CN116375889A - Rabies virus glycoprotein antigen, truncated body and application thereof - Google Patents
Rabies virus glycoprotein antigen, truncated body and application thereof Download PDFInfo
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Abstract
The invention discloses a rabies virus glycoprotein antigen, a truncated body and application thereof. The protein shown in the following M1): m1) the rabies virus glycoprotein is formed by connecting 20 th to 69 th amino acid residues from the N end and 210 th to 415 th amino acid residues from the N end, and the amino acid sequence of the rabies virus glycoprotein is shown as SEQ ID No. 1. According to the three-dimensional structure of the existing protein, the structure of rabies virus glycoprotein and antibody is analyzed, an antigen fragment with better stability and direct interaction with the antibody is selected, eukaryotic insect expression system is used for expression, the protein can be well combined with the antibody through immunological experiment verification, and the truncated protein antigen has strong affinity with the antibody and can be used for preparing a high-sensitivity diagnostic kit.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rabies virus glycoprotein antigen, a truncated body and application thereof.
Background
Rabies is an acute infectious disease of human and livestock caused by Rabies virus (Rabies virus, RV), and belongs to the class B infectious disease and the class II animal epidemic disease. Rabies virus is a single-stranded RNA virus, belongs to the genus rabies virus of the Rhabdoviridae, has bullet-shaped appearance, has spiral symmetry nucleocapsids and has envelope on the surfaces. Rabies virus is widely spread in wild mammals, carnivorous mammals are their natural hosts, and pet dogs and cats are the main infectious sources for human beings and domestic animals to develop rabies.
Rabies virus mainly attacks the nervous systems of humans and animals, and after the onset of disease, an effective treatment method is lacking, the death rate is close to 100%, and the current effective prevention and treatment means mainly comprise rabies vaccine inoculation and rabies virus serum injection. Developed countries have fewer cases of infection of humans by pets due to forced immunization of pets, and are mainly concerned with infection of wild animals.
The rabies vaccine is an effective method for protecting pets and humans and preventing rabies. Rabies virus vaccines are commonly used as antigens by inactivating/attenuating viruses or pseudoviruses. The level of protein antibodies in the blood of the pets is detected after immunization, and the resistance of the animals to virus strains can be determined. Daily monitoring of rabies virus antibody levels in pets is an important means for selecting vaccines, assessing the reasonability of immunization programs, grasping health status, and is also a main basis for timely vaccine injection. At present, diagnostic reagents aiming at rabies virus antibody level are uneven, and a rapid, sensitive, high-specificity, simple to operate, economical and practical diagnostic method is imperative to be established.
Major antigens of rabies virus include glycoprotein (G) antigen on the outer membrane of the virus and nucleoprotein (N) antigen on the inner layer. Glycoprotein G can bind to acetylcholine receptor in human and animal nervous system to make virus have neurotoxicity, and can generate neutralizing antibody and hemagglutination inhibiting antibody in vivo, and the neutralizing antibody has protective effect. Nucleoprotein N can produce complement-binding antibodies and precipitants in vivo, but has no protective effect. Thus, in selecting antigen proteins for detection antibodies, existing studies and patents have commonly used viral particles, glycoprotein G alone or glycoprotein G and nucleoprotein N in tandem. Glycoprotein G is typically selected for use as a truncated protein because of the transmembrane region at the C-terminus, which is costly to express and purify.
The development of structural biology has enabled scientists to more intuitively observe the interaction of viral antigen proteins with antibodies. At present, the structures of the rabies virus glycoprotein G and the nucleoprotein N are published, and the teams in China, the United states and the United kingdom respectively publish the complex structures of the rabies virus glycoprotein G and a monoclonal antibody light chain and heavy chain variable region, thereby providing important support for deeply understanding the combination of the glycoprotein G and the monoclonal antibody. As mentioned in the world animal health Organization (OIE), virus neutralization assay (VN) and enzyme linked immunosorbent assay (ELISA) are recommended methods for rabies monitoring antibody responses in vaccinated animals. The VN method needs to use living rabies virus and Vero cells, has good sensibility and specificity, but because the rabies virus is a highly pathogenic microorganism, the laboratory operation risk is high, and the VN method is often used as a verification test and is not suitable for clinical large-scale detection.
