CN114085274B - FIPV N recombinant protein and colloidal gold test strip for rapidly detecting FIPV infection - Google Patents
FIPV N recombinant protein and colloidal gold test strip for rapidly detecting FIPV infection Download PDFInfo
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- CN114085274B CN114085274B CN202111403374.4A CN202111403374A CN114085274B CN 114085274 B CN114085274 B CN 114085274B CN 202111403374 A CN202111403374 A CN 202111403374A CN 114085274 B CN114085274 B CN 114085274B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention provides a FIPV N recombinant protein and a colloidal gold test strip for rapidly detecting FIPV infection, belonging to the technical field of biological detection. The amino acid sequence of the FIPV N recombinant protein provided by the invention is shown as the sequence ID No. 1. The invention expresses FIPV N protein based on high conservation and excellent immunity of N protein by using a prokaryotic expression system, and a colloidal gold test strip is developed by utilizing the protein and polyclonal antibody obtained after the immune kittens so as to detect FIPV yin-yang serum, thereby laying a foundation for establishing a FIPV rapid diagnosis method.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a FIPV N recombinant protein and a colloidal gold test strip for rapidly detecting FIPV infection.
Background
Feline coronavirus (FIPV) is a high mortality disease in cats, occurring in cats worldwide, primarily through contact or transmission between cat populations via the digestive and respiratory tracts, and the primary target cells of FIPV are cat monocytes and macrophages and are capable of efficient infection and activation and replication in monocytes/macrophages, which can lead to inflammatory lesions including vasculitis, peritoneal effusions, and fibrosis and granulomatous inflammation.
FIPV has 4 major structural proteins including spike glycoprotein (S), membrane (M) protein, small envelope (E) protein, nucleocapsid (N) protein. Although the S protein is a FIPV neutralizing antigen, antibodies with neutralizing activity are produced. However, S protein has high variability and contains ADE epitopes, and thus cannot be used as a test. The N protein is also the main structural protein of FIPV, and researches show that the N protein has stronger antigenicity and can induce strong cellular immunity and humoral immunity, and the N gene of coronavirus is highly conserved relative to spike protein, and can be used as an excellent target gene for antigen-antibody detection. In the early stages of FIPV infection, high levels of anti-N protein antibodies can be produced in vivo. Therefore, the N protein can be used as a key index for early and rapid detection of FIPV infection.
At present, the rapid diagnosis product aiming at FIPV is lack, only RT-PCR can carry out rapid detection, but common pet hospitals do not have corresponding detection equipment, which greatly influences the discovery and diagnosis of illness state, so that the provision of a rapid, convenient and low-cost early rapid detection mode is a problem to be solved urgently in the research of the field.
Disclosure of Invention
The invention provides a PV N recombinant protein and a colloidal gold test strip for rapidly detecting FIPV infection, wherein the method is based on high conservation and excellent immunity of N protein, the FIPV N protein is expressed by a prokaryotic expression system, and the colloidal gold test strip is developed by utilizing the protein and polyclonal antibodies obtained after immunization of kittens so as to detect FIPV yin-yang serum, thereby laying a foundation for establishing a FIPV rapid diagnosis method.
In order to achieve the above object, the present invention provides a FIPV N recombinant protein, the amino acid sequence of which is shown as Seq ID No. 1.
The invention also provides a coding gene of the FIPV N recombinant protein according to the technical scheme.
The invention also provides a specific PCR primer for amplifying the coding gene according to the technical scheme.
The invention also provides application of the FIPV N recombinant protein in preparing an IELISA kit for rapidly detecting feline coronavirus infection.
The invention also provides a colloidal gold test strip for rapidly detecting the feline coronavirus infection, and the FIPV N recombinant protein is used as an antigen molecule to be detected by the test strip.
Preferably, the substrate and the adsorption layer are included; the adsorption layer sequentially comprises a sample pad, a colloidal gold mark pad, a nitrocellulose membrane and a water absorption pad from a sample adding end, wherein the sample pad and the colloidal gold mark pad are made of glass fiber cotton; the colloidal gold marked pad is adsorbed with FIPV N recombinant protein marked by colloidal gold; the nitrocellulose membrane is provided with a quality control line C and a detection line T; the water absorbing pad is made of water absorbing filter paper.
