CN109306007B - Anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineering antibody and application thereof - Google Patents

Abstract

The invention belongs to the technical field of antibody application, and particularly relates to a genetic engineering antibody for resisting a porcine reproductive and respiratory syndrome virus nsp4 protein and application thereof, wherein the genetic engineering antibody is shown as SEQ ID No.6 and SEQ ID No.8, the two proteins are mixed to form a Y-shaped antibody, the Y-shaped antibody can be specifically combined with a porcine reproductive and respiratory syndrome virus nsp4 protein, the antibody only has one epitope, has good affinity and high specificity, and can be prepared into an ELISA (enzyme-linked immunosorbent assay) reagent, a Western-Blot detection reagent, an IFA (enzyme-linked immunosorbent assay) detection reagent and the like of the porcine reproductive and respiratory syndrome virus. The antibody can be obtained by an artificial synthesis method, so that the gene engineering antibody of the porcine reproductive and respiratory syndrome virus nsp4 can be obtained more conveniently and simply, the defects of high preservation cost of hybridoma cells, easy loss of phenotype and the like are effectively overcome, and the animal welfare problem in ascites preparation is avoided.

Description

Anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineering antibody and application thereof

Technical Field

The invention belongs to the technical field of antibody application, and particularly relates to a gene engineering antibody for resisting porcine reproductive and respiratory syndrome virus nsp4 protein and application thereof.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), belonging to the genus of arterivirus of the family of the Togaviridae, is the causative agent of Porcine Reproductive and Respiratory Syndrome and can cause late abortion, premature birth and stillbirth in pregnant sows, and Respiratory diseases in piglets and fattening pigs. Since the beginning of 6 months in 2006, pigs with high-pathogenicity PRRSV as the main pathogeny 'unknown high fever syndrome' appear in succession in more than ten provinces of Hubei, Hunan, Jiangxi, Anhui and the like of China, and serious economic loss is caused to the pig industry of China.

Porcine reproductive and respiratory syndrome virus is a enveloped, single-stranded, positive-stranded RNA virus with a genome length of about 15kb, a 5 'cap and a 3' poly (A) structure. The full-length genome contains at least 10 open reading frames (ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5b, ORF6 and ORF7), and adjacent reading frames are partially overlapped. ORFs 2-7 encoding viral structural proteins are located at the 3' end of the porcine reproductive and respiratory syndrome virus genome. ORFs 2-4 encode the minor structural proteins of the virus, and ORFs 5, 6 and 7 encode the major structural proteins of the virus, which are envelope protein GP5, envelope matrix protein (M) and nucleocapsid protein (N), respectively. And ORF1a and ORF1b at the 5' end of the genome occupy about four fifths of the full length of the genome, and can produce 4 kinds of polyprotein, pp1a, pp1a-nsp2TF, pp1a-nsp2N and pp1ab, respectively. These polymeric proteins can translate into at least 16 non-structural proteins involved in viral replication. Wherein pp1a can encode at least 10 non-structural proteins (nsp 1-8) after being hydrolyzed, including 4 non-structural proteins with proteolytic enzyme activity, which are nsp1 alpha, nsp1 beta, nsp2 and nsp 4.

Wherein nsp4 has 3C-like serine protease activity and functions to cleave the precursor polyprotein encoded by porcine reproductive and respiratory syndrome virus, thereby producing other non-structural proteins that together comprise the viral replicase and transcriptase complex to effect viral replication and propagation. During long-term evolution, nsp4, in addition to acting as a protease to cleave polyproteins to produce non-structural proteins required for viral replication, can target the central tap molecule of the innate immune signaling pathway or kinases to evade the host immune response.

The antibody is globulin capable of being specifically combined with corresponding antigen, has an immune function, and is widely used in the fields of immunological analysis, radioimmunoassay, immune-oriented therapy and the like. In 1975, Kohler and Milstein successfully produced monoclonal antibodies using in vitro cell culture and screening techniques, which became a milestone in the field of antibody research. With the continuous and intensive research on antibodies, the antibodies are developed to the third generation of genetic engineering antibodies, people can reconstruct and assemble antibody molecules at the genetic level, the genetic engineering antibodies not only retain or increase the main biological activity of natural antibodies, but also overcome certain defects of monoclonal antibodies in preparation and application, and the monoclonal antibodies show strong momentum in the field of biotechnology.

