CN109136267B - BVDV Erns proteins and antibodies and methods for screening BVDV infected cattle - Google Patents
BVDV Erns proteins and antibodies and methods for screening BVDV infected cattle Download PDFInfo
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- CN109136267B CN109136267B CN201811095791.5A CN201811095791A CN109136267B CN 109136267 B CN109136267 B CN 109136267B CN 201811095791 A CN201811095791 A CN 201811095791A CN 109136267 B CN109136267 B CN 109136267B
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Abstract
The invention provides a BVDV E rns A protein and an antibody and a method for screening BVDV infected cattle, which relates to the technical field of biology, and the BVDV E rns Protein expressed using insect cell baculovirus expression vector system, protein expressed thereby and monoclonal antibody binding to the protein, and vector containing the same rns The kit of the protein and/or the monoclonal antibody can be widely applied to BVDV related research. When the cattle are immunized by the BVDV E2 subunit vaccine, the serum of the cattle has an antibody which is combined with the BVDV E2 protein but does not have an antibody which can be combined with the BVDV E2 rns Protein-bound antibodies. When the cattle are infected by the whole virus of the BVDV or immunized by the inactivated vaccine of the whole virus, the BVDV E and the BVDV E exist in the serum of the cattle rns Protein-bound antibodies. Therefore, the method judges whether the cattle immunized by the BVDV E2 subunit vaccine is infected by the BVDV by using the difference between the BVDV E2 subunit vaccine and is convenient and quick.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a BVDV E rns Proteins andantibodies and methods for screening cattle infected with BVDV.
Background
Bovine viral diarrhea virus was first discovered in new york state to be a bovine infectious disease with diarrhea, cough and fever as the main causes, called Bovine Viral Diarrhea (BVD). A disease of cattle with low incidence of disease and high mortality, with extensive ulceration of the gastrointestinal tract as the evident lesion, called Mucosal Disease (MD), was later discovered. In 1971, the American veterinary Association has collectively named Bovine Viral Diarrhea (BVD) and Mucosal Disease (MD) as bovine viral diarrhea/mucosal disease, abbreviated as BVD/MD.
Bovine Viral Diarrhea Virus (BVDV) is a representative species of Pestivirus genus (Pestivirus) of Flaviviridae family (Flaviviridae), and BVDV is antigen-related to Classical Swine Fever Virus (CSFV) and Border virus (BDV) of sheep within the genus, serologically cross-reactive, and capable of breaking through host specificity to cross-infect, so BVDV is not only one of the important pathogens of cattle, but also infects sheep, goats, pigs, deer and other ruminants.
BVDV virions are nearly spherical, have a diameter of about 40-60nm, a nucleocapsid diameter of 25-30nm, and have an envelope. The float density in CsCl was 1.14g/cm3, and the sedimentation coefficient S20=140. The virus was sensitive to ether, chloroform and ph 3.0. The virus is thermolabile, can be inactivated at 56 ℃, and MgCl2 has no protective effect on the virus. The virus is stable at low temperature, and the vacuum freeze-dried virus can be stored for a long time at the temperature of-60 to-70 ℃.
The BVDV genome is a single-stranded positive-strand RNA, has a genome size of about 12.5kb, and is divided into a 5 'untranslated region, an open reading frame region and a 3' untranslated region. Wherein the E2 protein is the main part for determining the antigenicity of the BVDV and is also the main part for binding with an anti-BVDV antibody, mediating the immune neutralization reaction and recognizing and adsorbing by host cells. The gene coding the E2 protein is positioned at 2077-3198 of ORF and consists of 374 amino acid residues. The non-glycosylated protein has a molecular weight of about 42kDa.
BVDV E2 subunit vaccines are about to come into the market, but an effective detection method for identifying whether immune vaccines generate antibodies or wild viruses infect to generate antibodies is not established. Moreover, BVDV-associated proteins are rarely studied in the current research, and BVDV-associated proteins and antibodies are not studied, so that the technical basis for establishing an effective detection method for identifying whether an immune vaccine generates antibodies or wild virus infects the antibodies is lacked.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for expressing BVDV E rns The expression vector of the protein relieves the lack of a vector capable of expressing BVDV E in the prior art rns The problem of carriers and systems for proteins.
The second purpose of the invention is to provide a BVDV E expressed by the expression vector rns The protein can be widely applied to BVDV related research, and lays a material foundation for BVDV related molecular biology experiments.
The third purpose of the invention is to provide a BVDV E rns The monoclonal antibody combined with the protein can be widely applied to BVDV related research, and lays a material foundation for BVDV-related molecular biological experiments.
The fourth purpose of the invention is to provide a kit, which comprises BVDV E rns Protein and/or BVDV E rns The protein-bound monoclonal antibody can be applied to various molecular biological experiments for researching BVDV.
The fifth purpose of the invention is to provide the BVDV E rns Protein, and the above BVDV E rns The application of the protein-bound monoclonal antibody or the kit in detection of BVDV is wide in application range, and is suitable for detecting BVDV and proteins contained in BVDV or antibodies resisting BVDV.
The sixth purpose of the invention is to provide a method for screening cattle infected by BVDV wild strains, which alleviates the problem of the prior art that a method for screening cattle infected by BVDV wild strains from cattle immunized by BVDV E2 subunit vaccine is lacked.
