CN109136267A - BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection - Google Patents
BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection Download PDFInfo
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- CN109136267A CN109136267A CN201811095791.5A CN201811095791A CN109136267A CN 109136267 A CN109136267 A CN 109136267A CN 201811095791 A CN201811095791 A CN 201811095791A CN 109136267 A CN109136267 A CN 109136267A
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Abstract
The present invention provides a kind of BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection, is related to field of biotechnology, the BVDV ErnsAlbumen is expressed using baculovirus insect cell system carrier system, the albumen of expression and with the protein bound monoclonal antibody, and include the ErnsAlbumen and/or the kit of monoclonal antibody can be widely used in the correlative study of BVDV.When ox is after BVDV E2 subunit vaccine is immune, can may be not present in the presence of the antibody in conjunction with BVDV E2 albumen in the serum of ox can be with BVDV ErnsThe antibody that albumen combines.When ox is by the totivirus infection of BVDV or after the inactivated vaccine of totivirus is immune, can exist in the serum of ox can be with BVDV ErnsThe antibody that albumen combines.Therefore the present invention using both between difference, come judge the ox after BVDV E2 subunit vaccine is immune whether by BVDV virus infection, it is convenient and efficient.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a BVDV ErnsProteins and antibodies and methods for screening BVDV infected cattle.
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.
BVDV (BVDV) is a representative species of the genus Pestivirus of the Flaviviridae family (Flaviviridae), and is antigenically related to the Classical Swine Fever Virus (CSFV) and the Border virus (BDV) of sheep, 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 density of the suspension in CsCl was 1.14g/cm3, and the sedimentation coefficient S20 was 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 the 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 BVDV and is also the main part for binding with anti-BVDV antibody, mediating the immune neutralization reaction and recognizing and adsorbing by host cells. The gene coding the E2 protein is located at position 2077 and 3198 of ORF and consists of 374 amino acid residues. The non-glycosylated protein has a molecular weight of about 42 kDa.
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 the generated 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 BVDVErnsThe expression vector of the protein relieves the lack of a vector capable of expressing BVDV E in the prior artrnsThe problem of carriers and systems for proteins.
The second purpose of the invention is to provide a BVDV E expressed by the expression vectorrnsThe protein can be widely applied to BVDV-related research, and lays a material foundation for BVDV-related molecular biological experiments.
The third purpose of the invention is to provide a BVDV ErnsThe 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 ErnsProtein and/or BVDV ErnsThe 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 ErnsProtein, and the above BVDV ErnsThe 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 strain, which alleviates the problem of lacking a method for screening cattle infected by BVDV wild strain from cattle immunized by BVDV E2 subunit vaccine in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
expression of BVDV ErnsAn expression vector of the protein, wherein the expression vector expresses E encoded by a sequence shown as SEQ ID NO.1rnsA protein; the expression vector is baculovirus;
further, the BVDV ErnsThe protein is expressed by an insect cell baculovirus expression vector system.
The invention also provides BVDV E expressed by the expression vectorrnsA protein;
further, the BVDV ErnsThe 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 methodrnsProtein-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 ErnsProteins and/or monoclonal antibodies as described above.
Further, the kit is an ELISA kit; the ELISA kit comprises BVDV E marked by a markerrnsA protein; and/or labeled with BVDV ErnsProtein-bound monoclonal antibodies;
further, the BVDV ErnsThe protein is enzyme-labeled protein; and/or, the BVDV ErnsThe monoclonal antibody of the protein is an enzyme-labeled antibody;
further, enzymes used in the enzyme labeling include horseradish peroxidase, acid phosphatase, alkaline phosphatase, β -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 antibodyA body detection kit; BVDV E in blocking ELISA antibody detection kitrnsProtein as coating plate antigen, the BVDV ErnsThe monoclonal antibody of the protein is an antibody marked by horseradish peroxidase;
further, the negative control in the kit is BVDV ErnsNegative sera.