Disclosure of Invention

Aiming at the problems, the invention provides a genetic engineering antibody for resisting the porcine reproductive and respiratory syndrome virus nsp4 protein, wherein the genetic engineering antibody is a protein peptide segment shown by SEQ ID NO.6 and SEQ ID NO. 8.

The invention also aims to provide application of the gene engineering antibody of the nsp4 protein of the porcine reproductive and respiratory syndrome virus. The genetic engineering antibody can be used for preparing a detection kit for porcine reproductive and respiratory syndrome virus.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant prepares an anti-porcine reproductive and respiratory syndrome virus nsp4 protein genetic engineering antibody aiming at a porcine reproductive and respiratory syndrome virus nsp4 protein, the antibody comprises protein peptide segments shown by SEQ ID No.6 and SEQ ID No.8, the two proteins are mixed to form a Y-shaped antibody which can be specifically combined with the porcine reproductive and respiratory syndrome virus nsp4 protein, and the antibody only has one epitope and has good affinity, high specificity and the same specificity property with a monoclonal antibody.

The protection scope of the invention also includes the polynucleotide for coding the protein peptide segments shown in SEQ ID NO.6 and SEQ ID NO.8, and the polynucleotide can be selected from SEQ ID NO.5 and SEQ ID NO. 7.

The protection scope of the invention also includes the variable region amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.4 contained in the genetic engineering antibody.

The invention also includes the polynucleotide for encoding the variable region polypeptide shown in SEQ ID NO.2 and SEQ ID NO.4, and the polynucleotide can be selected from SEQ ID NO.1 and SEQ ID NO. 3.

The application of the anti-porcine reproductive and respiratory syndrome virus nsp4 protein genetic engineering antibody in preparing the detection kit of the porcine reproductive and respiratory syndrome virus comprises preparing an ELISA reagent, a Western-Blot detection reagent, an IFA detection reagent and the like of the porcine reproductive and respiratory syndrome virus by utilizing protein peptide segments shown by SEQ ID NO.6 and SEQ ID NO. 8.

Compared with the prior art, the invention has the following advantages:

the invention provides a gene engineering antibody for resisting the nsp4 protein of the porcine reproductive and respiratory syndrome virus for the first time, which has strong affinity, can be used for experiments such as Western-Blot, IFA and the like, and provides materials and support for the establishment of basic research, epidemiological investigation and clinical diagnosis and identification methods of the porcine reproductive and respiratory syndrome virus.

The nucleotide sequence of the antibody can be obtained by an artificial synthesis method, so that the gene engineering antibody of the anti-porcine reproductive and respiratory syndrome virus nsp4 can be obtained more conveniently and simply, the defects of high preservation cost of hybridoma cells, easy loss of phenotype and the like are effectively overcome, and the animal welfare problem involved in ascites preparation is avoided.

Drawings

FIG. 1 is a schematic diagram of plasmid construction;

FIG. 2 is a graph for identifying the optimal time for determining antibody secretion by Western-Blot;

FIG. 3 is a schematic diagram of Western-Blot antibody identification and secretion.

FIG. 4 is a schematic diagram of IFA identification of secreted antibodies.

FIG. 5 is a schematic diagram of the results of Western-Blot experiments using the genetically engineered antibody prepared in the present invention and the monoclonal antibody of porcine reproductive and respiratory syndrome virus nsp4 protein as primary antibodies.

FIG. 6 is a schematic diagram of IFA detection of the genetically engineered antibody prepared according to the present invention as a primary antibody.

Detailed Description

The embodiments of the present invention are described below by specific examples, and the technical means used in the present invention are all methods known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. The reagents or materials of the invention, if not specifically described, are commercially available.

Example 1:

preparation of genetically engineered antibody against nsp4 protein of porcine reproductive and respiratory syndrome virus:

the genetic engineering antibody of the nsp4 protein for resisting the porcine reproductive and respiratory syndrome virus can be obtained by industrial synthesis according to amino acid sequences shown in SEQ ID NO.6 and SEQ ID NO. 8; or obtained by a conventional protein expression mode, and the invention adopts the following modes:

1) taking the sequence shown in SEQ ID NO.5 as a template, designing specific primers IgG1-HV-F1 and IgG1-HC-R to amplify the heavy chain full-length polynucleotide containing a leader sequence, which corresponds to a heavy chain region AH1 of the genetically engineered antibody; the sequence shown in SEQ ID NO.7 is used as a template, specific primers kappa-LV-F1 and kappa-LC-R1 are designed to amplify the full-length light chain polynucleotide containing a leader sequence, and the full-length light chain polynucleotide corresponds to a light chain region AL1 of the genetic engineering antibody.

