gL protein, belonging to the technical field of biological engineering. The invention discloses an establishment method of indirect ELISA based on gL protein detection IBR, which is used for carrying out prokaryotic expression and purification on gL protein of infectious rhinotracheitis virus as a coating antigen and establishing an indirect ELISA detection method of infectious bovine rhinotracheitis. The method has good sensitivity, specificity, repeatability and the like, can be used for large-scale clinical sample detection, and provides an important technical means for antibody level detection and quarantine control after the bovine infectious disease rhinotracheitis vaccine is immunized.)
1. An establishment method of indirect ELISA based on gL protein detection IBR is characterized by comprising the following specific steps:
(1) preparing IBRV recombinant gL protein, and purifying the protein to obtain purified protein;
(2) detecting an IBRV serum sample by taking the purified protein obtained in the step (1) as a coating antigen, and simultaneously determining the antigen coating concentration, the antigen coating condition, the serum dilution, the type of a sealing solution, the sealing condition, the serum incubation time, the secondary antibody dilution, the incubation condition and the color development time;
(3) determination of the cut-off value for indirect ELISA: critical value is mean value +2 × standard deviation; the average value is the average value of the negative serum indirect ELISA result, and the standard deviation is the standard deviation of the negative serum indirect ELISA result; indirect ELISA result determination criteria: the OD value of the IBRV serum sample is greater than or equal to the critical value and is judged to be positive, and the OD value of the IBRV serum sample is smaller than the critical value and is judged to be negative;
(4) the indirect ELISA method was analyzed for specificity, sensitivity and reproducibility.
2. The method for establishing the indirect ELISA for detecting IBR based on gL protein as claimed in claim 1, wherein the step (1) of preparing IBRV recombinant gL protein comprises the following steps:
1) PCR amplifying gL gene;
2) constructing a recombinant plasmid pET32 a-gL;
3) transforming E.coli DH5 alpha competent cells by the recombinant plasmid pET32a-gL, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting pET32a-gL plasmid, and sequencing;
4) transforming pET32a-gL plasmid into escherichia coli competence BL21(DE3), inducing by IPTG, and purifying to obtain recombinant gL protein;
5) recombinant gL protein was identified using SDS-PAGE and WesternBlot.
3. The method for establishing the indirect ELISA for detecting IBR based on gL protein according to claim 2, wherein the primer sequences used in the PCR amplification in the step 1) are as follows:
gL-F：5’-GAGGATCCCTGGCGGCGCTGCTGTGGCTCC-3’；SEQ ID NO.1；BamH I；
gL-R：5’-ATGAATTCCTAGCGGTAGATGCCGTCGCC-3’；SEQ ID NO.2；EcoR I。
4. use of the indirect ELISA method for the detection of IBR based on the gL protein according to any of claims 1 to 3 for the identification of the infectious disease rhinotracheitis in cattle.
Technical Field
The invention relates to the technical field of biological engineering, in particular to an establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detecting IBR (infectious bronchitis Virus) based on gL protein.
Background
Infectious Bovine Rhinotracheitis (IBR) is a contact Infectious disease caused by Infectious Bovine Rhinotracheitis Virus (IBRV), and is clinically characterized by severe respiratory tract infection, conjunctivitis, abortion, vulvovaginitis, balanitis and the like. Infected cattle often present recessive infection, and then other healthy cattle are continuously infected, when the immunity of cattle is low or stress occurs, acute infection can be developed, and a large amount of economic loss is caused, and the disease is classified as a B-type epidemic disease by the world animal health Organization (OIE), and is also one of the necessary diseases of the imported animals in China. In recent years, the domestic reports of the occurrence of IBR in cattle have increased rapidly. The reason for analyzing the method mainly comprises the following aspects: firstly, the cattle breeding base is not always a beef or milk producing place, cattle transportation is frequent in different regions, secondly, when semen is input from abroad, and animals or animal products such as embryos, cattle and the like have the condition of omission, thirdly, intensive cultivation causes the propagation speed of IBR to be accelerated, and the positive rate is increased.