The invention also provides the BVDV ErnsThe use of the protein, the monoclonal antibody or the kit in detection of 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 cattlernsProtein-bound antibodies;
if present in the bovine serum with said BVDV ErnsProtein-bound antibodies, then the cattle are cattle infected with a wild strain of BVDV;
the cattle are immunized by a 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 methodrnsProtein-bound antibodies;
wherein the enzyme-labeled antibody in ELISA is the antibody of claim 3 which binds to BVDV ErnsProtein-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 carrierrnsAdding the enzyme-labeled antibody into an ELISA reaction plate of the proteinBody, detection of OD after incubation and color development450Then according to OD450Calculating 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 the blocking rate of 30% -35% are suspicious samples;
further, the blocking rate is calculated according to the following method:
blocking rate ═ (OD average of negative control-OD average of sample)/OD average of negative control × 100%.
Compared with the prior art, the invention has the following beneficial effects:
the expression of BVDV E provided by the inventionrnsThe 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 inventionrnsThe protein has a neutralizing epitope thereon, and the generated neutralizing antibody has the capability of neutralizing BVDV and CSFV. ErnsHas the activity of binding dsRNA and 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 ErnsThe 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 ErnsProtein and/or BVDV ErnsProtein bindingThe monoclonal antibody of (3) 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 inventionrnsProtein, and the above BVDV ErnsThe 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 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 the cattle are immunized by the BVDV E2 subunit vaccine, the cattle serum has an antibody which is combined with BVDV E2 protein but does not have the antibody which can be combined with BVDV ErnsProtein-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 cattlernsProtein-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 testedrnsProtein-bound antibody to judge whether the cattle immunized by the BVDV E2 subunit vaccine are infected by the BVDV virus. 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 shows a graph E in example 1 of the present inventionrnsAnd (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 ErnsAn expression vector of the protein, wherein the expression vector expresses the E coded by the sequence shown as SEQ ID NO.1rnsA protein; the expression vector is baculovirus.
The BVDV E is expressed by adopting an insect cell baculovirus expression vector systemrnsThe 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 ErnsThe protein is expressed by an insect cell baculovirus expression vector system. Bacillosis of insect cellAn Insect cell baculovirus expression vector system (IC-BEVS) has the traditional advantages of a eukaryotic expression system and has the characteristic of high expression level of recombinant protein of the prokaryotic expression system, and has the advantages of good safety of ① baculovirus, short vector construction period of ② system compared with a mammalian cell expression system, capability of accommodating macromolecular insert fragments of ③ baculovirus, high-level expression of recombinant protein of ④ baculovirus expression system and capability of expressing a plurality of genes in the same cell of ⑤ Insect baculovirus expression vector system.
ErnsThe protein is one of the important structural proteins of BVDV, and can induce the organism to generate neutralizing antibodies, so the post-translational modification process of the protein in an expression system is very important, if the expressed protein lacks the post-translational modification process, the expressed protein has antigenicity difference with the natural protein, and an inclusion body is easily formed. E expressed by insect cell baculovirus expression vector systemrnsThe 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 for expression induces 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 experimental error can be reduced.
The invention also provides BVDV E expressed by the expression vectorrnsA protein. ErnsIs 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. ErnsHas the activity of binding dsRNA and 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 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 detectedrnsThe antibody to the protein can be used, for example, as a standard substance in molecular biology experiments, and can also be used, for example, as a preparationPreparation against BVDV ErnsThe 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 ErnsThe 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 inventionrnsProtein-bound monoclonal antibodies are antibodies obtained using conventional methods, e.g., using BVDV ErnsAfter the protein immunization of mice, the above antibody was obtained by preparing hybridoma cells. The method for preparing the monoclonal antibody is not limited in the invention as long as the monoclonal antibody can be used with the BVDV ErnsAnd (3) protein combination.
In some alternative embodiments of the invention, the BVDV E isrnsThe 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 Green488, Oregon Green 500, Oregon Green488, 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, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate or chelate, diamine, bispyanin, La Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.