The amplified fragments are respectively connected into the middle of EcoRI and BamHI enzyme cutting sites in the pABL vector, and 2 plasmids (H1 and L1) are successfully constructed by sequencing and identification, and the structures of the plasmids are shown in figure 1.

2) Plasmids H1 and L1 were co-transfected into HEK-293T cells according to Thermo Fisher

2000 instructions for transfection reagents the procedure was as follows: the heavy and light chain plasmids were added to 50. mu.l of Opti-MEM at 0.5ug each (in a 24-well plate), the transfection reagent was added at 2.5. mu.l, the mixture was allowed to act at room temperature for 15min, and the cells were incubated for 4-6h

FreeStyle^TM293Expression Medium。

Transfecting HEK-293T cells according to the pairing transfection method, changing liquid after 6h, collecting cell supernatants at 48h, 72h, 96h and 120h respectively, adding 5 xSDS-PAGE Loading Buffer, carrying out 10min at 100 ℃ in a boiling water bath, centrifuging 10min at 4 ℃ at 12000r/min, sucking the supernatants, carrying out SDS-PAGE electrophoresis, and carrying out gel preparation and gel pouring according to the use instruction of a BIO-RAD protein electrophoresis apparatus. The protein was then transferred to PVDF membrane. And (3) sealing the PVDF membrane, adding goat anti-mouse IgG-HRP, incubating for 1h at room temperature, washing the membrane for three times by TBST, adding a Super Signal Western Pico chemiluminescence substrate, observing in a chemiluminescence imaging system, photographing and storing the result, wherein the experimental result is shown in figure 2.

The results show that the antibody secretion is optimal after the cells are co-transfected by the plasmids H1 and L1 for 96H.

Using the above method, a large number of engineered antibodies (AH1 and AL1) expressed by HEK-293T cells were prepared and used as primary antibodies, and the antibody protein concentration was 500. mu.g/mL using a mouse immunoglobulin G (IgG) ELISA kit, and used in the following examples.

Example 2:

specificity identification of anti-porcine reproductive and respiratory syndrome virus nsp4 genetic engineering antibody:

specific identification (ELISA test):

1) purified Porcine Reproductive and Respiratory Syndrome Virus nsp4 Protein (obtained by eukaryotic expression of transfected cells) was coated at a concentration of 1. mu.g/mL on an enzyme plate by plasmid pCAGGS-nsp4 (ref: Tao R, Fang L, Bai D, Ke W, Zhou Y, Wang D, Xiao S.Porine reproducing and reproducing Virus non responsive Protein 4 Cleas Porine DCP1a To Attenue enzyme activity.J Immunol.2018Aug 29. pi: ji1701773.doi: 10.4049/jimunol.1701773.) overnight at 4 ℃. Three times of PBST washing were performed, 100. mu.L of the genetically engineered antibody obtained in example 1 as a primary antibody and the supernatant of HEK-293T cells not subjected to transfection as a control were incubated in an incubator at 37 ℃ for 1 hour per well, and 100. mu.L of 1: and (3) incubating the 4000 diluted goat anti-mouse IgG-HRP enzyme-labeled secondary antibody in an incubator at 37 ℃ for 1h, washing the secondary antibody with PBST for three times, adding a substrate, and developing for 10min in a dark place, wherein the result observation and analysis are performed after the development. The result shows that the antibody in the supernatant of the HEK-293T cell can interact with nsp4 protein of porcine reproductive and respiratory syndrome virus, the concentration of the genetically engineered antibody is high, and the OD630nm value still has 2.662+0.067 at 96h, so that the OD630nm value does not show time change and is more than 2.

Subsequently, the engineered antibody prepared in example 1 was diluted in two-fold and subjected to ELISA test, and the test results are shown in fig. 3. The results show that when the dilution of the genetically engineered antibody is greater than 1: 2560, the OD630nm value was less than 1.