Preferably, the FIPV N recombinant protein preparation method is as follows:
synthesizing a gene fragment with 1134bp by carrying out secondary mechanism and NLS nuclear localization signal analysis on N protein, and obtaining a recombinant N gene by carrying out PCR amplification on the gene fragment;
connecting the obtained recombinant N gene with a pET32a vector subjected to the same enzyme digestion by using a ligase, converting the recombinant N gene into an escherichia coli T7shuffle competent cell, screening positive clones through ampicillin resistance, extracting a PCR (polymerase chain reaction) identification positive bacterial liquid plasmid, and obtaining a positive recombinant plasmid pET32a-FIPV-N through enzyme digestion identification;
and (3) converting the positive recombinant plasmid pET32a-FIPV-N into E.coli BL21 competent cells, adding isopropyl-beta-D-thiogalactoside for induced expression and purification, and obtaining the FIPV N recombinant protein.
Preferably, the particle diameter of the colloidal gold adsorbed on the colloidal gold-labeled pad is 30-80nm, the proportion of the colloidal gold-labeled pad to the FIPV N recombinant protein is 20 mu g protein, 1mL of colloidal gold solution is added, and the spraying amount of the FIPV N recombinant protein in the colloidal gold-labeled pad is 6 mu L/cm.
Preferably, the quality control line C contains 1 mu L/cm of rabbit anti-cat coronavirus N protein polyclonal antibody, and the detection line T contains 1 mu L/cm of sheep anti-cat IgG antibody;
the distance between the detection line and the quality control line is 0.5cm, and the distances between the detection line and the nitrocellulose membrane are 1cm and 1.5cm respectively.
Preferably, the amount of FIPV N recombinant protein required for detection of FIPV in the colloidal gold test strip is as low as 2.46 μg/mL.
Preferably, the application method comprises the following steps:
collecting serum samples of cats to be detected, dripping the serum samples into a colloidal gold test strip sample pad, and horizontally placing the serum samples for 3-5min to observe results;
if the quality control line C and the detection line T at the nitrocellulose membrane show two red strips, the sample to be detected contains FIPV N recombinant protein;
if only the quality control line C shows a red band, the fact that FIPV N recombinant protein is not detected in the sample to be detected is indicated;
if the test strip does not show any strips, the detection operation is incorrect or the test strip fails.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention prokaryotic expresses a FIPV N truncated gene segment with a TEV enzyme cutting site,obtaining the soluble FIPV N target protein. The TEV cleavage site is a TrxA tag that dissolves the protein in PBS by promoting disulfide bond folding. Polyclonal antibodies are obtained from immunized experimental cats, and the time-free valence of 4 is 1:10 by ELISA determination 5 The FIPV N protein has good immunogenicity. In addition, the specific indirect immunofluorescence result shows that the structural characteristics of the FIPV N protein expressed by the prokaryote are similar to those of the natural N protein of the virus, and the FIPV antibody expression can be effectively detected.
2. According to the preparation method of the colloidal gold test paper, the colloidal gold test paper is assembled by coating a glass fiber film as a gold-labeled pad after marking N protein, and respectively coating a cat anti-N protein polyclonal antibody and a monoclonal antibody rabbit anti-cat FC monoclonal antibody as a quality control line and a detection line on a nitrocellulose film. The E colloidal gold detection result shows that the truncated FIPV N protein can be specifically combined with the polyclonal antibody of the naturally infected FIPV cat, the colloidal gold result is matched with the RT-PCR result, and the colloidal gold detection result can be used as an index of clinical diagnosis, thereby providing a rapid, convenient and low-cost detection mode for early rapid diagnosis.
3. The invention obtains the FPIV-N protein with high purity and content by the prokaryotic expression system expression with short period, cost saving and simple process. And the colloidal gold test strip is developed by utilizing the polyclonal antibody obtained after the protein and the immunized kittens. The test strip has higher accuracy and lays a foundation for establishing a FIPV rapid diagnosis method.
