CN116063410B - Mutant of rabies virus G protein and application thereof - Google Patents
Mutant of rabies virus G protein and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
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- G—PHYSICS
- G01—MEASURING; TESTING
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a mutant of rabies virus G protein and application thereof, belonging to the technical field of biology. The mutant of the rabies virus G protein provided by the invention is obtained by mutating the 313 th amino acid of the wild rabies virus G protein from lysine to arginine, mutating the 409 th amino acid from serine to cysteine and optionally replacing the signal peptide thereof. After 2 site mutations and optionally also replacement of the signal peptide, the stability of the obtained mutant of rabies virus G protein and the expression level in eukaryotic expression systems are both significantly improved. The mutant of the rabies virus G protein provided by the invention can be used for accurately detecting the concentration of rabies virus antibodies in serum, and has good specificity, so that the mutant is beneficial to the application of the mutant in the detection of quantitatively detecting the rabies virus antibodies and the objective and accurate evaluation of the immune effect of a rabies virus vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mutant of rabies virus G protein and application thereof, in particular to a mutant of rabies virus G protein for detecting rabies virus antibody and application thereof in qualitative and quantitative detection of rabies virus antibody.
Background
Rabies Virus (RV) belongs to the genus Lyssavirus (Rhabdoviridae) of the family Rhabdoviridae. The shape is in a bullet shape, the nucleocapsid is in spiral symmetry, the surface is provided with an envelope, and single-stranded RNA is contained in the envelope. Rabies virus is a pathogen that causes rabies, with two major antigens: one is glycoprotein antigen (G protein, etc.) on the outer membrane of virus, which can bind to acetylcholine receptor to make virus neurotoxic, and can generate neutralizing antibody and hemagglutination inhibiting antibody in vivo, and the neutralizing antibody has protective effect; the other is inner nuclear protein antigen (N protein, etc.), which can generate complement binding antibody and precipitant in vivo without protection.
Immunization with rabies virus vaccine is currently the only effective method for preventing and controlling rabies. The degree of antibody titer after immunization of a vaccine directly affects the resistance of a host (including dogs, cats, etc.) to viruses, wherein the higher the antibody titer, the higher the resistance in principle.
At present, various detection methods for antibody titer (neutralizing antibody activity) after rabies virus vaccine immunization are available, including ELISA (enzyme-linked immunosorbent assay), RFFIT (rfed fit) method, FAVN (faVN) method and the like. The RFFIT method is one of the detection methods recommended by the World Health Organization (WHO) and is most widely applied in evaluating the activity of an antibody for neutralizing rabies virus at present, and is recorded in 2015 edition pharmacopoeia. The method adopts the neutralization reaction of virus and serum to be detected, and adds cells to check the virus activity to detect the antibody titer, so that the accuracy is high. The FAVN method is also a method for detecting neutralizing antibody activity, and is similar to the RFFIT method in principle, except that the observed index is different, and is recommended by the international veterinary agency (OIE) for detecting animal serum rabies virus antibody activity. However, both RFFIT and FAVN methods are difficult to popularize, popularize and detect in the field because they involve cell culture and require manipulation in a specialized laboratory. The ELISA method is simple to operate and can be suitable for basic level and site use, but the enzyme-linked immunosorbent assay kit sold on the market at present is developed by adopting N protein and cannot represent the titer of the neutralizing antibody.
The colloidal gold immunochromatography technology is a novel immune labeling technology which uses colloidal gold as a tracer marker and is applied to antigen-antibody detection, is convenient to use, quick in reaction (can be completed within 15-20 minutes), does not involve professional operations such as cell culture and the like, is convenient for basic layer and field use, and can overcome the defects of RFFIT and FAVN methods in detecting rabies virus antibodies. For example, patent document CN113252893a discloses a method and a test strip (colloidal gold test strip) for rapid quantitative detection of rabies virus antibody by using rabies virus G protein, which uses rabies virus G protein to be adsorbed on a gold pad of the colloidal gold test strip, uses a mixture of mouse anti-canine secondary antibody and mouse anti-feline secondary antibody to coat a detection line, and uses a monoclonal antibody coating quality control line of rabies virus G protein, so that the concentration of rabies virus antibody can be accurately quantified, and the titer of neutralizing antibody can be well represented. However, when rabies virus G protein in a natural state is produced at present, the problems of low protein expression level, poor stability, easiness in precipitation and the like often exist, so that the application of the rabies virus G protein in detecting rabies virus antibodies is limited.
Disclosure of Invention
In view of one or more of the problems existing in the prior art, one aspect of the present invention provides a mutant of rabies virus G protein, which is characterized in that amino acid 313 of rabies virus G protein shown in SEQ ID NO. 1 is mutated from lysine to arginine, amino acid 409 is mutated from serine to cysteine, and the amino acid sequence of the first mutant is named as a first mutant, wherein the amino acid sequence of the first mutant is shown in SEQ ID NO. 2.