In some alternative embodiments, the enzyme label is typically, but not limited to, one or more of horseradish peroxidase, acid phosphatase, alkaline phosphatase, β -D-galactosidase, glucose oxidase, glucose starch, acetylcholinesterase, inorganic pyrophosphatase, luciferase, or catalase.
The invention also provides a kit, which comprises the BVDV ErnsProteins and/or said proteins and BVDV ErnsA protein-bound monoclonal antibody.
Proteins and antibodies are commonly used reagents in molecular biology experiments and thus comprise the BVDV ErnsProteins and/or said proteins and BVDV ErnsThe 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 markerrnsA protein; and/or BVDV E labeled with a labelrnsA monoclonal antibody to a protein.
For example, in an alternative embodiment, the ELISA kit comprises BVDV E for coating on a reaction platernsProteins, and BVDV E labelled with an enzymernsThe monoclonal antibody of the protein, the kit can detect the BVDV E in a sample to be detected by adopting a competition method ELISArnsThe antibody of (1);for example, in yet another alternative embodiment, the ELISA kit comprises an enzyme-labeled BVDV ErnsThe monoclonal antibody of the protein can detect BVDV E in a sample to be detected by adopting a double-antibody sandwich ELISA methodrnsA protein.
In some alternative embodiments, the BVDV E isrnsThe protein is enzyme-labeled protein; and/or, the BVDVErnsAlternatively, the enzyme used in the enzymatic 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.
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 kitrnsProtein as coating plate antigen, the BVDV ErnsThe 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 ErnsAntibody negative sera of (1).
The invention also provides the BVDV ErnsProtein, the above BVDV ErnsThe monoclonal antibody or the application of the kit in detection of 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 cattle immunized by the BVDVE2 subunit vaccine are infected with BVDV again;
the method comprises detecting the presence or absence of BVDV E in bovine serumrnsProtein-bound antibodies; if present in the bovine serum with said BVDV ErnsProtein-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 the antibody which can be combined with the BVDV E2 proteinrnsProtein-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 cattlernsProtein-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 testedrnsProtein-bound antibody to judge whether the cattle immunized by the BVDV E2 subunit vaccine are infected by the BVDV virus. 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 ELISArnsProtein-bound antibodies; wherein the antibody labeled by enzyme in ELISA is the antibody described in the specification and BVDV ErnsProtein-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 solutionrnsProtein ELISA reaction plate, then adding the protein ELISA reaction plateEnzyme-labeled antibody, incubation and color development, and detection of OD by enzyme-labeling instrument450Then according to OD450Calculating 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 ErnsProtein (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 ErnsIncubating the monoclonal antibody of the protein for 1h at room temperature, washing the PBST for 6 times, adding 100 mu l of substrate color development solution, and reacting for 5 minutes at room temperature; the reaction was terminated by adding 100. mu.l of 1mol/L hydrochloric acid to each reaction well.
The advantageous effects of the present invention will be further described with reference to preferred embodiments.
Example 1 vector construction
Experimental materials:
reagent: synthetic BVDV ErnsPrimers, Pfu DNA polymerase, dNTPs, PCR buffer,vector, NotI endonuclease, competent cells, QIAguick PCR purification kit, LB-kana culture solution and BaculodirectTMA Baculovir expression System kit, SF900II serum-free culture solution, a reagent required by Western-blot, Erns positive serum of bovine viral diarrhea and a rabbit anti-pig secondary antibody marked by FITC.
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 ErnsAmplification of the genes:
BVDV Ernsthe 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 templaternsThe 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 productcloningsite (cacc) and Kozak transcription initiation sequence containing ATG (ACCATGG).