Specific identification (Western-Blot):

(1) identification of the nsp4 protein of porcine reproductive and respiratory syndrome virus: plasmids pCAGGS-nsp4 (ref: Tao R, Fang L, Bai D, Ke W, Zhou Y, Wang D, Xiao S. Porcine reproduction and Respiroratory synthetic Virus No. regulatory Protein 4 clear porves DCP1a To Attenuate Its antibiotic activity. J Immunol.2018Aug 29.pii: ji1701773.doi: 10.4049/jimunol.1701773.) 6ug, 3ug, 1.5ug, 0.75ug and 0ug were transfected into HEK-293T cells (3.5cm petri dish) for eukaryotic expression, and a blank was set up and harvested after 36h, respectively. Western-Blot experiments were carried out using the engineered antibody prepared in example 1 as a primary antibody, and the results are shown in FIG. 4.

The result shows that the genetic engineering antibody provided by the invention can generate a specific band which is clear and single and has no redundant miscellaneous band like the monoclonal antibody, and the genetic engineering antibody provided by the invention can be specifically combined with the nsp4 protein of the porcine reproductive and respiratory syndrome virus and has the characteristic of high specificity of the monoclonal antibody.

(2) Identification of porcine reproductive and respiratory syndrome virus: MARC-145 cells were inoculated in a 3.5cm dish, and 1MOI of porcine reproductive and respiratory syndrome virus WUH3 strain was inoculated when the cells grew to a monolayer, and samples were collected at 12h, 24h, 36h, and 48h after inoculation, respectively, and the obtained samples were used as antigens. Western-Blot experiments were performed using the genetically engineered antibody prepared in example 1 and the nsp4 monoclonal antibody (prepared by fusing myeloma cells and spleen cells according to the conventional methods in the art) as a primary antibody, and the results are shown in FIG. 5.

The result shows that the genetically engineered antibody provided by the invention can react with porcine reproductive and respiratory syndrome virus, and the strip of the genetically engineered antibody is clear and single as that of a monoclonal antibody control group without other miscellaneous strips, which indicates that the genetically engineered antibody provided by the invention has the same high specificity and specificity as that of the monoclonal antibody.

Specificity identification (IFA detection)

Putting a cell slide in a 24-well plate, inoculating MARC-145 cells, inoculating WUH3 strains of porcine reproductive and respiratory syndrome virus with 1MOI when the cells grow to a monolayer, respectively collecting samples 12h, 24h, 36h and 48h after inoculation, and simultaneously setting up a non-virus cell control. After preparation of the cell samples, the cell culture medium was discarded and washed 3 times with PBS (1X, pH 7.4, same below), 1mL of PBS per well for 5min each time. The action is slow when adding and removing liquid, avoiding blowing up the cells. mu.L of 4% paraformaldehyde was added to the first well and the cells were fixed for 15 min. Adding methanol pre-cooled at-20 deg.C, 1 mL/well, permeabilizing cell for 10min, and washing with PBS 3 times. Adding sealing liquid 400 mul/hole, sealing for 45-60 min. The engineered antibody prepared in example 1 was added thereto at 250. mu.L/well, incubated at 37 ℃ for 1 hour, and washed 3 times with PBS. Fluorescein Isothiocyanate (FITC) labeled goat anti-mouse IgG (diluted 100 times with PBS) was added at 250. mu.L/well, the fluorescent secondary antibody was discarded after 1h exposure at 37 ℃, washed 3 times with PBS, and the procedure was carefully protected from light. And finally, sealing and observing by using a microscope. The experimental results are shown in FIG. 6.

The result shows that the genetic engineering antibody provided by the invention can identify the nsp4 protein of the porcine reproductive and respiratory syndrome virus, the specific fluorescence is increased along with the prolonging of time, and the genetic engineering antibody can have obvious fluorescence reaction with the porcine reproductive and respiratory syndrome virus.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

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Claims (4)

1. An anti-porcine reproductive and respiratory syndrome virus nsp4 protein genetic engineering antibody comprises protein peptide segments shown in SEQ ID NO.6 and SEQ ID NO. 8.

2. Polynucleotides encoding the peptide fragments of the proteins shown in SEQ ID NO.6 and SEQ ID NO. 8.

3. The polynucleotide of claim 2, represented by SEQ ID No.5 and SEQ ID No. 7.

4. Use of the antibody of claim 1 or the polynucleotide of claim 2 in the preparation of a kit for the detection of porcine reproductive and respiratory syndrome virus.

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