In some embodiments, the mutant of rabies virus G protein is a second mutant, which is named as a second mutant, wherein the signal peptide of the first mutant is further replaced by the signal peptide of the first mutant shown as SEQ ID NO. 2, and the amino acid sequence of the second mutant is shown as SEQ ID NO. 5.
In some embodiments, the mutant of rabies virus G protein further carries a tag for protein purification, optionally the tag comprises a His tag.
In another aspect, the invention provides a gene encoding a mutant of rabies virus G protein as described above.
In some embodiments, the use of the mutant of rabies virus G protein in the preparation of a kit, reagent or colloidal gold test strip for detecting rabies virus antibodies is also within the scope of the present invention.
In still another aspect, the present invention provides a colloidal gold test strip for detecting rabies virus antibodies, wherein a gold pad of the colloidal gold test strip is adsorbed with a mutant of the above-mentioned rabies virus G protein labeled with colloidal gold, and a detection line of the colloidal gold test strip is coated with the mutant of the above-mentioned rabies virus G protein.
In some embodiments, the quality control line of the colloidal gold test strip is coated with polyclonal antibody of rabies virus G protein.
In some embodiments, the concentration of the mutant of rabies virus G protein used to prepare the gold pad, the concentration of the mutant of rabies virus G protein used to coat the detection line, and the concentration of the polyclonal antibody used to coat the rabies virus G protein of the quality control line may be 0.5-2.0mg/ml, respectively.
In yet another aspect, the present invention provides a method for detecting rabies virus antibody, which comprises the steps of:
1) Dripping a serum sample to be detected on a sample pad of the colloidal gold test strip, and reacting for 15-25 minutes; and
2) And (3) detecting the colloidal gold test strip after the reaction in the step (1) by using an immunochromatography quantitative analyzer, and giving out the titer level of the rabies virus antibody in the serum sample to be detected.
Based on the technical scheme, the provided mutant of the rabies virus G protein is obtained by mutating the 313 th amino acid of the wild type rabies virus G protein from lysine to arginine, mutating the 409 th amino acid from serine to cysteine and optionally replacing the signal peptide thereof, and the results of the examples show that after the wild type rabies virus G protein is subjected to the above 2 site mutation and optionally replacing the signal peptide thereof, the stability (more stable and difficult to precipitate after protein purification) and the expression quantity of the mutant of the rabies virus G protein in a eukaryotic expression system are obviously improved, so that the mutant of the rabies virus G protein can be efficiently produced, can be stably stored for a long time, and is more beneficial to the application of the mutant in detecting rabies virus antibodies and the like compared with the wild type rabies virus G protein.
The results of the examples also show that when the mutant of the rabies virus G protein provided by the invention is used for preparing a colloidal gold test strip to quantitatively detect the rabies virus antibody concentration in serum (such as canine serum or feline serum) and has good detection accuracy and specificity, and the mutant of the rabies virus G protein provided by the invention has good consistency with a neutralizing antibody detection method (such as RFFIT and FAVN method), so that the mutant of the rabies virus G protein provided by the invention can be used for qualitatively and quantitatively detecting the rabies virus antibody titer level in serum, thereby being beneficial to evaluating the immune effect of a vaccine, and can be used for preparing reagents, kits or colloidal gold test strips for detecting the rabies virus antibody titer level, and the like.
Drawings
FIG. 1 is a diagram of an electrophoresis gel after purification of rabies virus recombinant G proteins (G (A) and G (C)) and after 7 days of standing.
FIG. 2 is a schematic diagram of a colloidal gold test strip.
FIG. 3 is a photograph of a test card assembled with a colloidal gold test strip.
Fig. 4 is a standard curve for the quantitative detection of rabies virus antibodies.
Fig. 5 is a two-dimensional code picture of rabies virus antibody quantitative detection.
Fig. 6 is a photograph of a specific assay of a colloidal gold test card for rabies virus antibody assay.
Fig. 7 is a photograph of clinical serum sample detection using a colloidal gold test card for rabies virus antibody detection.
Detailed Description
The invention aims to provide a mutant capable of efficiently expressing produced rabies virus G protein with improved stability, and provides an application of the mutant of rabies virus G protein in detecting rabies virus antibody level in serum by using a colloidal gold immunochromatography method.