Wherein the detailed PCR steps are as follows: taking the synthesized BVDV ErnsAs a template, 1. mu.l each of primers (10. mu.M), 5. mu.l of dNTPs (2.5mM), 5. mu.l of 10 XPCR buffer, 1. mu.l of Pfu polymerase, and 36. mu.l of double distilled water were added. The pre-denaturation at 95 ℃ for 3 min, followed by denaturation at 94 ℃ for 30 sec, annealing at 57 ℃ for 30 sec, and extension at 72 ℃ for 1 min for 35 cycles, and finally extension at 72 ℃ for 10 min. 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 QIAguickTMPCR purification kit, and the detailed procedures were referred to the purification kit operating manual. After purificationAfter confirmation by 1.0% agar gel electrophoresis, the OD value was measured at a wavelength of 260nm using a spectrophotometer, and the recovered DNA concentration was converted and stored in a refrigerator at-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 (1.2mol/L NaCl, 0.06mol/L MgCl)2)，1μlThe 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)cloning reaction to OneIn a TOP10chemical company E.coli (Invitrogen) action tube, the tube wall was gently tapped and mixed well, placed on ice for 30 minutes, heat-tapped in a water bath at 42 ℃ for 30 seconds, then the reaction tube was quickly returned to ice and cooled for 2 minutes, and then 250. mu.l of SOC culture solution which had been warmed up was added to the reaction tube, and placed in a 37 ℃ chamber and cultured with shaking at a rotation speed of 200r/min 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 from the front to the top, 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 ErnsScreening of recombinant transfer vectors: BVDV E was performed by PCR methodrnsPreliminary screening of recombinant transfer vectors. 24 single clones were picked up on LB-kana screening plates using a sterile 10. mu.l white pipette tip, eachInoculated into 1ml LB-kana culture solution, and cultured overnight in a shaking incubator at a constant temperature of 37 ℃ with shaking at a rotation speed of 200 r/min.
First, PCR reaction solution was prepared and divided into 24 tubes, each of which contained 0.5. mu.l each of BacERNS-F1 and BacERNS-R1 primer (10. mu.M), 2.5. mu.l dNTPs (2.5mM), 2.5. mu.l 10 XPCR buffer, 0.5. mu.l Pfu DNA polymerase, and 16.5. mu.l 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 a 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.
By QIAprepTMExtracting 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 RNase A100 mg/mL) to suspend thalli, transferring the mixed liquid into a 1.5mL microcentrifuge tube, adding 250 mu l of buffer P2, turning the mixture up and down for several times until the liquid becomes transparent and sticky, adding 350 mu l of buffer N3, slowly shaking the mixture for several times, and centrifuging the mixture 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. The recombinant vector was cleaved with the restriction endonuclease NotI, and then analyzed by electrophoresis on a 1.0% agarose gel at 100V. If the size of the recombinant vector is consistent with the expected size of the product, 2 extracted recombinant vectors are selected to obtainvector'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 BaculodirectTMBaculovir mus Expression System for LR recombination reaction. The specific operation is as follows: sterile manipulation 1. mu.l of recombinant transfer vector (100-300ng), 4. mu.lThe reaction buffer and 1. mu.l of double distilled water were added to 10. mu.l of Baculodirect, respectivelyTMLinear DNA (300 ng). Taking LR out of refrigerator at-70 deg.CAfter the enzyme mix was warmed on ice for 2 minutes, it was shaken 2 times for 2 seconds each time, and 4. mu.l of LR was takenThe 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 well62 wells were repeated with good (greater than 95% cell viability) Sf-9 cells. 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 lSF900II serum-free insect cell culture solution; transfection mixture B: 12 μ lreagent and 200 mu lSF900II serum-free insect cell culture solution. 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 were carefully washed with SF900II serum-free insect cell culture medium, and then A, B mixture was divided into two tubes, each tube was added with 800. mu.l of SF900II serum-free insect cell culture medium, and the tubes were inverted several times. Finally, the culture solution in the 6-well plate is pumped to the greatest extent, and A, B mixed solution is slowly added into the 2-well 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, 3ml of Sf-900 II SFM containing antibiotics and 100. mu.M ganciclovir was added to each well, and finally sealed with scotch tape and incubated in a constant temperature 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 ErnsHarvesting 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: DNA in the supernatant of Sf-9 insect cells infected for 4 days was extracted and amplified using primers BacERNS-F1 and BacERNS-R1, and the results are shown in FIG. 3, where lane 1 is infected Sf9 cells, lane M is 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 can show that a fragment with the size of the target gene is amplified, and the target gene exists in the infected sf9 cell, which preliminarily indicates that the infected sf9 is positive.