In summary, the existing detection method has various defects, and is urgent to screen out proper rabies virus antigens and antibodies by means of immunological principles, genetic engineering technology and the like, so that ELISA coated plates can be adopted as carriers for reaction, and the detection method is rapid, simple and convenient, can be used for batch detection and is more suitable for clinical detection.
Disclosure of Invention
Therefore, the embodiment of the invention provides rabies virus glycoprotein antigen, a truncated body and application thereof.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
the present invention provides a protein represented by the following M1): m1) connecting 20 th to 69 th amino acid residues from the N end of rabies virus glycoprotein and 210 th to 415 th amino acid residues from the N end of rabies virus glycoprotein;
the amino acid sequence of the rabies virus glycoprotein is shown as SEQ ID No. 1.
The invention also provides a protein or a truncated body, wherein the truncated body is shown in any one of the following (1) - (4):
(1) The amino acid sequence is a protein shown as SEQ ID No. 2;
(2) A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 2;
(3) A protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2;
(4) A protein which has more than 95% of identity with the amino acid sequence shown in SEQ ID No.2 and has the same function.
The invention also provides a coding gene of the protein or the truncated body thereof.
In one embodiment of the present invention, the above protein or truncated form of the coding gene is as shown in any one of the following 1) to 3):
1) A DNA molecule shown in SEQ ID No. 4;
2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in 1) and which codes for the protein described in M1) above or for the truncations described above;
3) A DNA molecule which has more than 95% identity with the DNA molecule defined in 1) or 2) and which encodes the protein described in M1) above or the truncations described above.
The present invention also provides any one of the following biological materials (a 1) to (a 4):
(a1) An expression cassette containing the above-mentioned coding gene;
(a2) Recombinant vectors containing the above-mentioned coding genes;
(a3) Recombinant bacteria containing the above-mentioned coding genes;
(a4) Transgenic cell lines containing the coding genes described above.
The invention also provides application of the protein or the truncated body thereof and/or the coding gene and/or the biological material in preparation of immunogen and/or antigen, or the immunogen or antigen is directed against rabies virus or rabies virus glycoprotein.
The invention also provides application of the protein and/or the protein or the truncated body thereof and/or the coding gene and/or the biological material as antigen in preparing antibodies against rabies virus;
or, the application of the protein or the truncated body thereof and/or the coding gene and/or the biological material in preparing products for preventing and/or treating diseases caused by rabies viruses;
or, the protein or the truncated body thereof and/or the coding gene and/or the biological material are applied to preventing and/or treating diseases caused by rabies virus.
The invention also provides the application of the protein or the truncated body thereof and/or the coding gene and/or the biological material in any one of the following (b 1) - (b 6):
(b1) Inhibiting viral infection;
(b2) Products that inhibit viral infection;
(b3) Inhibiting virus invasion;
(b4) A product that inhibits viral entry;
(b5) Detecting a viral antibody;
(b6) Detecting a product of a viral antibody;
the virus is rabies virus.
The invention also provides a product, the active ingredient of which is the protein or the truncations thereof;
the functions of the product are as follows (c 1) - (c 3):
(c1) Drugs or vaccines that inhibit viral infection;
(c2) Drugs or vaccines that inhibit viral entry;
(c3) Reagents for detecting viral antibodies;
the virus is rabies virus.
The invention finally provides a polyclonal antibody which is prepared by taking the protein or the truncated body thereof as an immunogen.
The embodiment of the invention has the following advantages:
according to the protein three-dimensional structure of the existing rabies virus glycoprotein, the structure of the rabies virus glycoprotein and the antibody is analyzed, an antigen fragment with better stability and direct interaction with the antibody is selected, and a eukaryotic insect expression system is used for expression, so that the rabies virus glycoprotein truncated protein is obtained. Through immunological experiments, the truncated protein antigen can be well combined with an antibody, has strong affinity with the antibody, and can be used for preparing a high-sensitivity diagnostic kit and establishing a high-sensitivity diagnostic method. Compared with the existing common antigen, the truncated antigen has better stability, higher expression quantity, lower cost and better specificity and sensitivity of detecting antibody level. The prepared Elisa antibody detection kit and antibody detection test strip have good effect, lower cost and strong specificity and stability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.