To achieve the above object, the inventors have made the following 2-site mutant modifications to wild-type rabies virus G protein: the 313 th amino acid of wild rabies virus G protein is mutated from lysine to arginine, the 409 th amino acid is mutated from serine to cysteine, and the surprise shows that the stability (more stable after protein purification and difficult precipitation) and the expression quantity in a eukaryotic expression system of the mutant of the obtained rabies virus G protein are obviously improved, and the mutant can still be used for detecting the rabies virus antibody level in serum; in addition, the inventor further carries out the following modification on the signal peptide on the basis of the mutation modification of the above 2 sites: the original signal peptide of the wild rabies virus G protein is replaced by the signal peptide shown as SEQ ID NO. 4, and the surprise shows that the stability and the expression quantity of the mutant of the rabies virus G protein are further improved. Based on the detection, the invention also provides a colloidal gold test strip for qualitative and/or quantitative detection of rabies virus antibodies.
The invention is illustrated in detail by the following specific examples in conjunction with the accompanying drawings. The terms "first" and "second" and the like are used herein to distinguish between similar objects and are not intended to be limited to a particular order or sequence or number of objects.
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
Examples detailed embodiments and specific operation procedures are given on the premise of the technical scheme of the present invention, and examples are helpful for understanding the present invention, but should not be construed as limiting the present invention.
Example 1: preparation and identification of rabies virus G protein and mutant thereof
1.1 materials:
plasmid pcDNA3.1, E.coli Mach1T1 competent cells, DL2000 DNA Marker, protein molecular mass standard were all purchased from Takara company; t4 DNA ligase, restriction endonuclease HindIII, ecoRI were all purchased from NEB; CHO-S cells were purchased from Invitrogen company; plasmid miniprep kit, DNA gel recovery kit, genome extraction kit were all purchased from Axygen company; rabbit polyclonal antibodies to rabies virus G protein were purchased from the company of arctigenin science, inc.
1.2 method:
1.2.1 Synthesis of the coding Gene for rabies Virus G protein, construction and identification of pcDNA3.1 (+) -G recombinant expression vector
And screening to obtain a sequence with high conservation degree of wild rabies virus G protein by using on-line analysis software of Genbank, wherein the sequence is shown as SEQ ID NO. 1 (GenBank accession number: AGN 94251.1). The inventors have made the following innovations through their efforts to explore: in order to improve the stability of the protein and the expression quantity in a eukaryotic expression system, mutating the 313 th position of an amino acid sequence shown in SEQ ID NO. 1 from lysine to arginine, mutating the 409 th position from serine to cysteine, and naming the obtained mutant mutated at the 2 positions as a first mutant, wherein the amino acid sequence of the first mutant is shown in SEQ ID NO. 2; in order to further increase the expression level in the eukaryotic expression system, the signal peptide of the first mutant shown in SEQ ID NO. 2 was further improved, and it was determined to replace the original signal peptide (MVPQALLFVP LLVFPLCFG, SEQ ID NO. 3) with a signal (MGWSCILFLVATATGVHSVPQALLFVPLLVFSLCFG, SEQ ID NO. 4) capable of further promoting secretory expression in the eukaryotic expression system, and the resulting mutant was named as a second mutant, the amino acid sequence of which is shown in SEQ ID NO. 5. A Blast alignment shows that the sequence 100% identical to the amino acid sequences shown in SEQ ID NO. 2 and SEQ ID NO. 5 does not exist in the database of Genbank et al.
In this example, his tags (HHHHH, as shown in SEQ ID NO: 6) facilitating the purification of the later protein were also designed after the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO: 5. The His-tagged amino acid sequences above were translated into nucleic acid sequences N1 (corresponding to SEQ ID NO: 1), N2 (corresponding to SEQ ID NO: 2) and N3 (corresponding to SEQ ID NO: 5), respectively, according to codon bias of the CHO-S host cell. The three nucleotide sequences (N1, N2 and N3) are respectively connected with a cloning vector pcDNA3.1 (+) to obtain pcDNA3.1 (+) -G recombinant expression vectors which are respectively named pcDNA3.1 (+) -G (A), pcDNA3.1 (+) -G (B) and pcDNA3.1 (+) -G (C), and are directly combined into complete genes by adopting a gene total synthesis mode, and the complete gene synthesis, connection and sequencing processes are all entrusted to be completed by the wuhan aokodingshui biotechnology Co.
1.2.2CHO-S cell electrotransformation
Mu.g of pcDNA3.1 (+) -G (A), pcDNA3.1 (+) -G (B) and pcDNA3.1 (+) -G (C) were combined with 1X 10, respectively 7 CHO-S cells were homogenized in 200. Mu.L electric shock cup, after 30ms electric shock at 1100v, the cell suspension after electric shock was removed in 30mLCD08 medium, 37℃and 5% CO 2 Shake flask culture at 65rpm and 125 mL.