ErnsProtein expression analysis: verification of BVDV E by Western-blotrnsExpression 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 addedrnsAnd (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 ErnsInoculating recombinant baculovirus into Sf-9 cells, culturing in a 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 primary anti-BVDVErnsPositive serum (1: 1000) was exposed for 1 hour, and FITC-labeled rabbit-anti-pig IgG secondary antibody was added to observe the presence or absence of a fluorescent signal 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 ErnsCoating protein (0.1 μ g/mL) on ELISA reaction plate, washing, sealing, adding 50 μ l/well of serum sample to be detected, incubating at room temperature for 1h, washing with PBST for 3-6 times, and adding horseradish peroxidase-labeled BVDV ErnsIncubating the monoclonal antibody of the protein for 1h at room temperature, washing the PBST for 6 times, adding 100 mu l of substrate color development solution, and reacting for 5 minutes at room temperature; the reaction was terminated by adding 100. mu.l of 1mol/L hydrochloric acid to each reaction well.
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 with the E2 subunit vaccine; hog cholera virus ErnsAntibody positive sera.
Placing the reaction plate in a microplate reader to detect OD of each reaction hole450Calculating the blocking rate;
the blocking rate is calculated according to the following formula: blocking rate ═ (OD average of negative control-OD average of sample)/OD average 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 are as follows: bovine serum E from immunization with inactivated BVDV vaccine (whole virus)rnsThe positive rate of antibody positivity is 93.7%;
bovine serum infected with BVDV holovirus ErnsThe positive rate of antibody positivity is 100%;
bovine serum E immunized with E2 subunit vaccinernsThe positive rate of antibody positivity is 0%;
hog cholera virus ErnsAntibody-positive serum ErnsThe 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; each reactionThe reaction was terminated by adding 100. mu.l of 1mol/L hydrochloric acid to the well. ErnsThe positive rate of the antibody is 15.5 percent,
feeding Erns antibody negative cattle separately, wherein 80 heads of immune cattle viral diarrhea/mucosal virus inactivated vaccine (whole virus), 80 heads of immune cattle viral diarrhea/mucosal virus E2 subunit vaccine, and another 9 heads of immune cattle viral diarrhea/mucosal virus E2 subunit vaccine, collecting blood after 35 days to measure ErnsThe 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 vaccine and non-immune group bovine Erns antibody is 0%, and the detection rate after virus attack is 100%, which proves that the detection method has good sensitivity and specificity, can be used for detecting bovine viral diarrhea/mucosal virus Erns antibody, 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 the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the 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
<211>31
<212>DNA
<213> Artificial sequence
<400>2
caccatggtt ggatccatga aaatagtgcc c 31
<210>3
<211>24
<212>DNA
<213> Artificial sequence
<400>3
tataagctta gccgcatatg ctcc 24
Claims (10)
1. Expression of BVDV ErnsThe expression vector of the protein is characterized in that the expression vector expresses the E encoded by the sequence shown as SEQ ID NO.1rnsA protein; the expression vector is baculovirus;
preferably, the BVDV ErnsThe protein is expressed by an insect cell baculovirus expression vector system.
2. BVDV E expressed by the expression vector of claim 1rnsA protein;
preferably, the BVDV ErnsThe protein is labeled with a label;
preferably, the label comprises a fluorescent label, an enzymatic label or a biotin label.