Drawings
FIG. 1 is a schematic diagram of verification of recombinant plasmid pET32a-FIPV N provided by the embodiment of the invention, wherein (A) the schematic diagram of the recombinant plasmid pET32a-FIPV N; (B) PCR verification of recombinant plasmid and enzyme digestion product; m: maker,1: FIPV N and pET32a ligation products;
FIG. 2 is a schematic diagram of the identification of FIPV N recombinant proteins provided by an embodiment of the present invention, wherein (A) is an SDS-PAGE protein gel; m: maker,1: inclusion body, 2:50mM imidazole eluent, 3:100mM imidazole eluent; (B) WB glue pattern; m: maker,1:50mM imidazole eluent recombinant N protein, 2:100mM imidazole eluent;
FIG. 3 shows a recombinant FIPV N protein provided in an embodiment of the present inventionSchematic of antibody titers, wherein: the abscissa number represents dilution factor, 1:500;2:1000;3:2000;4:4000;5:8000; 6:1.6X10 4 ；7:3.2×10 4 ；8:6.4×10 4 ；9:1.28×10 6 ；10:2.56×10 6 ；11:5.12×10 6 The method comprises the steps of carrying out a first treatment on the surface of the The ordinate represents the P/N values of serum at different dilutions;
FIG. 4 is a schematic diagram of indirect immunofluorescence assay FIPV N protein provided in an embodiment of the present invention, wherein: the upper part is an experimental group, and the lower part is a control group;
FIG. 5 is a schematic diagram of specificity and sensitivity verification and detection of a test strip according to an embodiment of the present invention, wherein (A) is a schematic diagram of a transmission electron microscope of colloidal gold particles; (B-E) colloidal gold test paper detects FIPV-N (B), positive sample (C), negative sample (D) and other disease samples (E) with different concentrations. (F) FIPV samples (left), other cat-related disease virus (right) PCR detection schematic.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Bacterial strain, plasmid, test animal and reagent
Female kittens of 8 weeks old were purchased from beijing verdeliwa laboratory animal technologies limited; FIPV strain is stored in a laboratory; CRFK cells were purchased from the general division (Shanghai) biotechnology limited; rabbit anti-cat FC mab was purchased from Abcam; TEV enzyme, pET32a plasmid, E.coli DH5 alpha and T7shuffle were purchased from Shandong Hertz biotechnology Co., ltd; SDS-PAGE protein gel kit was purchased from Yase Biotechnology Co., ltd; fetal bovine serum was purchased from beijing holly biotechnology liability company; DMEM cell culture broth was purchased from GIBCO company; the diabody solution was purchased from GE company; freund's adjuvant was purchased from Sigma company; premix taq, plasmid mini kit, gel Extraction, T4 DNA ligase and transfection reagents were all purchased from TaKaRa.
EXAMPLE 1 construction of FIPV N protein recombinant plasmid and prokaryotic expression
The protein secondary structure prediction software and the protein nuclear localization signal prediction software in Novopro online tool are adopted to carry out secondary structure analysis and Nuclear Localization Signal (NLS) analysis on the FIPV N protein sequence of 79-1146 strain (GeneBank ID: DQ 010921.1). FIPV N expression genes 151-1131bp are selected by combining with high antigen indexes and alpha-helix, beta-sheet and beta-corner enrichment regions to carry out codon optimization, and gene fragments with the size of 1134bp are synthesized. The his tag, trxA tag, bamHI and XhoI cleavage sites were synthesized and TEV cleavage sites were added after the upstream BamHI site, as shown in fig. 1A. The synthesized N gene was digested with BamHI and XhoI and ligated into the same digested pET32a vector. After the prokaryotic expression pET32a-FIPV N recombinant plasmid is digested by BamHI and XhoI, a target band can be observed at 1134bp, as shown in FIG. 1B. The sequencing result of the target band shows that the PCR product accords with the sequence of the recombinant plasmid, which indicates that the pET32a-FIPV N recombinant plasmid is successfully obtained by the research.
Furthermore, E.coli BL21 (DE 3) competent cells were transformed with recombinant plasmid pET32a-FIPV N, single colonies were picked up and inoculated with 20mL ampicillin (final concentration 100. Mu.g/mL) LB medium, shake-cultured at 37℃for 2h, ampicillin 2L LB medium was inoculated into the culture at 1:100 volume ratio, shake-cultured at 37℃until OD600 = 0.6-0.8, and 1mM IPTG was added thereto, and shake-cultured at 20℃for 9h for expression. The pellet was centrifuged at 4500rpm at 4℃and suspended in 80mL Tris-HCl (pH=7.4), and the pellet was broken and lysed by sonication in an ice bath. Centrifuging at 10000rpm at 4deg.C for 20min, collecting supernatant, and purifying to obtain FIPV N recombinant protein with amino acid sequence shown in Seq ID No. 1.