1.2.3 wet-transfer Western Blot detection
After 48h of shaking culture, the mixture is centrifuged at 4000rpm for 10min, the culture medium and the cell bodies are collected, 50 mu L of the culture medium is subjected to electrophoresis, and a proper amount of cell bodies are taken for reduction treatment and then 50 mu L of the cell bodies are subjected to reduction treatment. After SDS-PAGE, taking gel for transferring (300 mA/1 h), sealing for 1h after transferring, adding Anti-6His-HRP antibody for incubation for 1h, and performing color development treatment. The results showed that the target protein was expressed and was present in both cells and medium.
1.2.4 purification of rabies virus recombinant G protein
About 60mL of each cell culture (namely protein solution) transformed by the three recombinant plasmids is collected and subjected to Ni column affinity chromatography, and the method specifically comprises the following steps of:
(1) Filtering the protein solution by using a filter membrane with the diameter of 0.45 μm to obtain a protein sample, slowly adding the protein sample into a purification column, and balancing the column by using a balancing solution (PBS, pH 7.2) until the ultraviolet absorption reaches a stable baseline;
(2) Eluting: eluting with 5-10 times of column volume of eluent (PBS containing 0.5M Imidazole, pH 7.2), and collecting eluate;
(3) Selecting a chromatographic column HisTrap FF (5 mL (GE product number: 17531901), washing the column cleanly by using 0.1M NaOH, balancing by using a balancing solution, hanging nickel, balancing 4 column volumes, loading (eluent collected in step (2)), washing 3 column volumes by using the balancing solution after loading, washing 3 column volumes by using the eluent, eluting by using the eluent, collecting the eluent according to UV, washing 3 column volumes by using 0.1M NaOH, washing 3 column volumes by using water, and preserving the chromatographic column by using 20% ethanol. The affinity purified sample was concentrated by ultrafiltration using a 10KD ultrafiltration tube to 1 XPBS (containing 0.1% NaN) 3 ) Sterile filtration through a 0.22 μm filter, final volume of about 1-2ml, and storage at-20 ℃. The protein expressed by pcDNA3.1 (+) -G (A) recombinant plasmid is named as G (A) protein; the protein expressed by the pcDNA3.1 (+) -G (B) recombinant plasmid is named as G (B) protein; the protein expressed by pcDNA3.1 (+) -G (C) recombinant plasmid is named as G (C) protein.
1.2.5 concentration determination of recombinant G protein of rabies virus
The concentrations of the G (A) protein, the G (B) protein and the G (C) protein solution obtained in the step 1.2.4 were measured by a conventional BCA method, and the measurement results are shown in Table 1 below. It can be seen that the concentration of the G (B) and G (C) proteins is significantly higher than that of the G (A) protein, i.e., the expression levels of the G (B) and G (C) proteins are far higher than that of the G (A) protein, which indicates that the mutation of 2 sites of the wild-type rabies virus G protein in the step 1.2.1 and the further replacement of the signal peptide can significantly improve the expression levels of the mutant (G (B) proteins and G (C) proteins) in eukaryotic expression systems.
Table 1: content determination of day 0 after purification of recombinant G proteins expressed by three recombinant plasmids
1.2.6 determination of purity of rabies virus recombinant G protein
Conventional reducing SDS-PAGE electrophoresis was used, wherein the separation gel was 12% and the stacking gel was 8%, and the electrophoresis result was shown in FIG. 1, wherein M represents marker, lane 1 represents G (A) protein after purification for 0 day, lane 2 represents G (C) protein after purification for 0 day, lane 3 represents G (A) protein after purification for 7 days, and lane 4 represents G (C) protein after purification for 7 days. It can be seen that the purity of the recombinant G protein (G (A) protein and the purity of the recombinant G protein (C) protein) can meet the requirement after 0 days of purification, and only one obvious main band exists. The gel imaging analyzer software is adopted for judging that the purity of the two recombinant G proteins is more than or equal to 90 percent. In the experiment, the purity of the G (B) protein after purification for 0 day is also subjected to conventional reducing SDS-PAGE electrophoresis detection, and the result shows that the G (B) protein also has an obvious main band, and the purity is more than or equal to 90 percent.
1.2.7 stability observations of rabies virus recombinant G protein
Under aseptic conditions, 1ml of each of the three proteins G (A), G (B) and G (C) was placed in a sterile and transparent 1.5ml centrifuge tube, and the proteins were observed at 2-8deg.C, as shown in Table 2 below. As can be seen from table 2 below, the G (a) protein gradually precipitated with prolonged standing time, indicating that the protein was less stable; the G (B) and G (C) proteins are always in a clear and transparent state, and floccules, precipitates or turbidity phenomena do not appear, so that the G (B) and G (C) proteins are very stable, and the stability of the G (B) and G (C) proteins obtained after site-directed mutagenesis and optional signal peptide replacement is obviously improved. Three proteins were collected for 7 days, centrifuged, and the supernatant was collected and the protein concentration was measured again, and the results are shown in the following Table 3, and the purities of the G (A) and G (C) supernatants were exemplarily detected by electrophoresis according to the procedure of step 1.2.6 described above, and the results are shown in FIG. 1 as lane 3 and lane 4, wherein lane 3 represents the G (A) protein after 7 days of purification and lane 4 represents the G (C) protein after 7 days of purification.