FIG. 1 shows the result of protein sequence alignment of rabies virus glycoprotein G provided by the example of the present invention;
FIG. 2 shows the structure of a rabies virus glycoprotein G trimer binding antibody (PDB number 7U 9G) provided by the example of the present invention;
FIG. 3 shows the structure of an angled rabies virus glycoprotein G monomer binding antibody (PDB number 7U 9G) provided by the examples of the present invention;
FIG. 4 shows the structure of a rabies virus glycoprotein G monomer binding antibody (PDB number 7U 9G) at another angle provided in the inventive examples;
FIG. 5 is a diagram showing the result of electrophoresis after expression and purification of a rabies virus glycoprotein G truncate provided by the embodiment of the invention;
FIG. 6 shows the result of specific serum detection of the test strip prepared in the example of the present invention;
FIG. 7 shows the result of sensitive serum detection of the test strip prepared in the example of the present invention.
In the figure: 11-a second variable region structure; 12-a first domain; 13-a second domain; 14-first variable region structure.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, 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.
In the invention, pFast-Bac1 is purchased from Thermo company, and pFastBac-6p-1 is obtained after the tag is added by artificial transformation; DH10Bac engineering bacteria are purchased from Beijing qingke biotechnology Co., ltd; sf9 cells were purchased from Thermo company; synthetic truncate gene sequences company: wuhan Jin Kairui Bio-engineering Co., ltd.
Example 1, rabies virus glycoprotein G sequence alignment and structural analysis
The sequence of rabies virus glycoprotein G refers to the NP-056796.1 amino acid sequence of NCBI of the American biotechnology information center, and total 524 amino acid residues comprise 1-19 amino acid residues of a signal peptide and 459-476 amino acid residues of a transmembrane region, and the full-length corresponding nucleotide sequence is NC-001542.1 and 1575bp. The result of comparing this protein sequence with the data in the protein structure database PDB in NCBI Blast function is shown in FIG. 1, there are now a number of structures of rabies virus glycoprotein G which have been resolved, of which 4 have very high similarity to the full length of the sequence, near or about 90%, of which the 7U9G structural A chain is more 100% identical to the sequence, covering 439 of the 524 amino acid residues, which is the three-dimensional structure of the protein of the sequence.
The published trimeric structure of rabies virus glycoprotein G is shown in fig. 2, where the trimers of rabies virus glycoprotein G share 3 glycoprotein G polypeptide chains and 6 antibody light/heavy polypeptide chains. One glycoprotein G monomer was selected for analysis, and the different angles are shown in fig. 3 and 4, wherein the first domain 12 and the second domain 13 are two domains of glycoprotein G monomer, the first variable region structure 14 is an antibody light chain variable region structure, and the second variable region structure 11 is an antibody heavy chain variable region structure.
As can be seen from FIGS. 3 and 4, the first domains 12 of rabies virus glycoprotein G are 20-69aa and 210-415aa, which can directly interact with the light chain and the heavy chain of the antibody, and the structure comprises a direct interaction surface for binding the antibody, and the amino acid residues at positions 69 and 210 are very close. The second domain 13 is 70-209aa, which is a relatively large distance from the antibody, and is presumably related to stable trimer structure, as analyzed in FIG. 2.
In addition to the 7U9G structure, other rabies virus glycoprotein G complex structures with antibodies were analyzed, in the 8A1E structure, the first domain that directly interacted with the antibody comprised 20-69aa and 209-418aa; in the 6LGW structure, the first domain that binds directly to the antibody comprises 20-50aa and 281-414aa, the antibody-binding domains are essentially identical compared to the complex structure of the three rabies virus glycoproteins G.
By complex structural analysis of three rabies virus glycoproteins G, the present invention selects for the use of flexible random coil sequencesGGGGSGGGGSThe rabies virus glycoprotein G is connected with the amino acid residues from 20 to 69 of the N end and the amino acid residues from 210 to 415 of the N end to be used as a new antigen for expression and purification.