The key point for effectively controlling the spread of the infectious bovine rhinotracheitis virus is to establish a rapid and accurate detection method. For IBR detection, an iELISA method is often adopted, the kit is mostly imported, the cost is high, the number of kits made in China is small, the supply is seriously insufficient, and the problems of missed detection and the like exist. IBRV belongs to herpesviridae, alphaherpesviridae, has a capsule membrane and a large molecular weight, encodes more than 70 proteins, wherein the gL protein has a molecular weight of about 17kDa, has the main functions of mediating the invasion of viruses to host cells and the diffusion of the viruses among cells, and can induce the generation of antibodies.
Therefore, it is a problem to be solved by those skilled in the art to provide a method for establishing indirect ELISA for detecting IBR based on gL protein.
Disclosure of Invention
In view of the above, the present invention provides a method for establishing indirect elisa (iiisa) for IBR detection based on gL protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
an establishment method of indirect ELISA based on gL protein detection IBR comprises the following specific steps:
(1) preparing IBRV recombinant gL protein, and purifying the protein to obtain purified protein;
(2) detecting an IBRV serum sample by taking the purified protein obtained in the step (1) as a coating antigen, and simultaneously determining the antigen coating concentration, the antigen coating condition, the serum dilution, the type of a sealing solution, the sealing condition, the serum incubation time, the secondary antibody dilution, the incubation condition and the color development time;
(3) determination of the cut-off value for indirect ELISA: critical value is mean value +2 × standard deviation; the average value is the average value of the negative serum indirect ELISA result, and the standard deviation is the standard deviation of the negative serum indirect ELISA result; indirect ELISA result determination criteria: the OD value of the IBRV serum sample is greater than or equal to the critical value and is judged to be positive, and the OD value of the IBRV serum sample is smaller than the critical value and is judged to be negative;
(4) the indirect ELISA method was analyzed for specificity, sensitivity and reproducibility.
Further, the steps for preparing the IBRV recombinant gL protein in the step (1) are as follows:
1) PCR amplifying gL gene;
2) constructing a recombinant plasmid pET32 a-gL;
3) transforming E.coli DH5 alpha competent cells by the recombinant plasmid pET32a-gL, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting pET32a-gL plasmid, and sequencing;
4) transforming pET32a-gL plasmid into escherichia coli competence BL21(DE3), inducing by IPTG, and purifying to obtain recombinant gL protein;
5) recombinant gL protein was identified using SDS-PAGE and WesternBlot.
Further, the sequences of the primers used in the PCR amplification in step 1) are as follows:
gL-F：5’-GAGGATCCCTGGCGGCGCTGCTGTGGCTCC-3’；SEQ ID NO.1；BamHI；
gL-R：5’-ATGAATTCCTAGCGGTAGATGCCGTCGCC-3’；SEQ ID NO.2；EcoR I。
further, the indirect ELISA method for detecting IBR based on gL protein is applied to identifying infectious bovine rhinotracheitis.
According to the technical scheme, compared with the prior art, the invention discloses and provides the establishment method of indirect ELISA for detecting IBR based on gL protein, the iELISA method for detecting the bovine infectious rhinotracheitis serum antibody is established on the basis of gL protein prokaryotic expression, the iELISA method has good sensitivity, specificity, repeatability and the like, can be used for large-scale clinical sample detection, and provides an important technical means for antibody level detection and quarantine control after immunization of bovine infectious rhinotracheitis vaccines.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a SDS-PAGE analysis of recombinant proteins of the invention;
wherein, M: pre-dyeing a Marker with protein; 1: no load; 2: precipitation after purification; 3: supernatant fluid; 4: performing whole bacteria;
FIG. 2 is a Westernblotting analysis of recombinant proteins of the invention;
wherein, 1: no-load 2: a gL protein;
FIG. 3 is a diagram showing a normal distribution diagram of the negative sample detection according to the present invention;
FIG. 4 is a graph showing the results of the sensitivity test according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Bovine kidney cells (MDBK), IBRV were stored in this laboratory. Coli DH5 α competent cells and e coli bl21 competent cells, the pET-32a plasmid was purchased from tiangen biochemistry. Restriction enzymes BamHI, EcoR I, T4 ligase, purchased from Saimer Federation; IPTG inducer, Ni-Agarose His tag protein purification kit, murine anti-His tag monoclonal antibody and HRP-labeled rabbit anti-bovine IgG antibody, which are purchased from Shunkang in the century. IBRV negative positive serum is purchased from China veterinary medicine inspection institute, and HRP-labeled rabbit anti-bovine-IgG is purchased from Beijing Boaosen biotechnology, Inc.; bovine serum albumin was purchased from Biosharp and horse serum was purchased from hangzhou biotechnology, ltd. 171 clinical bovine serum were stored in the laboratory and collected from Shijiazhuang, Tangshan, Chengde, Gallery, 28390; (county). Positive bovine sera for infectious bovine rhinotracheitis, bovine viral diarrhea, bovine brucellosis and bovine foot and mouth disease type O are all provided by the laboratory. Bovine infectious rhinotracheitis antibody detection kit (gB) was purchased from IDEXX, usa.