As can be seen from table 3, after 7 days of standing, the concentration of G (a) protein was significantly reduced, while the concentration of G (C) protein was not greatly changed; as can be seen from FIG. 1, the purity of the G (A) protein is obviously reduced after the G (A) protein is placed for 7 days, and the purity of the G (C) protein is not greatly changed, which indicates that the stability of the G (C) protein obtained after site-directed mutagenesis and signal peptide replacement is obviously improved. In the experiment, the purity of the G (B) protein which is placed for 7 days after purification is subjected to conventional reducing SDS-PAGE electrophoresis detection, and the result shows that the purity change is small, and the stability of the G (B) protein obtained after site-directed mutagenesis is obviously improved.
Table 2: stability observation of three recombinant proteins at 2-8deg.C
Table 3: measurement of protein concentration on day 7 after purification
| Protein name | Protein concentration | Status of | Remarks |
| G(A) | 0.34mg/ml | Precipitation | Wild type |
| G(B) | 0.91mg/ml | Clear and transparent | Mutant (wild type signal peptide) |
| G(C) | 1.30mg/ml | Clear and transparent | Mutant (replacement signal peptide) |
To further exclude the cause of microbial contamination, 0.1ml of each of the three protein solutions placed on the seventh day was again added to LB nutrient broth, and shaking culture was performed at 37℃for 7 days, and no occurrence of microorganisms was found. The results indicate that the precipitation and concentration decrease of the G (A) protein is due to its own nature (low stability), whereas the G (B) and G (C) proteins are greatly improved in stability as well as in expression due to mutation of 2 sites and optionally substitution of the signal peptide.
Example 2: preparation and use of colloidal gold test strip for detecting rabies virus antibody
2.1 preparation of colloidal gold solution: 155mL of purified water was placed on a magnetic heating stirrer and heated until boiling. 5mL of 1% chloroauric acid solution was added, and after boiling, 6mL of freshly prepared 1% trisodium citrate solution was added with constant stirring, and heating and boiling were continued for 5 minutes. Stopping heating, cooling to room temperature (15-25deg.C), and storing at 2-8deg.C in dark place. Should be a red clear liquid, free of turbidity and surface suspensions;
2.2 labelling of recombinant G (C) proteins: 1mL of the colloidal gold solution was placed in a centrifuge tube, transferred to a triangular flask, and 15. Mu.L of 0.2mol/L potassium carbonate was added. 10. Mu.g of the recombinant G (C) protein purified in example 1 was added to the colloidal gold solution, and the mixture was rapidly and uniformly mixed and left at room temperature (15-25 ℃) for 30 minutes. 10. Mu.L of 20% bovine serum albumin solution was added, mixed well and equilibrated at room temperature (15-25 ℃) for 5 minutes. 10 mu L of 20% PEG20000 solution is added, mixed evenly and equilibrated at room temperature (15-25 ℃) for 30 minutes to obtain gold-labeled antibody solution. The gold-labeled antibody solution was transferred to a centrifuge tube, centrifuged at 10000r/min for 10 minutes, the supernatant carefully aspirated, and discarded. To the precipitate, 100. Mu.L of Jin Biaofu solution (boric acid buffer containing 2% sucrose, 1% casein, 0.5% BSA, 0.1Triton X100, 0.1% SDS) was added, and the mixture was homogenized, and the mixture was stored at 2-8deg.C in the dark to obtain a colloidal gold-labeled recombinant G (C) protein. Should be a red clear liquid, free of turbidity and surface suspensions;
2.3 preparation of gold pad: clean glass cellulose membranes were immersed in gold pad treatment solution (boric acid buffer containing 1% sucrose, 0.5% BSA, 0.1Triton X100). The glass cellulose film was fully soaked for 3 minutes. Taking out, and drying in a drying chamber (the relative humidity is less than or equal to 30%) overnight (16-24 hours). Uniformly mixing the colloidal gold-labeled recombinant G (C) protein obtained in the step 2.2 with Jin Biaofu solution according to a ratio of 1:1, so that the concentration of the colloidal gold-labeled recombinant G (C) protein is about 1.0mg/ml (for example, 0.5-2.0 mg/ml), uniformly spraying the mixture on the soaked glass cellulose film at a speed of 8 mu L/cm by using a gold spraying film drawing instrument, and drying. The gold pad has uniform color and no smudge and crease;