Rabies virus glycoprotein G full-length amino acid sequence (SEQ ID No. 1):
MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCWRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSGGETGL。
rabies virus glycoprotein G full length nucleotide sequence (SEQ ID No. 3):
ATGGTTCCGCAGGCCCTGCTGTTTGTTCCGCTGCTGGTGTTTCCGCTGTGTTTTGGTAAATTTCCGATTTATACCATCCCGGATAAACTGGGCCCGTGGAGCCCGATTGATATTCATCATCTGAGTTGTCCGAATAATCTGGTTGTGGAAGATGAAGGCTGCACCAATCTGAGTGGTTTTAGTTATATGGAACTGAAAGTTGGCTATATTAGTGCAATTAAAATGAACGGCTTCACCTGTACCGGCGTGGTGACCGAAGCCGAAACCTATACCAATTTTGTTGGTTATGTGACCACCACCTTTAAACGCAAACATTTTCGTCCGACCCCGGATGCATGTCGTGCAGCCTATAATTGGAAAATGGCAGGCGATCCGCGCTATGAAGAAAGCCTGCATAATCCGTATCCGGATTATCATTGGCTGCGTACCGTGAAAACCACCAAAGAAAGTCTGGTTATTATTAGCCCGAGTGTTGCCGATCTGGACCCTTATGATCGTAGCCTGCATAGTCGCGTGTTTCCGGGCGGTAATTGTAGCGGTGTGGCCGTTAGCAGCACCTATTGCAGCACCAATCATGATTATACCATTTGGATGCCGGAAAATCCGCGCCTGGGCATGAGTTGTGATATTTTTACCAATAGTCGCGGTAAACGTGCCAGCAAAGGCAGTGAAACCTGCGGTTTTGTGGATGAACGTGGTCTGTATAAAAGCCTGAAAGGCGCCTGTAAACTGAAACTGTGTGGTGTGCTGGGTCTGCGTCTGATGGATGGCACCTGGGTGGCCATGCAGACCAGTAATGAAACCAAATGGTGTCCGCCGGGCCAGCTGGTGAATCTGCATGATTTTCGCAGCGATGAAATTGAACATCTGGTTGTGGAGGAACTGGTGAAAAAACGCGAAGAATGTCTGGATGCACTGGAAAGCATTATGACCACCAAAAGCGTTAGTTTTCGTCGTCTGAGCCATCTGCGCAAACTGGTGCCGGGCTTTGGTAAAGCCTATACCATTTTTAATAAGACCCTGATGGAAGCAGATGCACATTATAAAAGCGTTCGCACCTGGAATGAAATTATTCCGAGCAAAGGCTGCCTGCGCGTGGGTGGTCGTTGTCATCCGCATGTGAATGGTGTGTTTTTTAATGGCATTATTCTGGGTCCGGATGGCAATGTTCTGATTCCGGAAATGCAGAGTAGTCTGCTGCAGCAGCATATGGAACTGCTGGTTAGTAGTGTGATTCCGCTGATGCATCCGCTGGCCGATCCGAGCACCGTTTTTAAAAATGGTGATGAAGCAGAAGATTTCGTGGAAGTTCATCTGCCGGATGTTCATGAACGCATTAGCGGTGTGGATCTGGGTCTGCCGAATTGGGGTAAATATGTGCTGCTGAGCGCAGGTGCACTGACCGCACTGATGCTGATTATTTTTCTGATGACCTGCTGGCGCCGTGTGAATCGTAGCGAACCGACCCAGCATAATCTGCGCGGTACCGGCCGCGAAGTTAGTGTTACCCCGCAGAGCGGCAAAATTATTAGTAGCTGGGAAAGTTATAAGAGCGGTGGTGAAACCGGTCTGTAA。
rabies virus glycoprotein G20-69aa+210-415 aa truncate amino acid sequence (underlined is the random coil of the ligation) (SEQ ID No. 2):
KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYGGGGSGGGGSIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLM。
nucleotide sequence encoding rabies virus glycoprotein G20-69aa+210-415 aa truncate protein (SEQ ID No. 4): (underlined is the connected random coil):
AAATTTCCGATTTATACCATCCCGGATAAACTGGGCCCGTGGAGCCCGATTGATATTCATCATCTGAGTTGTCCGAATAATCTGGTTGTGGAAGATGAAGGCTGCACCAATCTGAGTGGTTTTAGTTATATGGAACTGAAAGTTGGCTATGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCATTTTTACCAATAGTCGCGGTAAACGTGCCAGCAAAGGCAGTGAAACCTGCGGTTTTGTGGATGAACGTGGTCTGTATAAAAGCCTGAAAGGCGCCTGTAAACTGAAACTGTGTGGTGTGCTGGGTCTGCGTCTGATGGATGGCACCTGGGTGGCCATGCAGACCAGTAATGAAACCAAATGGTGTCCGCCGGGCCAGCTGGTGAATCTGCATGATTTTCGCAGCGATGAAATTGAACATCTGGTTGTGGAGGAACTGGTGAAAAAACGCGAAGAATGTCTGGATGCACTGGAAAGCATTATGACCACCAAAAGCGTTAGTTTTCGTCGTCTGAGCCATCTGCGCAAACTGGTGCCGGGCTTTGGTAAAGCCTATACCATTTTTAATAAGACCCTGATGGAAGCAGATGCACATTATAAAAGCGTTCGCACCTGGAATGAAATTATTCCGAGCAAAGGCTGCCTGCGCGTGGGTGGTCGTTGTCATCCGCATGTGAATGGTGTGTTTTTTAATGGCATTATTCTGGGTCCGGATGGCAATGTTCTGATTCCGGAAATGCAGAGTAGTCTGCTGCAGCAGCATATGGAACTGCTGGTTAGTAGTGTGATTCCGCTGATG。