In order to solve the technical problems, the invention adopts the following technical scheme:
expression of BVDV E rns An expression vector of the protein, wherein the expression vector expresses E encoded by a sequence shown as SEQ ID NO.1 rns A protein; the expression vector is baculovirus;
further, the BVDV E rns The protein is expressed by an insect cell baculovirus expression vector system.
The invention also provides a BVDV E expressed by the expression vector rns A protein;
further, the BVDV E rns The protein is labeled with a label;
further, the label comprises a fluorescent label, an enzyme label or a biotin label.
The invention also provides a BVDV E prepared by the method rns Protein-bound monoclonal antibodies;
further, the monoclonal antibody is labeled with a label;
further, the label comprises a fluorescent label, an enzyme label or a biotin label.
The invention also provides a kit, which comprises the BVDV E rns Proteins and/or monoclonal antibodies as described above.
Further, the kit is an ELISA kit; the ELISA kit comprises BVDV E marked by a marker rns A protein; and/or labeled with BVDV E rns Protein-bound monoclonal antibodies;
further, the BVDV E rns The protein is enzyme-labeled protein; and/or, the BVDV E rns The monoclonal antibody of the protein is an enzyme-labeled antibody;
further, enzymes used in the enzyme labeling include horseradish peroxidase, acid phosphatase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucose starch, acetylcholinesterase, inorganic pyrophosphatase, luciferase, or catalase;
furthermore, the ELISA kit also comprises an antigen coated plate, a positive control, a negative control, a sample diluent, a washing solution, a developing solution and a stop solution.
Further, the ELISA kit is a blocking ELISA antibody detection kit; BVDV E in the blocking ELISA antibody detection kit rns Protein as coating plate antigen, the BVDV E rns The monoclonal antibody of the protein is an antibody marked by horseradish peroxidase;
further, the negative control in the kit is BVDV E rns Negative sera.
The invention also provides the BVDV E rns The protein, the monoclonal antibody or the kit can be used for detecting BVDV.
The invention also provides a method for screening cattle infected by BVDV wild strain, which comprises detecting whether BVDV E as described in claim 2 exists in the serum of the cattle rns Protein-bound antibodies;
if present in the bovine serum with said BVDV E rns Protein-bound antibodies, then the cattle are cattle infected with a wild strain of BVDV;
the cattle are immunized by BVDV E2 subunit vaccine;
further, the detection is carried out by using the kit.
Further, the presence or absence of BVDV E according to claim 2 in serum is detected by ELISA method rns Protein-bound antibodies;
wherein the enzyme-labeled antibody in ELISA is the antibody of claim 3 which binds to BVDV E rns Protein-bound monoclonal antibodies;
further, the enzyme-labeled antibody is a horseradish peroxidase-labeled antibody.
Further, the ELISA method is a blocking ELISA detection method;
further, the blocking ELISA detection method comprises the following steps: incubating the sample to be tested in the BVDV E-coated carrier rns Adding the enzyme-labeled antibody into an ELISA reaction plate of protein, incubating and developing, and detecting OD 450 Then according to OD 450 Calculating the blocking rate of the sample; wherein the content of the first and second substances,
the sample with the blocking rate of 0-30% is a negative sample;
the sample with the blocking rate of 35% -100% is a positive sample;
samples with the blocking rate of 30% -35% are suspicious samples;
further, the blocking rate is calculated according to the following method:
blocking rate = (mean OD of negative control-mean OD of sample)/mean OD of negative control × 100%.
Compared with the prior art, the invention has the following beneficial effects:
the expression of BVDV E provided by the invention rns The expression vector of the protein is expressed by using an insect cell baculovirus expression vector system, and has the advantages of high safety, short vector construction period and high protein expression level.
E expressed by the expression vector provided by the invention rns The protein has a neutralizing epitope thereon, and the generated neutralizing antibody has the capability of neutralizing BVDV and CSFV. E rns Has the activity of binding dsRNA to RNase, which is necessary for inhibiting dsRNA-mediated signaling. Therefore, the protein is an important protein in the experiment for researching BVDV. The protein can be applied to various molecular biological experiments through further modification, and lays a material foundation for BVDV-related molecular biological experiments.
The invention provides the BVDV E rns The monoclonal antibody combined with the protein can be widely applied to BVDV related research, and lays a material foundation for BVDV related molecular biological experiments.
The kit provided by the invention comprises BVDV E rns Protein and/or BVDV E rns The protein-bound monoclonal antibody can be applied to various molecular biological experiments for researching BVDV, such as immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay or immunofluorescence technology.
The BVDV E provided by the invention rns Protein, and the above BVDVE rns The application of the protein-bound monoclonal antibody or the kit in detection of BVDV is wide in application range, and is suitable for detecting BVDV and proteins contained in BVDV or anti-BVDV antibodies.