2.4 preparation of nitrocellulose coating film: NC film was cut into strips of 20mm by 30cm, and a PVC plate (30 cm by 6 cm) was taken out, with three double-sided adhesive tapes of unequal widths on the plate, with the narrow side on top. And uncovering the double-sided adhesive tape at the middle position of the PVC plate, and attaching the cut blank NC film to the adhesive tape with the right side facing upwards. Marking with a marker, marking the PVC plate with a blank film, and marking the film with a metal spraying film marking instrument. The pinhead of the spot film is fixed above the NC film and slightly contacts the NC film, the quality control line (C line) is 0.7cm away from the upper end of the film, and the detection line (T line) is 0.7cm away from the quality control line (C line). A quality control line (line C) was prepared by diluting a rabbit polyclonal antibody (available from Beijing Xin Ke Anxing Co., ltd.) to about 1.0mg/mL (e.g., 0.5-2.0 mg/mL) with PBS (0.1 mol/L, pH 7.2); after the G (C) protein (purified in example 1) was diluted to about 1.0mg/ml (e.g., 0.5-2.0 mg/ml), a test line (T line) was prepared. The speed of the metal spraying film drawing instrument is set to be 1.0 mu L/cm, and the drawn film plate is placed in a drying chamber (the relative humidity is less than or equal to 30 percent) to be dried overnight (16-24 hours). After drying, the appearance should be white, the front surface of the film is glossy, the film should be immediately soaked in water, and the surface of the film should not be damaged;
2.5 preparation of sample pad: the glass cellulose film is taken and placed in sample pad treatment solution (disodium hydrogen phosphate: 0.58%, monopotassium phosphate: 0.052%, sucrose: 1.0%, tween-20:0.5ml/l, surfactant: 1.0 ml/l) for complete soaking. Placing the completely soaked sample pad on a clean drying net, and placing the sample pad in a drying chamber (the relative humidity is less than or equal to 30%) for drying (16-24 hours). After drying, the surface of the sample pad should be complete and clean, the texture should be uniform, and the thickness and width should be consistent;
2.6 test strip assembly: under the condition that the relative humidity is less than or equal to 30 percent, according to the structural schematic diagram of the colloidal gold test strip shown in the figure 2, a gold pad is stuck on the PVC backboard stuck with the NC film, and the upper edge of the gold pad is stuck along the lower edge of the nitrocellulose film and exceeds the lower edge of the film by 0.1-0.2cm; attaching the sample pad under the gold pad, overlapping the sample pad and the gold pad by 0.1-0.2cm, and flattening; the double-sided adhesive tape on the uppermost side of the bottom plate, to which the sample pad is attached, is uncovered, and the water absorbing paper is attached above the nitrocellulose membrane, so that the water absorbing pad and the nitrocellulose membrane are overlapped by 0.1-0.2cm, and flattened. Trimming the assembled plate, and cutting into test strips with the width of 3.0 mm. Loading into a plastic card to press the test card; and then the test card is filled into an aluminum foil bag together with 1 package of drier, and the inner package is completed by heat sealing, and the physical photo of the obtained test card is shown in figure 3.
2.7 preparation of test strip standard curve:
2.7.1 mixing 3 parts of serum of dogs and cats immunized by rabies virus vaccine in equal volume, detecting the neutralizing antibody titer by military veterinary institute of medical science and research (OIE rabies reference laboratory), and calculating the average antibody titer to be 24.2U/ml after 3 times of detection. The sample to be measured was diluted to 16U/ml with 10mM BS solution at pH7.2, and then continued to be diluted with 10mM BS solution at pH7.2 at a multiple ratio (16U/ml, 8U/ml, 4U/ml, 2U/ml, 1U/ml, 0.5U/ml, 0.25U/ml and 0.125U/ml).
2.7.2 at 15-25 deg.C, sucking 70. Mu.L of the sample to be tested prepared in step 2.7.1, dripping into the sample-adding hole (S hole of the test card shown in FIG. 3) of the test card obtained in step 2.6, and standing horizontally for 20 min. The KRD-105 immunochromatographic quantitative analyzer was started to detect the T-line gray value, and the results are shown in Table 4 below.
Table 4: correspondence between different antibody titers and gray values
| Valence (U/ml) | 16 | 8 | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 |
| Gray scale value | 500 | 320 | 181 | 100 | 55 | 30 | 18 | 10 |
2.7.3 the gray values shown in the table 4 are input into the KRD-105 immunochromatographic quantitative analyzer data software (respectively corresponding to the titers of the corresponding samples to be tested) to generate a standard curve, the result is shown in fig. 4, and the standard curve is converted into a two-dimensional code which can be identified by the KRD-105 immunochromatographic quantitative analyzer, as shown in fig. 5.