EXAMPLE 2 expression, purification and antigen Activity verification of rabies Virus glycoprotein G truncate protein
According to the results of the structure prediction of example 1, amino acid residues 20 to 69 and amino acid residues 210 to 415 of rabies virus glycoprotein G were selected by flexible random coilGGGGSGGGGSAnd (5) carrying out expression purification on the truncated protein formed by connection.
The gene sequence corresponding to the truncated body is synthesized in a gene company, the gene sequence is constructed on a pFastBac-6p-1 modified vector containing a GST tag by a molecular cloning method, the vector is transformed into DH10Bac engineering bacteria after being identified correctly, blue white spot screening is carried out, white spot identification is selected, plasmid extraction is carried out, and the Bacmid of the stem of the recombinant insertion target fragment is obtained and is transfected into insect Sf9 cells. Lesions were observed after 72 hours, first generation virus P1 was collected, sf9 cells continued to be infected, and P2 and P3 generation viruses were collected. The P3 generation virus is used to infect normal Sf9 insect cells, and the expression of the target protein rabies virus glycoprotein G truncate protein is carried out. After the cells were collected by centrifugation, the target protein was purified by GST affinity chromatography, and then the target protein was eluted after cleavage of the tag by adding PreScission Protease protease to the column.
Insect cells infected with viruses for 72 hours were collected, centrifuged at 2000rpm for 10 minutes, suspended in buffer A (50 mM Tris, pH8.0, 150mM NaCl, 1mM DTT), placed in a 50ml glass beaker in an ice bath, and disrupted using an ultrasonic cell disruptor.
The disruption of insect cells was selected at 250W power, 3s of ultrasound per cycle, 9s of stop, total time per round for 3min, 2 rounds total. Centrifuging the cracked suspension at a low temperature and high speed centrifuge at 12000rpm for 20min at 4deg.C, and continuing the subsequent purification experiment of the supernatant.
And (3) under the low temperature condition of 0-4 ℃, allowing the supernatant after centrifugation to flow through a GST affinity chromatography column, and repeating the combination for 3-4 times to enable the target protein to be fully combined on the affinity chromatography column. The impurity protein was washed off with 50ml of buffer A, and the mixture was digested with PreScission protease (sigma) at 4℃for 12 hours. Finally, the GST-tagged protein of interest is eluted with buffer A. The purification results were examined using polyacrylamide gel electrophoresis (SDS-PAGE). The result of identifying by SDS-PAGE shows that the expressed rabies virus glycoprotein G truncated protein is shown in figure 5, and the target protein band is near the 25kDa position, so that the purity is higher, and the target protein rabies virus glycoprotein G truncated protein is obtained.