Example 2FIPV N protein purification and characterization
The supernatant protein sample collected in example 1 was applied to a nickel column equilibrated with 50mM Tris-HCl beforehand, incubated for 1h to allow the protein to bind well to the nickel column, after which the sample was allowed to slowly flow out, the effluent was collected and loaded twice. Proteins were eluted and collected with 5 column volumes of imidazole at 50mM and 100mM concentrations, respectively; the collected proteins were transferred to an ultrafiltration tube, concentrated by ultrafiltration at 4℃and 3000r/min, 8. Mu.l of the concentrated proteins were added, respectively, and 2. Mu.l of 5 Xprotein loading buffer were added, mixed well and denatured for 10min, and 8. Mu.l of samples were taken, respectively, for SDS-PAGE identification.
SDS-PAGE showed that the band of interest appeared at about 55kDa in inclusion bodies, 50 and 100mM imidazole elution concentrate (FIG. 2A). And, after 100mM eluting the concentrate, a purer FIPV N protein was obtained. In addition, western blot results showed that 100mM imidazole-eluted FIPV N protein was able to bind specifically to anti-his secondary antibody (fig. 2B), indicating successful acquisition of purified FIPV N protein.
Example 3FIPV N protein quantification
8 gradient diluted BSA protein standard samples, 25. Mu.L, respectively designated as A, B, C, D, E, F, G, H, were taken and FIPV N protein samples to be tested, 25. Mu.L, were sequentially added to the microplate. 200 mu L of working solution is added into each hole, and the mixture is vibrated on a vibrator for 30s at room temperature so as to be fully and uniformly mixed. Sealing the microplate with a self-sealing bag, and sealing at 37deg.C for 30min. And cooling the 96-well plate to room temperature, reading the absorbance value of the sample at 562nm by using an enzyme-labeled instrument, and calculating the concentration of the protein to be detected according to the obtained linear equation. N protein was diluted in serial 2 at concentrations of 27.27, 13.56, 5.65, 3.83, 2.46, 0.63 and 0.31. Mu.g/mL, respectively.
EXAMPLE 4 animal immunization
Healthy FIPV negative 8-week-old female cats are selected for the experiment, vein blood collection is carried out on the negative cats before immunization, and serum is separated to serve as negative control. 100 mug of purified protein is added with Freund's complete adjuvant with equal volume for emulsification, and is injected into the rear neck and the rear waist of a cat respectively, and 0.5mL of separated serum is collected by vein after one week for preservation. After one week, 100. Mu.g of purified protein was emulsified with incomplete adjuvant and subjected to a second immunization injection, and after one week, 0.5mL of separated serum was collected intravenously and stored. After one week, 100. Mu.g of purified protein was emulsified with incomplete adjuvant, and 4 immunization injections were performed, 30mL of blood was collected one week and serum was isolated and stored at-20℃for use.
EXAMPLE 5 preparation of polyclonal antibodies and potency detection
Coating a 96-well ELISA plate with 5 mug/mL FIPV N protein, 100 mug/well, and overnight at 4 ℃; blocking was performed with 3% BSA in PBS for 2h at 37 ℃. After 5 washes with PBST, serum samples diluted in a 2-fold gradient were added, 100. Mu.L/well and incubated for 1h at 37 ℃. PBST wash 5 times followed by 1:5000 dilution of HRP-labeled rabbit anti-cat FC secondary antibody, 100. Mu.L/well, incubation in 37℃incubator for 1h. After 5 times of PBST washing, 100. Mu.L/well TMB single-color development solution was added for 10min, and 100. Mu.L/Kong Zhongzhi solution was added. The OD450 value was read with a microplate reader and the polyclonal antibody titer was calculated.
ELISA results showed that the antibody titer of the 1-serum was similar to that of the control group, and almost no antibody could be detected. As the number of immunizations increased, the antibody titers increased significantly (fig. 3). 2 at 32,000-fold dilution, 3 at 74,000-fold dilution and 4 at 5.92×10 5 Generates effective antibody titer (P/N) when diluted in multiple>2). Furthermore, the prepared FIPV N antigen detects the antibody content in the positive FIPV sample which is confirmed, which indicates that the recombinant FIPV N has stronger immunogenicity. In addition, the prepared FIPV N antigen detected the antibody content in the confirmed FIPV positive samples, indicating that the prepared antigen was effective.
Example 6 Indirect immunofluorescence
CRFK cells were plated in 6-well plates and when CRFK monolayers grew to about 70% they were infected with FIPV, the negative control was normal cells. The cells were transfected in an incubator at 37℃for 2h, and negative control wells were added with DMEM medium containing 2% fetal bovine serum. 5% CO at 37 DEG C 2 After incubation in the incubator for 12-24h, the supernatant was aspirated and washed 3 times with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 15min, permeabilized with 0.5% Triton X-100 at room temperature for 6min, and then blocked with 3% BSA at room temperature for 1h. The prepared cat anti-N polyclonal antibody was purified with 3% bsa according to 1:500 dilution, 4 ℃ overnight. PBST was washed 3 times, and 1:5000 dilutions of FITC-labeled rabbit anti-cat FC, cassette incubated for 1h and washed 3 times with PBST. Finally, the nuclei were stained with DAPI for 3min, and the results were observed under a fluorescent inverted microscope after washing.