2.7.4 the kit for detecting rabies virus antibody is obtained by taking 20 test cards which are completely packaged in an inner package, loading the test cards into a paper box, printing a two-dimensional code shown in fig. 5, a product name, a batch number, an expiration date, notes and the like into a complete label, and attaching the complete label to the paper box.
2.8 use of colloidal gold test card for rabies virus antibody detection
2.8.1 taking out the serum of the dogs and cats to be tested as a sample to be tested, and placing the sample at room temperature for balancing for 20 minutes.
2.8.2 removal of colloidal gold kit for rabies virus antibody detection (obtained in step 2.7.4 above), and equilibration at room temperature for 20 minutes.
2.8.3 taking out the complete test card packaged in the kit, tearing the aluminum foil bag, and putting the test card on a laboratory table or table flatly.
2.8.4 70. Mu.l of the sample to be measured is taken by a 100-200. Mu.l pipette, carefully dropped into the well of the test card, and allowed to stand for about 20 minutes (e.g., 15-25 minutes).
2.8.5 start KRD-105 immunochromatography quantitative analyzer, scan the two-dimensional code on the kit with the two-dimensional code scanner of instrument own, get into the detection interface.
2.8.6 test card is inserted into the sample holes of the KRD-105 immunochromatography quantitative analyzer one by one after reacting for 20 minutes, and then is clicked for detection, and the rabies virus antibody titer of the sample to be detected is automatically detected and calculated by the instrument, wherein the unit is U/ml.
Example 3: evaluation of quality of colloidal gold test card for rabies virus antibody detection
3.1 evaluation of specificity
The specificity of the product (colloidal gold test card for rabies virus antibody detection) was evaluated by detecting serum samples containing relevant, similar virus antibodies including encephalitis B virus, canine distemper virus, canine parvovirus, feline calicivirus using the colloidal gold test card for rabies virus antibody detection prepared in example 2.
Materials and methods: 100-1000. Mu.l each of serum positive for antibodies containing the following viruses was collected: encephalitis B virus, canine distemper virus, canine parvovirus, feline parvovirus, and feline calicivirus (collected and prepared by Beijing standard Chizehui biotechnology Co., ltd.). The above serum samples were tested according to the procedure "use of colloidal gold test card for 2.8 rabies virus antibody detection" in example 2, and the test results are shown in table 5 and fig. 6 below.
As can be seen from the detection results shown in the following Table 5 and FIG. 6, the results of detecting the serum sample by using the colloidal gold test card for detecting the rabies virus antibody provided by the invention are all negative (the detection value is less than 0.5U/ml, and the judgment is negative), which indicates that the colloidal gold test card for detecting the rabies virus antibody provided by the invention can not detect the antibody of the virus related to or similar to the rabies virus, and has good specificity.
Table 5: specificity detection results
Note that: a test value <0.5U/ml was judged as negative.
3.2 comparing the quantitative detection result of the test card with the detection result of the neutralizing antibody
The total of 20 dogs and cats were collected from the Beijing animal epidemic prevention control center, and about 1ml each. Each was divided into 2 parts, each of which was 0.5ml. One of them was sent to OIE rabies reference laboratory (military medical institute of veterinary research) for detection of neutralizing antibodies; the other test was performed according to the "use of colloidal gold test card for 2.8 rabies virus antibody detection" procedure in example 2. The results of the two detection methods are shown in Table 6 below.
As can be seen from the results shown in Table 6, the same serum sample was tested using a colloidal gold test card made of the G (C) protein prepared in example 1, and the consistency with the test results of the neutralizing antibodies was very high (y= 1.0632x-0.4002, R 2 = 0.9871), the colloidal gold test card provided by the invention can replace a neutralizing antibody detection method to a certain extent to detect rabies virus antibodies, and is more convenient to use and higher in detection efficiency compared with the neutralizing antibody detection method.
Table 6: comparison of results of two detection methods of 20 parts of dog/cat serum
3.3 colloidal gold test strip for detecting rabies virus antibody, test card and appearance character of kit
Test strip: preserving at 2-30deg.C for 18 months or longer;
test card: the aluminum foil bag should be sealed well, has smooth surface and no crack, and is internally provided with a bag of drying agent; the plastic shell of the test card should not be damaged, the surface is smooth, the sample hole and the detection window should be clean, no foreign matters exist, the detection window should be moderate in position, and the C, T mark is clear;
the kit comprises: the label and the mark are complete, and the label should be clear with the information such as product name, batch number, validity period, two-dimensional code and the like.