The purified rabies glycoprotein G truncate protein was used to verify antigen activity using the Elisa method. Coating purified rabies virus glycoprotein G truncated protein 1 mu G/ml,100 mu l/hole, coating overnight at 4 ℃, blocking 0.1% gelatin for 2h at 37 ℃, washing the plate 3 times with 250 mu l/hole of PBST washing liquid (0.1%), adding 100 mu l/hole of diluted clinical rabies virus positive serum, incubating 1 h at 37 ℃ (positive control group is rabies virus positive serum, negative control group is rabies virus negative serum, three replicates are performed in each group), washing the plate 3 times with 250 mu l/hole of PBST washing liquid (0.1%), adding 100 mu l/hole of HRP-labeled rabbit anti-canine secondary antibody (diluted with PBS according to 1:10000) after beating, reacting for 30 min at 37 ℃, washing the plate 3 times again, adding TMB color developing liquid (commercialized) 100 mu l/hole after beating, developing at room temperature for 10min, finally adding 0.5M sulfuric acid, 50 mu l/hole, terminating the reaction, and measuring OD by an enzyme marker 450nm Values. The results are shown in Table 1, and the rabies virus glycoprotein G truncated protein can be specially generated with clinical serum, positive control and negative controlThe specific reaction has better antigen activity.
TABLE 1 results of antigen Activity verification of rabies Virus glycoprotein G truncate protein
| Positive control | Negative control | Clinical serum |
| 1.623 | 0.055 | 1.522 |
| 1.705 | 0.063 | 1.476 |
| 1.562 | 0.058 | 1.604 |
Example 3 Elisa antibody detection kit of rabies virus glycoprotein G truncated protein
1. Preparation and detection steps of an Elisa antibody detection kit with rabies virus glycoprotein G truncated protein as an antigen coating:
an indirect Elisa antibody detection kit prepared by taking rabies virus glycoprotein G truncated protein prepared in the embodiment 2 as an antigen is used for detecting the ELISA detection kit of rabies virus antibody level in dog serum, wherein the concentration of the rabies virus glycoprotein G truncated protein coated and purified in the detection kit is 1 mug/ml.
The rabies virus glycoprotein G short protein antigen coating plate is taken out from the detection kit, the serum to be detected is diluted 100 times by the sample diluent on the serum dilution plate (198 mu l of sample diluent is mixed with 2 mu l of serum to be detected), 100 mu l of mixed solution is taken and added into the ELISA plate, and meanwhile, 2 holes are respectively added to positive serum and negative serum, and 100 mu l of each hole is used.
The samples in each well were gently shaken, covered with a plate cover, and incubated at 37℃for 30 minutes.
The solution in the wells was discarded, 250 μl of working wash solution was added to each well, repeated 3 times, and the solution was thoroughly dried after the final plate wash.
Mu.l of enzyme-labeled antibody was added to each well, the coated plate was covered with a sealing plate membrane, and incubated at 37℃for 30 minutes.
The solution in the wells was discarded, 250 μl of working wash solution was added to each well, repeated 3 times, and the solution was thoroughly dried after the final plate wash.
Mu.l of substrate solution was added to each well, the coated plate was covered with a sealing plate membrane and incubated at room temperature for 10 minutes.
The reaction was stopped by adding 50. Mu.l of stop solution to each well.
Determination of the sample and absorbance (OD) against 450 nm wavelength 450nm ). Positive control OD 450nm The value is more than or equal to 0.6, negative control OD 450nm The value is less than or equal to 0.2, and the test is true.
Interpretation criteria: S/P= (sample to be tested OD) 450nm Mean-negative control OD 450nm Mean value)/(positive control OD 450nm Mean-negative control OD 450nm Mean value). S/P is more than or equal to 0.3, the sample is positive, and the serum antibody of the sample to be detected reaches the protection level; S/P is less than 0.3, the sample is negative, and the serum antibody of the sample to be detected does not reach the protection level.