The method for screening cattle infected by the BVDV wild strain can screen cattle infected by the BVDV wild strain from cattle immunized by the BVDV E2 subunit vaccine. When cattle are immunized by BVDV E2 subunit vaccine, the cattle serum will have antibody binding to BVDV E2 protein but not have antibody capable of combining with BVDV E2 rns Protein-bound antibodies. When the cattle are infected by the whole virus of the BVDV or immunized by the inactivated vaccine of the whole virus, the BVDV E and the BVDV E exist in the serum of the cattle rns Protein-bound antibodies. The present invention therefore makes use of the difference between the presence of BVDV E and the absence of BVDV E in the serum of the sample to be tested rns And (3) judging whether the cattle immunized by the BVDV E2 subunit vaccine is infected by the BVDV virus or not by the antibody combined by the proteins. The method utilizes a molecular biological method, samples only need to collect bovine serum, and whether the samples providing the serum are infected by the BVDV virus can be detected by utilizing the specific reaction between the antigen and the antibody, so the method has the advantages of simple operation and high flux.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of the construction of an expression vector provided in example 1 of the present invention;
FIG. 2 shows the amplified target gene for vector construction provided in example 1 of the present invention;
FIG. 3 shows a fragment of a gene of interest amplified from Sf-9 insect cells infected with day 4 according to example 1 of the present invention;
FIG. 4 is a drawing of the present inventionExample 1 in E rns And (3) detecting the result of Western-blot detection of protein expression.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments and the accompanying drawings, and it is obvious that the described embodiments are some, not all embodiments of the present invention. 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.
The invention provides a method for expressing BVDV E rns An expression vector of the protein, wherein the expression vector expresses the E coded by the sequence shown as SEQ ID NO.1 rns A protein; the expression vector is baculovirus.
The BVDV E is expressed by adopting an insect cell baculovirus expression vector system rns The protein and baculovirus belong to Baculoviridae (Baculoviridae), are divided into nucleopolyhedrovirus and granulosis virus, and the virion is rod-shaped and is a virus with a capsule membrane and capable of specifically infecting arthropods. Baculovirus can accommodate insertion of large fragments of foreign DNA and is an ideal vector for expression of large fragments of DNA.
In some preferred embodiments, the BVDV E rns The protein is expressed by an insect cell baculovirus expression vector system. An Insect cell baculovirus expression vector system (IC-BEVS) has the traditional advantages of a eukaryotic expression system and has the characteristic of high expression quantity of recombinant protein of a prokaryotic expression system. The insect cell baculovirus expression vector system has the following advantages: (1) baculovirus has good safety. (2) Compared with a mammalian cell expression system, the system has a short vector construction period. (3) Baculoviruses can accommodate inserts of macromolecules. (4) Baculovirus expression systems achieve high levels of recombinant protein expression. (5) The insect baculovirus expression vector system has the ability to express multiple genes simultaneously in the same cell.
E rns The protein is one of the important structural proteins of BVDV, and can induce the organism to generateAnd antibodies, and thus the process of post-translational modification of proteins in expression systems is important, and if the expressed protein lacks post-translational modification processing, the expressed protein will be antigenically different from the native protein and will readily form inclusion bodies. E expressed by insect cell baculovirus expression vector system rns The protein can obtain the protein with similar natural structure, thereby having the similar antigen epitope with the natural protein. The protein obtained by using the insect cell baculovirus expression vector system to express is used for inducing an antibody generated by an organism to be close to a natural antibody, so that the experiment and the application in molecular biology are facilitated, and the experiment error can be reduced.
The invention also provides BVDV E expressed by the expression vector rns A protein. E rns Is a highly conserved protein in the BVDV encoding protein, and has a neutralizing epitope thereon, and the generated neutralizing antibody has the capability of neutralizing BVDV and CSFV. E rns Has the activity of binding dsRNA and RNase, which is necessary for inhibiting dsRNA-mediated signaling. Therefore, the protein is a very important protein in the experiment for researching BVDV. The protein can also be applied to various molecular biological experiments through further modification, for example, the protein can be used for typing or quantitatively detecting anti-BVDV E in a sample to be detected rns The antibody to the protein can be used, for example, as a standard in molecular biology experiments, and can also be used for preparing anti-BVDV E rns The antigen of the antibody of the protein, and the like, and the protein lays a material foundation for BVDV-related molecular biology experiments.
The invention also provides a BVDV E rns The monoclonal antibody combined with the protein can be widely applied to the research related to BVDV, lays a material foundation for the molecular biological experiments related to BVDV, and can be applied to immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay or immunofluorescence technology. The BVDV E of the invention rns Protein-bound monoclonal antibodies are antibodies obtained using conventional methods, e.g., using BVDV E rns After the protein immunization of mice, the above antibody was obtained by preparing hybridoma cells. The invention does not limit the preparation method of the monoclonal antibody, but onlyTo be compatible with the BVDV E rns And (4) combining the proteins.
In some alternative embodiments of the invention, the BVDV E is rns The protein and/or the monoclonal antibody is labeled with a label. In some alternative embodiments, the label may be, for example, but not limited to, a fluorescent label, an enzymatic label, or a biotin label.
In some alternative embodiments, the fluorescent label is typically, but not limited to, labeled with a fluorescent dye as follows: alexa 350, alexa 405, alexa 430, alexa 488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, p-aminobenzoic acid, perylene, p-butyl acetate, and mixtures thereof cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate, diamine, bispyanin, la Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.
