CN116854784A - Canine coronavirus N protein antigen truncate and application thereof - Google Patents
Canine coronavirus N protein antigen truncate and application thereof Download PDFInfo
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Abstract
The invention discloses a canine coronavirus N protein antigen truncated body and application thereof, wherein the amino acid sequence of the antigen truncated body is shown as SEQ ID No. 2. According to the invention, the structure of the canine coronavirus nucleocapsid protein N protein is analyzed according to the existing and predicted protein three-dimensional structure, fragments with better stability are selected, the fragments are expressed by using an escherichia coli prokaryotic expression system, the activity of the antigen protein is high through the verification of an immunological experiment, the antigen protein can be well combined with an antibody, the prepared ELISA antibody detection kit has the advantages of low production cost of antigen raw materials, good specificity and high sensitivity, and the kit can be applied to the canine coronavirus antibody detection in clinical canine serum samples.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a canine coronavirus N protein antigen truncated body and application thereof.
Background
Canine coronaviruses currently lack effective therapeutic agents, canine octa vaccines include coronavirus vaccines, but because of the high mutation rate of coronaviruses, there are multiple types of vaccines that are difficult to develop sufficient protection and are rarely used. By enhancing the feeding management, sanitary measures are strictly implemented to prevent diseases, and symptomatic treatment measures are adopted after the diseases occur, so that the occurrence of the diseases is reduced. The symptoms and epidemiology of canine coronavirus are similar to rotavirus infection, and often have mixed infection with rotavirus, canine parvovirus and the like, so that diagnosis is difficult, and a rapid, sensitive, high-specificity, simple-operation, economical and practical diagnosis method needs to be established.
The canine coronavirus mainly comprises nucleocapsid protein N, membrane protein M, spike protein S, small membrane protein E and the like. Nucleocapsid protein N is involved in viral nucleocapsid formation, mediating host cell immunity, and is relatively conserved. Membrane protein M plays a major role in the viral budding, assembly and maturation process. Spike protein S recognizes and interacts with host receptors, determines infectivity and host extent, stimulates the host to produce neutralizing antibodies, and is susceptible to mutation. The diagnosis and research of canine coronaviruses is currently mainly directed to the nucleocapsid protein N protein.
At present, the research on coronaviruses mainly focuses on viruses such as SARS, MERS and covd-19 related to human diseases, and the research on canine coronaviruses is relatively few. The canine coronavirus N protein sequence alignment shows that the 2-segment N protein structure sequence of the human coronavirus HCoV-NL63 in the published structure has the highest similarity, and the full-length N protein sequence of the COVID-19 is similar. The structure of the two coronavirus N proteins comprises an N-segment structural domain NTD and a C-end structural domain CTD, the full length of the N protein of HCoV-NL63 is not screened to obtain protein crystals, and the two structural domain fragments are respectively crystallized and structurally analyzed, so that the result can provide reference and support for the research of canine coronaviruses.
The serological detection method of the canine coronavirus mainly comprises an enzyme-linked immunosorbent assay and a colloidal gold immunochromatography. The ELISA has the advantages of being quick, simple, low in cost and capable of automatically detecting in batches. The colloidal gold immunochromatography is similar to the ELISA principle, can directly observe results by naked eyes, does not need an instrument, and is more suitable for detecting single samples. The establishment of both methods requires the provision of specific antigen proteins, and the diagnosis products with high sensitivity and strong specificity are obtained through process optimization.
Therefore, the invention provides a high-quality raw material for establishing a canine coronavirus antibody detection method by screening the canine coronavirus N protein antigen truncate with high activity.
Disclosure of Invention
Therefore, the embodiment of the invention provides a canine coronavirus N protein antigen truncated body and application thereof.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
the invention provides a canine coronavirus N protein antigen truncated body, and the amino acid sequence of the antigen truncated body is shown as SEQ ID No. 2.
The invention also provides a coding gene of the canine coronavirus N protein antigen truncated body, and the coding gene is a DNA molecule shown as SEQ ID No. 4.
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 the canine coronavirus N protein antigen truncated body and/or the antigen truncated body coding gene and/or the application of the biological material in antigen preparation.
The invention also provides a detection reagent for the canine coronavirus antibody, and the active ingredient of the detection reagent is the canine coronavirus N protein antigen truncated body.
Preferably, the detection reagent is an ELISA antibody detection reagent or a fluorescent microsphere antibody detection reagent or a colloidal gold antibody detection reagent.
The embodiment of the invention has the following advantages:
according to the invention, the structure of the canine coronavirus nucleocapsid protein N protein is analyzed according to the existing and predicted protein three-dimensional structure, fragments with better stability are selected, the fragments are expressed by using an escherichia coli prokaryotic expression system, the activity of the antigen protein is high through immunological experiments, the antigen protein can be well combined with an antibody, the prepared ELISA antibody detection kit has the advantages of low antigen raw material production cost, good detection effect, high specificity and stability, and the kit can be applied to canine coronavirus antibody detection in clinical canine serum samples.