2. Elisa antibody detection kit specificity test
And detecting the canine parvovirus CPV, the canine distemper virus CDV, the canine infectious hepatitis virus CAV I standard antibody positive serum and the rabies virus RABV antibody positive serum by using the prepared kit. The detection results are shown in the following table 2, and the S/P value of the serum is less than 0.3 except that the S/P value of the rabies virus standard antibody positive serum is obviously more than 0.3, so that the detection kit meets the judgment standard of negative serum, and has good specificity.
TABLE 2 specific serum assay results
3. Sensitivity test of Elisa antibody detection kit
The rabies virus standard antibody serum (4.0 IU/ml) is respectively diluted to 2.0, 1.5, 1.0, 0.8, 0.5, 0.3 and 0.1 IU/ml, and the detection is simultaneously carried out by using the detection kit for the Elisa antibody prepared by the invention and the detection kit for the outsourced rabies virus Elisa antibody. The detection results are shown in Table 3, and the detection kit of the Elisa antibody can detect 0.5 IU/ml of rabies standard antibody positive serum, and the outsourcing kit can also detect 0.5 IU/ml of rabies virus standard antibody positive serum, so that the detection method of the rabies virus Elisa antibody has good sensibility.
TABLE 3 sensitive serum assay results
Elisa antibody detection kit coincidence rate test
By using the Elisa antibody detection kit and the outsourcing rabies virus Elisa antibody detection kit of the invention to detect 35 canine serum samples simultaneously, the results are compared, the coincidence rate of detection of the Elisa antibody detection kit and the outsourcing kit sample of the invention is 100%, the detection kit and the outsourcing kit have good adaptability, and the specific detection results are shown in tables 4 and 5.
TABLE 4 sample compliance comparison of Elisa antibody detection kit of the present invention
TABLE 5 sample compliance rates of Elisa antibody detection kit and outsourcing kit of the present invention
Example 4 colloidal gold antibody test strip coated with rabies virus glycoprotein G truncated protein
1. The colloidal gold antibody detection test strip prepared by taking the rabies virus glycoprotein G truncated protein prepared in the embodiment 2 as an antigen is a test strip for detecting the rabies virus antibody level in the canine serum or the canine plasma by adopting a double-antigen sandwich method based on an immunochromatography platform, and the detection process by using the test strip comprises the following steps of:
and taking out the rabies virus colloidal gold antibody detection test strip from the kit, and recovering to room temperature.
And adding 10 mu l of canine serum or plasma into the sample diluent, and shaking and mixing uniformly to obtain the liquid to be tested.
And (5) adding 80 μl of the liquid to be tested into the sample hole of the test strip, reacting for 15 minutes, and judging the result by naked eyes.
Determination criteria: the color development of the line C shows that the test strip has effective result and the test is established; developing by a C line and a T line, and indicating that rabies virus antibodies in a sample to be tested reach a protection level; the color of the C line and the T line are not developed, which indicates that the rabies virus antibody in the sample to be tested does not reach the protection level, and the immunity is suggested to be enhanced.
2. Specificity test
And detecting canine parvovirus CPV, canine distemper virus CDV, canine infectious hepatitis virus CAV I, canine coronavirus CCV standard antibody positive serum and rabies virus RABV antibody positive serum by using a rabies virus colloidal gold antibody detection test strip. The results are shown in the following figures, except that the T line of the rabies virus standard antibody positive serum is colored, all the T lines of the rest serum are not colored, and the judgment standard of negative serum is met, so that the specificity of the method is good, as shown in figure 6.