IFA results showed that, as shown in fig. 4, both the experimental group and the control group can observe round nuclei, and the experimental group generated specific punctate fluorescence after treatment with purified cat anti-N polyclonal antibody, and no fluorescence was generated in the blank, indicating that purified cat anti-N polyclonal antibody can specifically recognize N protein in CRFK infected with FIPV. It follows that the purified feline anti-N polyclonal antibody can specifically bind FIPV and that FIPV N protein is predominantly in the nucleus of infected cells.
Example 7 preparation of FIPV N colloidal gold test paper
The cat coronavirus antibody detection test paper consists of a PVC supporting bottom plate, a sample pad, a gold mark pad, a nitrocellulose membrane (NC membrane) and a water absorption pad 5. Colloidal gold particles are prepared by adopting a trisodium citrate reduction method: 100mL of ultrapure water was placed in a 500mL beaker, 1mL of 10mg/mL of chloroauric acid was added and heated to boil, 1.6mL of 10mg/mL of trinna citrate was added under heating and stirring, and the color change was observed until the heating was stopped when the color no longer changed. Naturally cooling, and then using ultrapure water to fix the volume to 100mL, and preserving at 4 ℃. The prepared colloidal gold is wine red, transparent in color, free of sediment and floaters. The colloidal gold is observed to be round particles under a transmission electron microscope, the particle diameter is about 30-80nm (figure 5A), and the method is suitable for preparing colloidal gold test paper.
40mL of colloidal gold was added with 80. Mu.L of 0.2mol/L K 2 CO 3 480. Mu.L of recombinant protein of feline coronavirus N (diluted with 1mg/mL of ultrapure water) was added dropwise under stirring, and the mixture was stirred uniformly and reacted at room temperature for 70 minutes. 4mL of 50mg/mL casein solution is added dropwise under stirring, the mixture is reacted for 10min at room temperature after being stirred uniformly, the mixture is centrifuged for 25min at 12000r/min, and the gold mark-FIPV N protein coupling substance precipitate is prepared after the previous step is discarded. The precipitate was treated with 8mL of protein preservation solution (30 g/L sucrose, 10g/L casein, 10 g/LTtion X-100, 30g/L Na) 2 B 4 O 7 .10H 2 O) after re-suspending, preserving at 4 ℃ for standby. Spraying gold-labeled N protein on a glass fiber membrane according to the amount of 6 mu L/cm, drying at 45 ℃ for 1h, adding a drying agent, and sealing and preserving. Primary antibodies (sheep anti-cat IgG Fc) and secondary antibodies (rabbit anti-cat coronavirus N protein polyclonal antibodies) are respectively sprayed on NC films by using an HM3030 three-dimensional film drawing instrument according to the scribing amount of 1 mu L/cm and are used as detection lines (T line) and quality control lines (C line), and are dried at 42 ℃ for 2 hours, and are sealed and stored by adding a drying agent.
Example 8FIPV N colloidal gold test paper sample detection
8FIPV positive samples and 8 negative samples were collected in a pet hospital, and 1 serum sample of Feline Pestivirus (FPV), feline herpesvirus type 1 (FHV-1), feline Calicivirus (FCV), feline leukemia (FeLV) and the like were each collected, and a chromogenic reaction was performed using a test strip.
The colloidal gold test strips prepared by coupling the colloidal gold particles with N proteins at concentrations of 27.27, 13.56, 5.65, 3.83, 2.46, 0.63 and 0.31. Mu.g/mL showed that antibodies in serum could be detected at concentrations of 2.46. Mu.g/mL, but not at concentrations of 0.63. Mu.g/mL, indicating high sensitivity of the strips (FIG. 5B). All 8FIPV positive samples appeared double-lined, while FIPV negative samples appeared only C-lined (fig. 5C and 5D). In addition, the test strip is used for detecting cat serum after infection of Feline Pestivirus (FPV), feline herpes 1 type rash virus (FHV-1), feline Calicivirus (FCV) and feline leukemia (FeLV), and the test strip can not detect diseases such as Feline Pestivirus (FPV), feline herpes 1 type rash virus (FHV-1), feline Calicivirus (FCV) and feline leukemia (FeLV), which shows that the test strip has better specificity (figure 5E). To verify whether cats harvested serum successfully infected FIPV, feline Pestivirus (FPV), feline herpes type 1 virus (FHV-1), feline Calicivirus (FCV) and feline leukemia (FeLV), each virus was verified for expression in serum using PCR (fig. 5F). The result shows that the collected serum contains the corresponding virus, and the accuracy of the control group is verified, so that the specificity of the test strip is more strongly demonstrated.