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 N protein of canine coronavirus provided by the embodiment of the invention;
FIG. 2 shows the published N protein structure of coronaviruses according to an embodiment of the invention;
FIG. 3 shows the structure of the alpha fold predicted canine coronavirus N protein provided by the embodiment of the invention;
FIG. 4 is a diagram showing the result of electrophoresis after the expression and purification of the truncated NTD 27-157aa of the N protein of canine coronavirus provided by the embodiment of the invention.
In the figure: a is HCoV-NL63 NTD (5N 4K); b is HCoV-NL63 CTD (5 EPW); c is COVID-19N (8 FD 5).
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 present invention, the truncated gene sequence company is synthesized: wuhan Jin Kairui Bio-engineering Co., ltd; BL21 (DE 3) competent cells: purchased from beijing all gold biotechnology limited; pET-32a expression vector: purchased from beijing solebao technologies limited.
Example 1, canine coronavirus N protein sequence alignment and structural analysis
The sequence of the canine coronavirus nucleocapsid protein N protein refers to WDW25666.1 amino acid sequence of NCBI of the American biotechnology information center, and has 395 amino acid residues, no signal peptide and transmembrane region, and the full-length corresponding nucleotide sequence is OQ198019.1, 1188bp. The result of the alignment of this protein sequence in NCBI Blast function with the structural data in the protein structural database PDB is shown in FIG. 1. The first line covers the full length of the COVID-19 (SARS-CoV 2) N protein full length structure 8FD5, homology 28.65%. The two fragments of HCoV-NL63 with the highest homology are 38.82% of the homology of NTD domain 5N4K and 38.05% of CTD domain 5EPW, and have certain reference value.
The structure of HCoV-NL63 NTD (5N 4K) is shown in part A of FIG. 2, the middle is 3 antiparallel beta sheets, and irregular curly and spherical sulfate ions are distributed on two sides. The HCoV-NL63 CTD (5 EPW) structure is shown in part B of FIG. 2, with multiple alpha helices on one side and 2 longer beta sheets on the other side of the overall structure. The full length structure of covd-19N (8 FD 5) is shown in figure 2, part C, with the left half of the NTD domain being 2 β sheets wrapped around many random coils and the right CTD domain being predominantly an α -helix. The secondary structure distribution of the N proteins of HCoV-NL63 and COVID-19 has a certain similarity.
The method utilizes the free structure display software ChimeraX developed by UCSF of san Francisco division of California university to provide the alpha fold structure prediction function, inputs the 395 th amino acid residue sequence of the N protein of the canine coronavirus, uses the alpha fold software to calculate and predict the three-dimensional structure of the protein, and finally obtains the prediction structure shown in figure 3. The prediction structure is predicted and built based on the existing structure information, the left side NTD domain is formed by surrounding the random coil and small alpha helix by 3 beta sheets at the center, the right side CTD domain is formed by distributing two sides of a plurality of alpha helices and 2 beta sheets, and the two sides are similar to the secondary structure distribution of the published structure. In this structure, there is no stable interaction between the two domains of NTD and CTD, the relative position is not fixed, the overall length structure is presumed to be unstable, and the NTD and CTD alone have stable spatial structures.
By comparing the NTD and CTD truncations of resolved HCoV-NL63, we selected 27-157aa of the canine coronavirus N protein as N-terminal domain NTD and 245-346aa as C-terminal domain CTD for purification respectively for subsequent experimental verification.