In some alternative embodiments, the enzyme label is typically, but not limited to, one of the following classes of enzyme labels: horseradish peroxidase, acid phosphatase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucose starch, acetylcholinesterase, inorganic pyrophosphatase, luciferase or catalase.
The invention also provides a kit, which comprises the BVDV E rns Proteins and/or said proteins and BVDV E rns A protein-bound monoclonal antibody.
Proteins and antibodies are commonly used reagents in molecular biology experiments and thus comprise the BVDV E rns Proteins and/or said proteins and BVDV E rns The kit of protein-bound monoclonal antibodies has a variety of uses, for example, in immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay, or immunofluorescence techniques.
In some alternative embodiments, the kit is an ELISA kit; the ELISA kit comprises BVDV E marked by a marker rns A protein; and/or BVDV E labeled with a label rns A monoclonal antibody to a protein.
For example, in an alternative embodiment, the ELISA kit comprises BVDV E for coating on a reaction plate rns Proteins, and BVDV E labelled with an enzyme rns The monoclonal antibody of the protein, the kit can detect the BVDV E in a sample to be detected by adopting a competition method ELISA rns The antibody of (1); for example, in yet another alternative embodiment, the ELISA kit comprises an enzyme-labeled BVDV E rns The monoclonal antibody of the protein can detect BVDV E in a sample to be detected by adopting a double-antibody sandwich ELISA method rns A protein.
In some alternative embodiments, the BVDV E is rns The protein is enzyme-labeled protein; and/or, the BVDV E rns The monoclonal antibody of the protein is an enzyme-labeled antibody. Alternatively, the enzyme used in the enzyme label may be, for example, but not limited to, horseradish peroxidase, acid phosphatase, alkaline phosphatase, β -D-galactosidase, glucose oxidase, glucose starch, acetylcholinesterase, inorganic pyrophosphatase, luciferase, or catalase. It is understood that the present invention is not limited to the kind of enzyme in the enzyme-labeled antibody in ELISA, as long as it complies with the principle of ELISA assay, and horseradish peroxidase-labeled antibody is preferably used.
In some alternative embodiments, the kit further comprises an antigen-coated plate, a positive control, a negative control, a sample diluent, a washing solution, a developing solution, and a stop solution.
In some alternative embodiments, the ELISA kit is a blocking ELISA antibody detection kit; BVDV E in blocking ELISA antibody detection kit rns Protein as coating plate antigen, the BVDV E rns The monoclonal antibody of the protein is an antibody marked by horseradish peroxidase;
the kit also comprises a negative control, wherein the negative control is BVDV E rns Antibody negative sera of (1).
The invention also provides the BVDV E rns Protein, the above BVDV E rns The use of the monoclonal antibody or the kit in detecting BVDV. In some alternative embodiments, the use comprises detecting whether a test sample is infected with BVDV; whether a sample to be detected generates an antibody against BVDV after being immunized by the BVDV full-virus fire-fighting vaccine; whether the antibody related to the BVDV exists in the sample to be tested, and the like.
The invention also provides a method for screening cattle infected by the BVDV wild strain, which is used for detecting whether the cattle immunized by the BVDV E2 subunit vaccine is infected with BVDV again;
the method comprises detecting the presence or absence of BVDV E in bovine serum rns Protein-bound antibodies; if present in the serum of cattle with said BVDV E rns Protein-bound antibody, then the cattle is cattle infected with a wild strain of BVDV.
When the cattle are immunized by the BVDV E2 subunit vaccine, the serum of the cattle has an antibody which is combined with the BVDV E2 protein but does not have an antibody which can be combined with the BVDV E rns Protein-bound antibodies. When the cattle are infected by the whole virus of the BVDV or immunized by the inactivated vaccine of the whole virus, the BVDV E and the BVDV E exist in the serum of the cattle rns Protein-bound antibodies. The present invention thus makes use of the difference between the two, namely the presence or absence in the serum of the sample to be tested, of a compound which can be compared with BVDV E rns Protein-bound antibodies to determine whether the vaccine has been immunized with the BVDV E2 subunit vaccineWhether the cattle is infected by BVDV virus again. The method utilizes a molecular biological method, samples only need to collect bovine serum, and whether the samples providing the serum are infected by the BVDV virus can be detected by utilizing the specific reaction between the antigen and the antibody, so the method has the advantages of simple operation and high flux.
In some alternative embodiments, the presence or absence of BVDV E in serum is determined by ELISA rns Protein-bound antibodies; wherein, the antibody labeled by enzyme in ELISA is the antibody described in the specification and BVDV E rns Protein-bound monoclonal antibodies; the enzyme-labeled antibody is preferably a horseradish peroxidase-labeled antibody.
In a preferred embodiment, the ELISA method is a blocking ELISA detection method. In some alternative embodiments, the method comprises the steps of: incubating the sample to be tested and the negative control in the BVDV E-coated solution rns Adding the enzyme-labeled antibody into an ELISA reaction plate of the protein, incubating and developing, and detecting OD by an enzyme-labeling instrument 450 Then according to OD 450 Calculating the blocking rate of the sample; wherein, the sample with the blocking rate of 0-30% is a negative sample; the sample with the blocking rate of 35% -100% is a positive sample; samples with a blocking rate of 30% -35% were suspicious samples.