3. BVDV E according to claim 2rnsProtein-bound monoclonal antibodies;
preferably, the monoclonal antibody is labeled with a label;
preferably, the label comprises a fluorescent label, an enzymatic label or a biotin label.
4. A kit comprising the BVDV E of claim 2rnsA protein and/or a monoclonal antibody according to claim 3.
5. The kit of claim 4, wherein the kit is an ELISA kit; the ELISA kit comprises BVDV E marked by a markerrnsA protein; and/or labeled with BVDV ErnsProtein-bound monoclonal antibodies;
preferably, the BVDV ErnsThe protein is enzyme-labeled protein; and/or, the BVDV ErnsThe monoclonal antibody of the protein is an enzyme-labeled antibody;
preferably, the enzyme used in the enzyme label comprises horseradish peroxidase, acid phosphatase, alkaline phosphatase, β -D-galactosidase, glucose oxidase, glucose starch, acetylcholinesterase, inorganic pyrophosphatase, luciferase or catalase;
preferably, the ELISA 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.
6. The kit of claim 5, wherein the ELISA kit is a blocking ELISA antibody detection kit; the blocking ELISA antibody detection reagentBVDV E in BoxrnsProtein as coating plate antigen, the BVDV ErnsThe monoclonal antibody of the protein is an antibody marked by horseradish peroxidase;
preferably, the negative control in the kit is BVDV ErnsNegative sera.
7. A BVDV E of claim 2rnsUse of a protein, a monoclonal antibody according to claim 3 or a kit according to any one of claims 4 to 6 for the detection of BVDV.
8. A method of screening cattle infected with a wild strain of BVDV, comprising detecting the presence of BVDV E according to claim 2 in the serum of cattlernsProtein-bound antibodies;
if present in the bovine serum with said BVDV ErnsProtein-bound antibodies, then the cattle are cattle infected with a wild strain of BVDV;
the cattle are immunized by a BVDV E2 subunit vaccine;
preferably, the assay is an assay using the kit of any one of claims 4-6.
9. The method according to claim 8, wherein the presence of BVDV E according to claim 2 in serum is detected by ELISArnsProtein-bound antibodies;
wherein the enzyme-labeled antibody in ELISA is the antibody of claim 3 which binds to BVDV ErnsProtein-bound monoclonal antibodies;
preferably, the enzyme-labeled antibody is a horseradish peroxidase-labeled antibody.
10. The method of claim 9, wherein the ELISA method is a blocking ELISA detection method;
preferably, the blocking ELISA detection method comprises the following steps: will be testedIncubation of samples on BVDV E-coated samplesrnsAdding the enzyme-labeled antibody into an ELISA reaction plate of protein, incubating and developing, and detecting OD450Then according to OD450Calculating 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 the blocking rate of 30% -35% are suspicious samples;
preferably, the blocking rate is calculated as follows:
blocking rate ═ (OD average of negative control-OD average of sample)/OD average of negative control × 100%.
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| CN111057132A (en) * | 2019-10-16 | 2020-04-24 | 四川农业大学 | Bovine viral diarrhea virus E0 protein amino acid and preparation method thereof |
| CN115724957A (en) * | 2022-10-21 | 2023-03-03 | 北京纳百生物科技有限公司 | Heavy chain and light chain variable regions of monoclonal antibody against bovine viral diarrhea virus NS3 protein |
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| CN111057132A (en) * | 2019-10-16 | 2020-04-24 | 四川农业大学 | Bovine viral diarrhea virus E0 protein amino acid and preparation method thereof |
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| CN115724957A (en) * | 2022-10-21 | 2023-03-03 | 北京纳百生物科技有限公司 | Heavy chain and light chain variable regions of monoclonal antibody against bovine viral diarrhea virus NS3 protein |
| CN115724957B (en) * | 2022-10-21 | 2024-11-19 | 北京纳百生物科技有限公司 | Heavy and light chain variable regions of monoclonal antibodies against NS3 protein of bovine viral diarrhea virus |
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