3. Sensitivity test
The standard antibody serum (4.0 IU/ml) of rabies virus is respectively diluted to 2.0, 1.5, 1.0, 0.8, 0.5, 0.3 and 0.1 IU/ml, and the detection is simultaneously carried out by using the research method and the outsourced rabies virus Elisa antibody detection kit. The results are shown in the following table, the method established in the embodiment can detect 0.5 IU/ml rabies virus antibody positive serum, and the outsourcing kit can also detect 0.5 IU/ml rabies virus antibody positive serum, which indicates that the colloidal gold antibody detection test strip established in the embodiment has good sensitivity, as shown in fig. 7.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The protein shown in the following M1):
m1) connecting 20 th to 69 th amino acid residues from the N end of rabies virus glycoprotein and 210 th to 415 th amino acid residues from the N end of rabies virus glycoprotein;
the amino acid sequence of the rabies virus glycoprotein is shown as SEQ ID No. 1.
2. A protein or a truncate, said truncate being as set forth in any one of (1) - (4) below:
(1) The amino acid sequence is a protein shown as SEQ ID No. 2;
(2) A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 2;
(3) A protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2;
(4) A protein which has more than 95% of identity with the amino acid sequence shown in SEQ ID No.2 and has the same function.
3. A gene encoding the protein of claim 1 or the protein of claim 2 or a truncate thereof.
4. The coding gene of claim 3, wherein the gene is,
the coding gene of the protein of M1) or the protein truncated body of claim 2 in claim 1 is as shown in any one of the following 1) to 3):
1) A DNA molecule shown in SEQ ID No. 4;
2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in 1) and which encodes a protein according to M1) of claim 1 or a truncate according to claim 2;
3) A DNA molecule having more than 95% identity to the DNA molecule defined in 1) or 2) and encoding a protein according to M1) of claim 1 or a truncating of claim 2.
5. A biological material of any one of the following (a 1) to (a 4):
(a1) An expression cassette comprising the coding gene of claim 3 or 4;
(a2) A recombinant vector comprising the coding gene of claim 3 or 4;
(a3) A recombinant bacterium comprising the coding gene of claim 3 or 4;
(a4) A transgenic cell line comprising the coding gene of claim 3 or 4.
6. Use of a protein according to claim 1 and/or a protein according to claim 2 or a truncate thereof and/or a coding gene according to claim 3 or 4 and/or a biomaterial according to claim 5 for the preparation of an immunogen and/or an antigen or which is directed against rabies virus or rabies virus glycoprotein.
7. Use of a protein according to claim 1 and/or a protein according to claim 2 or a truncate thereof and/or a coding gene according to claim 3 or 4 and/or a biomaterial according to claim 5 as an antigen for the preparation of antibodies against rabies virus;
or, the use of a protein according to claim 1 and/or a protein according to claim 2 or a truncate thereof and/or a coding gene according to claim 3 or 4 and/or a biological material according to claim 5 for the preparation of a product for the prevention and/or treatment of diseases caused by rabies virus;
or, the use of a protein according to claim 1 and/or a protein according to claim 2 or a truncate thereof and/or a coding gene according to claim 3 or 4 and/or a biological material according to claim 5 for the prophylaxis and/or treatment of diseases caused by rabies virus.
8. Use of a protein according to claim 1 and/or a protein according to claim 2 or a truncate thereof and/or a coding gene according to claim 3 or 4 and/or a biomaterial according to claim 5 in any one of the following (b 1) - (b 6):
(b1) Inhibiting viral infection;
(b2) Products that inhibit viral infection;
(b3) Inhibiting virus invasion;
(b4) A product that inhibits viral entry;
(b5) Detecting a viral antibody;
(b6) Detecting a product of a viral antibody;
the virus is rabies virus.
9. A product comprising as an active ingredient the protein of claim 1 or the protein of claim 2 or a truncate thereof;
the functions of the product are as follows (c 1) - (c 3):
(c1) Drugs or vaccines that inhibit viral infection;
(c2) Drugs or vaccines that inhibit viral entry;
(c3) Reagents for detecting viral antibodies;
the virus is rabies virus.
10. A polyclonal antibody prepared using the protein of claim 1 or the protein of claim 2 or a truncated form thereof as an immunogen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116854784A (en) * | 2023-09-05 | 2023-10-10 | 北京纳百生物科技有限公司 | Canine coronavirus N protein antigen truncate and application thereof |
| CN116854784B (en) * | 2023-09-05 | 2023-11-17 | 北京纳百生物科技有限公司 | Canine coronavirus N protein antigen truncate and application thereof |
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