In some preferred embodiments, the blocking rate is calculated as follows:
blocking rate = (OD average of negative control-OD average of sample)/OD average of negative control × 100%.
In an alternative embodiment, the blocking ELISA assay is as follows: coated BVDV E rns Protein (0.1 mu g/mL), after washing and sealing, adding the serum to be detected and negative control into each 50 mu l/hole, incubating for 1h at room temperature, and washing for 6 times by PBST; adding horseradish peroxidase labeled BVDV E rns Incubating the monoclonal antibody of the protein at room temperature for 1h, washing by PBST for 6 times, adding 100 mu l of substrate color development solution, and reacting for 5 minutes at room temperature; 100. Mu.l of 1mol/L hydrochloric acid was added to each reaction well to terminate the reaction.
The advantageous effects of the present invention will be further described below with reference to preferred embodiments.
Example 1 vector construction
Experimental materials:
reagent: synthetic BVDV E rns Primers, pfu DNA polymerase, dNTPs, PCR buffer,
vector, notI endonuclease, competent cell, QIAguick PCR purification kit, LB-kana culture solution and Baculodirect TM A Baculovir Expression System kit, SF900II serum-free culture solution, a reagent required by Western-blot, erns positive serum for bovine viral diarrhea and a FITC labeled rabbit anti-pig secondary antibody.
Cell: sf-9 insect cells were purchased from Invitrogen Life technologies, USA
The flow of the construction method of the bovine viral diarrhea Erns recombinant baculovirus is shown in figure 1.
A)BVDV E rns Amplification of the gene:
BVDV E rns the BVDV E of the gene is amplified by high-fidelity PfuDNA polymerase by taking a synthesized sequence shown as SEQ ID NO.1 as a template rns The gene and the amplification primer comprise BacERNS-F1 and BacERNS-R1, wherein BacERNS-F1 has a sequence shown as SEQ ID NO.2, bacERNS-R1 has a sequence shown as SEQ ID NO.3,
the primer sequences are shown below:
BacERNS-F1:5’-CACCATGGTTGGATCCATGAAAATAGTGCCC-3’；
BacERNS-R1:5’-TATAAGCTTAGCCGCATATGCTCC-3’。
in addition, in this embodiment, the primer introduces into the 5' end of the PCR product
Cloningsite (CACC) and Kozak transcription initiation sequence containing ATG (ACCATGG).
Wherein the detailed PCR steps are as follows: taking the synthesized BVDV E rns As a template, 1. Mu.l each of primers (10. Mu.M), 5. Mu.l of dNTPs (2.5 mM), 5. Mu.l of 10 XPCR buffer, 1. Mu.l of Pfu polymerase, and 36. Mu.l of double distilled water were added. Pre-changing at 95 deg.CDenaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute for 35 cycles, and final extension at 72 ℃ for 10 minutes. After the reaction, electrophoresis analysis was performed on 1.0% agarose gel at 100 volts, and the electrophoresis results are shown in FIG. 2, in which lane 1 is a gene template, lane 2 is a negative control, and lane 3 is a DNA Marker 2000. The size of the target gene is about 946bp, and a fragment with the size of the target gene can be successfully amplified according to the graph 2. The target fragment was recovered using QIAguick (TM) PCR purification kit, and the detailed procedures were referred to the purification kit manual. After purification, the DNA was confirmed by 1.0% agarose gel electrophoresis, and the OD value was measured at a wavelength of 260nm using a spectrophotometer, and the concentration of the recovered DNA was converted and stored in a refrigerator at a temperature of-20 ℃ or lower for further use.
B) Construction of baculovirus transfer vectors
a)
Cloning reaction: the experimental procedures were referenced to the Invitrogen operating manual. The reaction system is as follows: 0.5-4. Mu.l of purified blunt-end PCR product (containing 5-10ng DNA), 1. Mu.l of saline solution (1.2 mol/L NaCl,0.06mol/L MgCl) 2 )，1μl />
The carrier was added with double distilled water to make the total volume 6. Mu.l. After mixing, the mixture was left at room temperature for 15 minutes, and the reaction tube was placed on ice for further use.
b) And (3) plasmid transformation: taking 2. Mu.l of the solution obtained in step a)
The cloning reaction is added to One->
TOP10chemical company E.coli (Invitrogen) action tube, gently tap the tube wall to mix well, place on ice for 30 minutes, heat shock in 42 ℃ water bath for 30 seconds, then quickly place the reaction tube back on ice to cool for 2 minutes, then add 250. Mu.l of SOC culture medium that has been warmed up to room temperature to place in 37 ℃ at 200r/minThe culture was incubated with shaking for 60 minutes. 50 mul of the bacterial liquid is evenly smeared on an LB-kana screening plate (containing 50 mu g/ml kanamycin), the plate is placed for 30 minutes with the front side upwards, the culture dish is inverted after the bacterial liquid is completely absorbed by the culture medium, and the bacterial liquid is cultured in an incubator at 37 ℃ overnight.
c)BVDV E rns Screening of recombinant transfer vectors: BVDV E was performed by PCR method rns Preliminary screening of recombinant transfer vectors. 24 single clones were picked up on LB-kana selection plates with a sterile 10. Mu.l white pipette tip, inoculated into 1ml of LB-kana culture medium, and cultured overnight with shaking at 200r/min in a 37 ℃ incubator with shaking.