Example 4: clinical serum sample detection
This example uses the colloidal gold test card for rabies virus antibody detection prepared in example 2 to detect serum samples from 3 dogs and 2 cats 30 days after immunization with rabies virus vaccine. Wherein the serum sample is obtained by immunizing 3 dogs and 2 cats with rabies virus vaccine at Beijing animal epidemic disease prevention control center, and collecting whole blood after 30 days of immunization to separate serum. The collected serum samples were then tested according to example 2, "use of colloidal gold test card for 2.8 rabies virus antibody detection". The detection results are shown in the following table 7 and fig. 7.
As can be seen from the detection results shown in the following Table 7 and FIG. 7, the results of detecting the serum samples by using the colloidal gold test card for detecting rabies virus antibodies provided by the invention are positive, namely, the rabies virus antibody titer is more than 0.5U/ml, which indicates that the colloidal gold test strip for detecting rabies virus antibodies in clinical serum (such as canine serum or feline serum) can be used for detecting rabies virus antibodies.
Table 7: detection results of clinical serum samples
| Clinical serum sample numbering | Detection result (U/ml) | |
| Canine | ||
| 1 | 12.50 | Positive and |
| Dog | ||
| 2 | 10.13 | Positive and |
| Canine | ||
| 3 | 16.2 | Positive and |
| Cat | ||
| 1 | 1.26 | Positive and |
| Cat | ||
| 2 | 14.42 | Positive and negative |
Example 5: comparative analysis of the present method (colloidal gold chromatography detection method) and neutralizing antibody method
According to the results shown in 3.2 and 4 in the embodiment 3, the immunochromatography quantitative analyzer matched with the colloidal gold test card provided by the invention can quantitatively detect the rabies virus antibody titer in serum, and the colloidal gold test card provided by the invention is adopted for detecting the same serum sample, has high consistency with the result obtained by the neutralizing antibody detection method, and has very simple operation when detecting the rabies virus antibody titer in serum compared with the neutralizing antibody detection method (such as FAVN, RFFIT and the like, which has slightly complicated operation and needs professional personnel to be completed in a professional laboratory), so that the colloidal gold test card provided by the invention is more suitable for rapid detection on a base layer or site, and is beneficial to popularization. The differences between the two types of methods are specifically shown in Table 8 below.
Table 8: the method (colloidal gold chromatography detection method) is compared with the neutralizing antibody detection method
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A mutant of rabies virus G protein is characterized in that the 313 th amino acid of the rabies virus G protein shown in SEQ ID NO. 1 is mutated from lysine to arginine, the 409 th amino acid is mutated from serine to cysteine, and the mutant is named as a first mutant, and the amino acid sequence of the first mutant is shown in SEQ ID NO. 2.
2. A mutant of rabies virus G protein is characterized in that a signal peptide of a first mutant shown in SEQ ID NO. 2 is replaced by a signal peptide shown in SEQ ID NO. 4, the mutant is named as a second mutant, and the amino acid sequence of the second mutant is shown in SEQ ID NO. 5.
3. The mutant of rabies virus G protein according to claim 1 or 2, which further carries a tag for protein purification.
4. A mutant rabies virus G protein according to claim 3, wherein the tag comprises a His tag.
5. A gene encoding the mutant of rabies virus G protein of any one of claims 1-4.
6. Use of a mutant of rabies virus G protein of any one of claims 1-4 in the preparation of a kit, reagent or colloidal gold test strip for detecting rabies virus antibodies.
7. A colloidal gold test strip for detecting rabies virus antibodies, wherein a gold pad of the colloidal gold test strip is adsorbed with a mutant of rabies virus G protein according to any one of claims 1 to 4 labeled with colloidal gold, and a detection line of the colloidal gold test strip is coated with the mutant of rabies virus G protein according to any one of claims 1 to 4.
8. The colloidal gold test strip according to claim 7, wherein the polyclonal antibody of rabies virus G protein is coated on the quality control line of the colloidal gold test strip.
9. The colloidal gold test strip according to claim 8, wherein the concentration of the mutant of the colloidal gold-labeled rabies G protein for preparing the gold pad, the concentration of the mutant of the rabies G protein for coating the detection line, and the concentration of the polyclonal antibody of the rabies G protein for coating the quality control line are respectively 0.5-2.0mg/ml.
10. A method for detecting a rabies virus antibody for non-disease diagnostic purposes comprising the steps of:
1) Dropping a serum sample to be tested onto a sample pad of the colloidal gold test strip according to any one of claims 7 to 9, and reacting for 15 to 25 minutes; and
2) And (3) detecting the colloidal gold test strip after the reaction in the step (1) by using an immunochromatography quantitative analyzer, and giving out the titer level of the rabies virus antibody in the serum sample to be detected.
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| CN113252893A (en) * | 2021-06-23 | 2021-08-13 | 北京市动物疫病预防控制中心 | Method for rapidly and quantitatively detecting rabies virus antibody by applying rabies virus G protein, encoding gene of G protein and test paper |
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