Sequence listing
<110> Shenzhen Hertz Life science technology Co., ltd
<120> FIPV N recombinant protein and colloidal gold test strip for rapidly detecting FIPV infection
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 377
<212> PRT
<213> FIPV N recombinant protein
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MNKPTTLGTRGTNNESKPLRFDGKIPPQFQLEVNRSRNNSRSGSQSRSVSRNRSQSRGRH 180
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TLTHTYYLPKDDAKTSQFLEQIDAYKRPSEVAKDQRQRRSRSKSADKKPEELSVTLVEAY 360
TDVFDDTQVEMIDEVTN 377
Claims (1)
1. The colloidal gold test strip for detecting cat coronavirus infection is characterized by comprising a bottom plate and an adsorption layer;
the adsorption layer sequentially comprises a sample pad, a colloidal gold mark pad, a nitrocellulose membrane and a water absorption pad from a sample adding end, wherein the sample pad and the colloidal gold mark pad are made of glass fiber cotton; the colloidal gold marked pad is adsorbed with FIPV N recombinant protein marked by colloidal gold; the nitrocellulose membrane is provided with a quality control line C and a detection line T; the water absorption pad is made of water absorption filter paper; the amino acid sequence of the FIPV N recombinant protein is shown as SEQ ID NO. 1;
the particle diameter of the colloidal gold adsorbed on the colloidal gold mark pad is 30-80nm, the proportion of the colloidal gold mark pad and the FIPV N recombinant protein is 20 mug protein, 1mL of colloidal gold solution is added, and the spraying amount of the FIPV N recombinant protein in the colloidal gold mark pad is 6 mug/cm; the amount of FIPV N recombinant protein required by the detection of the FIPV in the colloidal gold test strip is as low as 2.46 mug/mL;
the quality control line C contains 1 mu L/cm of rabbit anti-cat coronavirus N protein polyclonal antibody, and the detection line T contains 1 mu L/cm of sheep anti-cat IgG antibody;
the distance between the detection line and the quality control line is 0.5cm, and the distances between the detection line and the nitrocellulose membrane are 1.0cm and 1.5cm respectively;
the preparation method of the FIPV N recombinant protein comprises the following steps:
synthesizing a gene fragment with 1134bp by carrying out secondary mechanism and NLS nuclear localization signal analysis on N protein, and obtaining a recombinant N gene by carrying out PCR amplification on the gene fragment;
connecting the obtained recombinant N gene with a pET32a vector subjected to the same enzyme digestion by using a ligase, converting the recombinant N gene into an escherichia coli T7shuffle competent cell, screening positive clones through ampicillin resistance, extracting a PCR (polymerase chain reaction) identification positive bacterial liquid plasmid, and obtaining a positive recombinant plasmid pET32a-FIPV-N through enzyme digestion identification;
converting positive recombinant plasmid pET32a-FIPV-N into E.coliBL21 competent cells, adding isopropyl-beta-D-thiogalactoside for induced expression and purification to obtain FIPV N recombinant protein;
the using method of the colloidal gold test strip comprises the following steps:
collecting serum samples of cats to be detected, dripping the serum samples into a colloidal gold test strip sample pad, and horizontally placing the serum samples for 3-5min to observe results;
if the quality control line C and the detection line T at the nitrocellulose membrane show two red strips, the sample to be detected contains FIPV N recombinant protein;
if only the quality control line C shows a red band, the fact that FIPV N recombinant protein is not detected in the sample to be detected is indicated;
if the test strip does not show any strips, the detection operation is incorrect or the test strip fails.
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| N [Feline coronavirus];匿名;《NCBI，GenBank: AAY32599.1》;第1页 * |
| 猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达;王元红等;《浙江农业学报》;第32卷(第3期);第406-414页 * |
| 王元红等.猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达.《浙江农业学报》.2020,第32卷(第3期),第406-414页. * |
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