Firstly, PCR reaction liquid is prepared and divided into 24 tubes, each tube of PCR reaction liquid contains 0.5 mul of BacERNS-F1 and BacERNS-R1 primer (10 mul), 2.5 mul of dNTPs (2.5 mM), 2.5 mul of 10 XPCR buffer,0.5 mul of Pfu DNA polymerase and 16.5 mul of double distilled water. Mu.l of the bacterial solution was used as a template for PCR reaction.
The PCR reaction scheme is as described above. After the reaction, the reaction was analyzed by electrophoresis on 1.0% agar gel at 100 volts. If expected products appear, 4-5 corresponding bacterial liquids are selected, inoculated to 3ml of LB-Kana culture solution in a proportion of 1 per mill, and subjected to shaking culture overnight in a constant temperature shaking incubator at 37 ℃ at a rotating speed of 200 r/min.
Using QIAprep TM Extracting the recombinant vector by Spin Plasmid Kit: centrifuging 3mL of bacterial liquid at the rotating speed of 10000r/min for 4 minutes, removing supernatant, adding 250 mu l of buffer P1 (containing 100mg/mL of RNase A) to suspend thalli, transferring the mixed liquid into a 1.5mL microcentrifuge tube, adding 250 mu l of buffer P2, turning the microcentrifuge tube up and down for several times until the liquid becomes transparent and sticky, adding 350 mu l of buffer N3, slowly shaking the microcentrifuge tube for several times, and centrifuging the microcentrifuge tube at the rotating speed of 12000r/min for 10 minutes. Sucking supernatant, adding into QIAprep spin column, centrifuging at 12000r/min for 1 min, discarding filtrate, adding 750 μ l buffer PE into column to wash off excessive salts, centrifuging at 12000r/min for 1 min, discarding filtrate, centrifuging for 2 min to avoid liquid residue, placing column into a new microcentrifuge tube, adding 20 μ l double distilled water, standing for 1 min to fully dissolve vector DNA, centrifuging at 12000r/min for 1 min, collecting filtrate, and storing at-20 deg.C or below. Recombinant vectorAfter the body was cleaved with the restriction enzyme NotI, the body was analyzed by electrophoresis on a 1.0% agarose gel at 100 volts. If the size of the recombinant vector is consistent with the expected size of the product, 2 extracted recombinant vectors are selected to obtain
vector's own primer (M13F/M13R) was used for sequencing verification.
d) Preservation of recombinant transfer vector: inoculating the E.coli strain containing the recombinant transfer vector into 5ml LB-kana culture solution (containing 50. Mu.g/ml kanamycin), placing in a constant temperature shaking incubator at 37 ℃, shaking and culturing at the rotation speed of 200r/min for 16 hours, adding 20% glycerol, subpackaging, and storing at the temperature below 70 ℃.
C) LR recombination reactions
After the recombinant transfer vector is verified to be correct by sequencing, the nucleic acid concentration of the extracted recombinant vector is measured at the wavelength of 260nm by a spectrophotometer. Then Baculodirect TM Baculovir mus Expression System for LR recombination reaction. The specific operation is as follows: sterile 1. Mu.l of recombinant transfer vector (100-300 ng), 4. Mu.l
The reaction buffer and 1. Mu.l of double distilled water were added to 10. Mu.l of Baculodirect, respectively TM Linear DNA (300 ng). Taking the LR out of the refrigerator at-70 DEG C>
After the enzyme mix had been warmed on ice for 2 minutes, it was shaken 2 times for 2 seconds and 4. Mu.l of LR->
The enzyme mix is added to the mixture and the tube wall is tapped several times to mix it evenly without vigorous shaking to avoid the baculovirus DNA breaking. After 18 hours at 25 ℃ the LR recombination reaction was interrupted by adding 2. Mu.l of protease K solution and allowing to react at 37 ℃ for 10 minutes.
D) Transfection
6-well cell culture plates were inoculated with 1.5X 10 cells per well 6 Has good activity(cell viability > 95%) Sf-9 cells, repeat 2 wells. The cells were incubated at 26-28 ℃ for 1 hour to allow the cells to attach to the bottom of the plate. Before transfection, the following mixed solution is prepared, namely a transfection mixed solution A:20 μ l LR recombination reaction solution (containing recombined baculovirus DNA) and 200 μ l SF900II serum-free insect cell culture solution; transfection mixture B:12 μ l
reagent and 200. Mu.l SF900II serum-free insect cell culture medium. The transfection mixture A and B were mixed, and the tube wall was gently tapped and allowed to stand at room temperature for 45 minutes. The adhered Sf-9 cells are carefully cleaned by SF900II serum-free insect cell culture solution, then the A and B mixed solution is divided into two tubes, 800 mul of SF900II serum-free insect cell culture solution is added into each tube, and the tubes are turned upside down for a plurality of times. And finally, pumping the culture solution in the 6-hole plate as much as possible, and slowly adding the mixed solution A and the mixed solution B into the 2-hole plate along the tube wall. After sealing the cell culture plate, the plate was incubated at 26-28 ℃ for 5 hours. The mixture was removed by suction, 3ml of Sf-900 II SFM containing antibiotics and 100. Mu.M ganciclovir was added to each well, and finally sealed with cello tape and incubated in a thermostatic incubator at 26-28 ℃ for 5 days. The baculovirus gene which is not successfully recombined has a thymine kinase gene of herpes simplex virus, and the DNA cannot be copied in a culture solution added with ganciclovir, so that the generation of the non-recombined baculovirus is reduced.