Example 1 construction of recombinant eukaryotic expression plasmids and purification of protein expression
(1) Primer synthesis and synthesis
Based on the IBRV whole genome sequence (NC-001847) registered by GenBank, the Primer premier5.0 software is used to design corresponding primers of IBRV gL protein, and the Primer sequences are as follows:
gL-F：5’-GAGGATCCCTGGCGGCGCTGCTGTGGCTCC-3’；SEQ ID NO.1；BamHI；
gL-R：5’-ATGAATTCCTAGCGGTAGATGCCGTCGCC-3’；SEQ ID NO.2；EcoR I；
the primer synthesis service is provided by Shanghai Biotechnology, Inc.
(2) Construction of recombinant eukaryotic expression plasmid and expression and purification of protein
The extracted IBRV DNA is used as a template, the gL gene is amplified through PCR, and the PCR reaction system is as follows: 2 XPCR Mix 25 uL, template DNA 1 uL, gL-F1 uL, gL-R1 uL, deionized water 22 uL, total 50 uL. The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 50-62 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; final extension at 72 ℃ for 10 min. And recovering and purifying a PCR product, carrying out double enzyme digestion on the gL gene by using BamHI and EcoR I, cloning to a pET-32a expression vector to construct a recombinant plasmid pET32a-gL, transforming E.coli DH5 alpha competent cells, and carrying out sequencing identification by a biological engineering (Shanghai) company Limited after colony PCR identification.
Wherein, the primer sequences used for colony PCR identification are as follows:
P1：5’-TAATACGACTCACTATAGGG-3’；SEQ ID NO.3；
P2：5’-GCTAGTTATTGCTCAGCGG-3’；SEQ ID NO.4。
after PCR identification and verification, carrying out double enzyme digestion verification on plasmids extracted by amplification culture of positive colonies, obtaining a 628bp target fragment after enzyme digestion, wherein the size is consistent with the expected size, and a sequencing result shows that no mutation and code shift exist, thereby indicating that the pET-32a-gL expression vector is correctly constructed.
Transforming pET32a-gL plasmid into escherichia coli competence BL21(DE3), inducing for 5h by 1mmol/L IPTG, centrifuging, washing bacterial sludge by precooled PBS, finally resuspending by PBS with proper volume, ultrasonically crushing, centrifuging, collecting supernatant and precipitate, respectively adding protein sample loading buffer solution, boiling, and performing SDS-PAGE and solubility analysis; the protein is expressed by verification, and the expression form is inclusion body. The pellet after ultrasonication was purified by affinity chromatography using an inclusion body purification (Ni-NTA) kit, and the purification effect was analyzed by SDS-PAGE.
The whole mycoprotein is added with protein loading buffer solution to be boiled, and subjected to SDS-PAGE and membrane transfer, and WesternBlot detection is carried out by using standard positive serum (diluted by 1: 100) as a primary antibody and using rabbit anti-bovine IgG (diluted by 1: 5000) marked by HRP as a secondary antibody.
The SDS-PAGE results (FIG. 1) showed that a protein band of 37ku was obtained, corresponding to the expected size, and that purified protein was obtained, and the recombinant gL protein was successfully obtained. The Westernblot identification result (figure 2) shows that the prokaryotic expression recombinant gL protein can react with IBR positive serum and can be used for establishing an iELISA method.
Example 2 optimization and establishment of Indirect ELISA method
And (3) taking IBRV positive serum and IBRV negative serum, and establishing an iELISA method by taking the purified recombinant gL protein as an antigen to detect an IBRV serum antibody sample. In the process, the optimal antigen coating concentration, antigen coating conditions, serum dilution, the type of the blocking solution, blocking conditions, serum incubation time, secondary antibody dilution, incubation conditions and color development time are determined. Determination of OD of sample450nmThe value is obtained. The optimal condition is determined by the highest positive OD value/negative OD value (P/N value), positive serum OD450nmNot less than 1, negative serum OD450nmSmaller values, corresponding to conditions optimal for elisa.