E)BVDV E rns Harvesting and characterization of recombinant baculovirus
a) Collecting virus liquid into a 50ml centrifuge tube, centrifuging at the rotating speed of 3000r/min for 5 minutes to remove cell debris, adding 10% fetal calf serum into the virus liquid to reduce the damage of protease possibly to the virus, and then storing the virus liquid at the temperature of below 4 ℃ in a dark place for later use or storing the virus liquid at the temperature of below 70 ℃ for a long time. For primary separation after transfection, the cells were filtered through a 0.45 μm filter and then placed in 1.5ml centrifuge tubes.
b) Virus identification:
identification of the genes: the DNA in the supernatant of Sf-9 insect cells infected for 4 days is extracted, and the DNA in the supernatant is amplified by using primers BacERNS-F1 and BacERNS-R1, and the result is shown in figure 3, wherein lane 1 is the infected Sf9 cells, lane M is a DNA Marker200, lane 2 is a negative control, lane 3 is a cell control, and lane 4 is a positive control. As can be seen from FIG. 3, after the nucleic acid of the infected sf9 cell is extracted, and is subjected to reverse transcription and PCR amplification, the electrophoresis result shows that a fragment with the size of the target gene is amplified, and the target gene exists in the infected sf9 cell, which primarily indicates that the infected sf9 is positive.
E rns Protein expression analysis: verification of BVDV E by Western-blot rns Expression and reactogenicity of the protein. Firstly, SDS-PAGE is carried out, then protein bands on SDS-PAGE gel are transferred to PVDF membrane, the PVDF membrane is placed in PBST containing 5 percent skim milk powder, the PBST is sealed for 30 minutes at room temperature and is washed for 5 times, and primary anti-BVDV E is added rns And (3) reacting positive serum at room temperature for 1 hour, washing for 5 times, adding a rabbit anti-pig secondary antibody containing horseradish peroxidase markers, reacting at room temperature for 1 hour, washing for 5 times, and performing DAB (digital audio broadcasting) coloration in a dark room. The results are shown in FIG. 4, in which lane 1 is a cell control, lane 2 is an expressed protein, and lane M is a protein Marker. As can be seen from FIG. 4, the protein expression was subjected to a western-blot assay, and the result showed that a band of about 35kDa was expected, indicating that the expressed protein was reactogenic.
Immunofluorescence: the well-constructed BVDV E rns Inoculating recombinant baculovirus into Sf-9 cell, culturing in constant temperature incubator at 26-28 deg.C for 7 days, washing each well with PBST 6 times after 7 days, adding 100 μ l formaldehyde, fixing for 10 min, adding anti-BVDV E rns Positive serum (1: 1000) was exposed for 1 hour, FITC labeled rabbit anti-porcine IgG secondary antibody was added, and presence or absence of fluorescent signal was observed under an inverted fluorescence microscope.
Example 3 method for screening cattle infected with BVDV wild strain for blocking ELISA
The method comprises the following steps: mixing BVDV E rns Coating protein (0.1. Mu.g/mL) on ELISA reaction plate, washing and sealing, adding 50. Mu.l/well of serum sample to be detected, incubating at room temperature for 1h, washing for 3-6 times by PBST, adding horseradish peroxidase-labeled BVDV E rns Incubating protein monoclonal antibody at room temperature for 1h, washing by PBST for 6 times, adding substrate developing solution 100. Mu.l, 5 minutes at room temperature; 100. Mu.l of 1mol/L hydrochloric acid was added to each reaction well to terminate the reaction.
Wherein each sample to be detected comprises the following samples: bovine serum immunized with BVDV inactivated vaccine (whole virus); bovine serum infected with BVDV whole virus; bovine serum immunized against the E2 subunit vaccine; hog cholera virus E rns Antibody positive sera.
Placing the reaction plate in a microplate reader to detect OD of each reaction hole 450 Calculating the blocking rate;
the blocking rate is calculated according to the following formula: blocking rate = (mean OD of negative control-mean OD of sample)/mean OD of negative control × 100%.
The sample with the blocking rate of 0-30% is a negative sample;
the sample with the blocking rate of 35% -100% is a positive sample;
samples with the blocking rate of 30% -35% are suspicious samples;
the results were as follows: bovine serum E of immunized BVDV inactivated vaccine (whole virus) rns The positive rate of antibody positivity is 93.7%;
bovine serum infected with BVDV Total Virus E rns The positive rate of antibody positivity is 100%;
bovine serum E for immunization of E2 subunit vaccines rns The positive rate of antibody positivity is 0%;
hog cholera virus E rns Antibody-positive serum E rns The positive rate of antibody positivity was 0%.
Example 4 application of the method for screening cattle infected with BVDV wild strains
Screening and collecting 200 parts of bovine serum before immunization, coating purified Erns protein (0.1 mu g/mL), washing and sealing, adding 50 mu l/hole of serum to be detected, incubating at room temperature for 1h, and washing by PBST for 6 times; adding enzyme-labeled monoclonal antibody, incubating at room temperature for 1h, washing for 6 times by PBST, adding 100 mu l of substrate color development solution, and reacting at room temperature for 5 minutes; the reaction was terminated by adding 100. Mu.l of 1mol/L hydrochloric acid to each reaction well. E rns The positive rate of the antibody is 15.5 percent,
the Erns antibody negative cattle were kept separately, and 80 of them were immunized with inactivated bovine viral diarrhea/mucosal virus vaccine (whole bovine viral diarrhea/mucosal virus)Virus), 80 immune bovine viral diarrhea/mucosal virus E2 subunit vaccines, another 9 non-immune, blood sampling after 35 days for E rns The antibody is detected according to the method, the antibody positive rate of the immune bovine viral diarrhea/mucosal virus inactivated vaccine (whole virus) Erns is 93.7 percent, and the detection rate after virus attack is 100 percent; the positive rate of immune bovine viral diarrhea/mucosal virus E2 subunit vaccines and non-immune group bovine Erns antibodies is 0%, and the detection rate after virus attack is 100%, so that the detection method has good sensitivity and specificity, can be used for detecting the bovine viral diarrhea/mucosal virus Erns antibodies, and can be used for screening cattle infected by BVDV wild strains.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Tiankang biological products Ltd
<120> BVDV Erns protein and antibody and method for screening BVDV infected cattle
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 921
<212> DNA
<213> Artificial sequence
<400> 1
atgaaaatag tgcccaaaga gtctgagaaa gacagcaaga ctaaaccgcc agatgctacg 60
atagtggtag atggagttaa ataccaggta aagaagaagg gaaaagtcaa gagtaaaaat 120
acgcaggacg gtttatatca taacaaaaat aagccgccag aatcacgcaa gaaactagag 180
aaagcattat tggcatgggc aatattggct gcagtcttga ttcaagttac aatgggtgaa 240
aatataacac agtggaacct acaggataat gggacagaag ggatacaacg ggcaatgttc 300
caaagggggg tgaacagaag tctacacggg atctggccag gaaaaatctg tacaggtgtc 360
ccttcccatc tagccaccga tatggaacta aaaacgatcc atggtatgat ggatgcaagt 420
gaaaagacca actatacgtg ttgcagactt caacgccatg agtggaacaa gcatggttgg 480
tgcaactggt acaatattga accttggatt ttactcatga atagaaccca agccaatctt 540
actgagggtc aaccaccaag agaatgcgcg gtcacttgta ggtatgatag agatagtgat 600
ctgaatgtgg taacacaagc tagagatagc cccacaccat tgacaggctg taagaaaggg 660
caaaattttt cttttgcagg catattgatg cggggtccct gcaacttcga aatagctgcg 720
agcgatgtgt tgttcaaaga agatgaatgc accagcatgt ttcaggatac tgcgcattac 780
ctcgttgacg ggatgaccaa ttccctggaa agtgccagac aagggaccgc taaactgacg 840
acctggttag gcaagcagct tgggatacta ggaaaaaaat tggaaaacaa gagcaagaca 900
tggtttggag catatgcggc t 921
<210> 2
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<212> DNA
<213> Artificial sequence
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caccatggtt ggatccatga aaatagtgcc c 31
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tataagctta gccgcatatg ctcc 24
Claims (1)
1. BVDV E rns Use of a protein in the preparation of a kit for identifying whether a bovine to which a BVDV E2 subunit vaccine is administered is BVDV infection;
codingBVDV E rns The nucleotide sequence of the protein is shown as SEQ ID NO. 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965348A (en) * | 2009-04-28 | 2013-03-13 | 辉瑞大药厂 | Bovine viral diarrhea-mucosal virus with modified erns protein |
| US9291624B2 (en) * | 2011-08-24 | 2016-03-22 | Zoetis Services Llc | Vaccine diagnostics |
| CN108456663A (en) * | 2018-03-26 | 2018-08-28 | 中国农业科学院兰州兽医研究所 | 1 type bovine viral diarrhea virus sample particle of one kind and its preparation and application |
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| CN102965348A (en) * | 2009-04-28 | 2013-03-13 | 辉瑞大药厂 | Bovine viral diarrhea-mucosal virus with modified erns protein |
| US9291624B2 (en) * | 2011-08-24 | 2016-03-22 | Zoetis Services Llc | Vaccine diagnostics |
| CN108456663A (en) * | 2018-03-26 | 2018-08-28 | 中国农业科学院兰州兽医研究所 | 1 type bovine viral diarrhea virus sample particle of one kind and its preparation and application |
Non-Patent Citations (2)
| Title |
|---|
| Cloning, sequencing, and in vitro expression of glycoprotein gp48 of a noncytopathogenic strain of bovine viral diarrhea virus;Ilse U. Silva-Krott et al.;《Veterinary Microbiology》;19941231;第39卷;摘要，第6页图2 * |
| Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system;Grego, E. et al.;《Journal of veterinary diagnostic investigation》;20071231;第19卷(第1期);摘要 * |
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