The optimum conditions are as follows: the antigen coating concentration is 0.773 mug/mL, and the coating is carried out overnight at 4 ℃; blocking for 1h at 37 ℃ by using 3% BSA as a blocking solution; serum dilution 1:20, incubation at room temperature for 30 min. The rabbit anti-bovine IgG-HRP secondary antibody dilution is 1:5000, incubation is carried out for 30min at 37 ℃, and the reaction is terminated after TMB color development is carried out for 5min at 37 ℃.
Example 3 determination of the Indirect ELISA cut-off value
30 negative sera were randomly selected to determine the cut-off value for the indirect ELISA method. The negative serum was tested by indirect ELISA as described in example 2 to determine OD450nmValues and mean and standard deviation were calculated. The formula for the threshold is: critical value is mean +2x standard deviation.
According to the measured OD450nmValues were calculated as mean and standard deviation of 30 sera, mean 0.2101 and standard deviation 0.1562, i.e. cutoff 0.5225; namely the indirect ELISA method based on gL protein, OD, established by the invention450nmA positive result was found when the value was 0.5225 or more, and a negative result was found when the value was 0.5225 or less. The results are shown in FIG. 3.
Example 4 Indirect ELISA specificity assay
The established indirect ELISA method is used for detecting the infectious bovine rhinotracheitis virus, bovine viral diarrhea, bovine brucellosis and bovine foot and mouth disease virus O type positive bovine serum, and the specificity of the method is determined.
The result shows that the detection result of the IBRV positive serum is positive, and the other detection results are negative. The method is shown to have stronger specificity, and only has good reaction on IBRV positive serum, but has no reaction on other serum.
Example 5 Indirect ELISA sensitivity assay
Diluting the IBRV positive serum by 1: 20-1: 2560 according to a 2-fold ratio, carrying out iELISA test detection according to the conditions of the embodiment 2, and determining the sensitivity of the method.
The results of detection of IBRV-positive serum by gradient dilution of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560 and the like are shown in FIG. 4. The results of FIG. 4 show that the detection result of the positive serum is still positive after the positive serum is diluted by the highest dilution of 1:640, which indicates that the method has higher sensitivity.
Example 6 Indirect ELISA reproducibility test
Coating two different batches of enzyme-labeled plates, randomly selecting 4 negative and positive serum samples, diluting, and detecting with selected optimized conditions, wherein each group is provided with 3 parallel repeats, and the OD is determined450nmAnd carrying out statistics, calculating a variation coefficient, and evaluating the repeatability of the indirect ELISA method in batches and among batches.
Detecting 4 Negative serum samples (Negative1-4) and 4 Positive serum samples (Positive1-4) by using two different batches of enzyme label coated antigens, and calculating the coefficient of variation: the intra-batch variation coefficient of the negative serum sample is 1.9-6.2%, the inter-batch variation coefficient is 2.6-4.0%, the intra-batch variation coefficient of the positive serum sample is 2.9-7.7%, and the inter-batch variation coefficient is 2.7-4.6% (table 1). The result shows that the method has better repeatability.
TABLE 1 repeatability test of recombinant proteins gL
Example 7 Indirect ELISA clinical sample detection
171 parts of serum stored in the laboratory is detected by the IBRV indirect ELISA method established by the invention, and the total positive rate of the IBRV antibody is analyzed.
171 parts of serum collected from Hebei province, wherein the serum is positive for 70 parts and negative for 101 parts; the detection is carried out by the iELISA method established by the invention, the result is shown in table 2, the detection method established by the invention detects 71 parts of positive serum and 100 parts of negative serum, and the positive detection rate is 97.14 percent (68/70, namely the iELISA method established by the invention detects the number of the positive serum of the positive sample, divided by the total number of the positive serum), and the negative detection rate is 97.03 percent (98/101, namely the iELISA method established by the invention detects the number of the negative serum of the negative sample, divided by the total number of the negative serum); 69 parts of positive serum and 102 parts of negative serum are detected by the IDEXX kit, the positive detection rate is 92.86 percent, and the negative detection rate is 96.04 percent. The result shows that the detection method established by the invention has higher accuracy.
TABLE 2 clinical sample test results
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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