The canine coronavirus N protein full-length amino acid sequence (SEQ ID No. 1):
MSSNVSWADQVDAANRRQRSRSRGRSQNRTNASIPLSWFTSIIDESNGNFTSLMPPSGVPTGMGTAAQQCGYWYRAPTFYQVRRGKRVPLPPVWYFYFLGTGPHSNAAYGTAMDGVFWVKTKNGQIDPKSIKALGVRDSGTDPRRANIPNLPEGLRVSVPNASRPQSRAQSQTRSQNNSRASSASRNGSRASSVDRTKEDLKAVVAQLLSEMGVNKAFKQNQTQSQSKKKQKGSTPAATPHPNQEGKPVWKKKPNKEETVTQCFGPRSDSKNFGDADLIRLGVDDPRFKTVSYYAPGASASLFDSMVTVTDGSDGKKRVTFHTTIEVDPTKPGFEVFMAQIDAFKKPATFQQTQNFWETQASTQNSITDYFRGTTPGAGGSAVEIETFEMTDETK。
canine coronavirus N protein full-length nucleotide sequence (SEQ ID No. 3):
ATGAGTTCTAATGTCTCCTGGGCCGACCAAGTTGACGCAGCTAATCGCCGGCAACGTTCTCGATCCAGGGGCAGATCTCAAAACAGAACCAATGCTTCAATTCCGTTGTCTTGGTTTACCTCAATCATTGATGAGTCTAATGGAAACTTCACTAGTCTTATGCCTCCTAGTGGTGTACCTACTGGTATGGGCACTGCAGCTCAGCAGTGTGGTTACTGGTATCGTGCGCCAACCTTCTATCAGGTGCGCCGCGGTAAAAGAGTACCTCTACCTCCTGTATGGTACTTCTACTTTTTGGGTACTGGTCCTCATTCTAATGCCGCTTACGGAACAGCAATGGATGGCGTTTTCTGGGTTAAGACGAAAAATGGTCAGATTGACCCTAAGTCTATTAAAGCTCTTGGTGTACGTGACAGTGGTACAGATCCTAGGCGCGCTAACATACCTAACCTTCCTGAAGGGTTGCGTGTGAGCGTTCCTAATGCTTCCAGACCACAATCTAGGGCTCAGTCTCAGACAAGGTCCCAGAATAATTCACGTGCTTCCTCTGCCAGCAGAAATGGTTCCAGAGCTTCCAGCGTTGATAGGACTAAGGAGGATCTTAAGGCTGTTGTTGCTCAACTTCTTTCAGAGATGGGTGTAAATAAGGCTTTCAAGCAGAATCAGACTCAGTCCCAGTCTAAGAAGAAACAGAAGGGCTCCACTCCTGCTGCTACCCCCCACCCTAATCAGGAGGGCAAACCAGTTTGGAAGAAAAAGCCTAACAAGGAAGAAACTGTTACGCAGTGCTTTGGCCCTCGCAGTGACAGTAAAAATTTTGGTGATGCAGATTTGATCAGACTCGGTGTTGATGATCCGCGTTTTAAGACCGTTTCATACTACGCACCGGGTGCTTCTGCTTCCTTGTTTGATTCCATGGTAACTGTGACTGATGGCTCAGATGGTAAAAAGAGAGTGACGTTTCACACCACAATTGAGGTTGATCCCACAAAACCGGGATTTGAAGTGTTTATGGCTCAGATTGATGCTTTCAAGAAACCTGCAACCTTCCAGCAGACTCAGAATTTTTGGGAGACTCAGGCCTCAACACAGAATAGTATCACTGACTATTTTAGAGGCACTACTCCTGGTGCGGGTGGTTCTGCAGTTGAAATTGAAACTTTTGAAATGACTGATGAAACTAAGTGA。
EXAMPLE 2 canine coronavirus N protein truncate expression, purification and antigen Activity validation
Based on the results of similar structural comparison and N protein structure prediction in example 1, truncations of two domains of NTD 27-157aa and CTD 245-346aa of canine coronavirus N protein were selected for expression purification, respectively. Corresponding nucleotide sequences are respectively synthesized in gene companies, the nucleotide sequences are constructed on pET-32a expression vectors by a molecular cloning method, BL21 (DE 3) competent cells are transformed after correct identification, and prokaryotic expression attempts of two truncations are respectively carried out. The results showed that both truncations of NTD 27-157aa and CTD 245-346aa gave soluble expressed proteins. The result of electrophoresis of the NTD 27-157aa protein truncated body is shown in FIG. 4, and a target band is arranged at the size of about 32kDa, so that the purity is high, and a small amount of impurities are contained.
The antigen activity was verified by indirect ELISA method using the purified NTD 27-157aa and CTD 245-346aa protein truncations of the canine coronavirus N protein, respectively. The indirect ELISA method comprises the following steps:
(1) Coating: the purified N protein truncations of NTD 27-157aa and CTD 245-346aa of canine coronavirus were diluted to 1. Mu.g/ml, 100. Mu.l/Kong Baobei, respectively, in 96-well ELISA plates and coated overnight at 4 ℃.
(2) Closing: blocking was performed with blocking solution containing 0.1% BSA, 200. Mu.l/well, blocking for 2h at 37 ℃.
(3) Washing: plates were washed 3 times with 250 μl/well of PBST wash (0.1%) and finally spin dried.
(4) Incubation resistance: diluted canine coronavirus clinical serum was added, 100 μl/well, and incubated at 37deg.C for 30min (where positive control was canine coronavirus positive serum, negative control was canine coronavirus negative serum, and three replicates were performed for each group).
(5) Washing: plates were washed 3 times with 250 μl/well of PBST wash (0.1%) and finally spin dried.
(6) Secondary antibody incubation: HRP-labeled rabbit anti-canine secondary antibody (10000-fold diluted in PBS) was added to 100 μl/well and incubated at 37deg.C for 30min.
(7) Washing: plates were washed 3 times with 250 μl/well of PBST wash (0.1%) and finally spin dried.
(8) Color development: TMB color development (commercial) was added at 100. Mu.l/well and developed at 37℃for 10min.
(9) And (3) terminating: the reaction was stopped by adding 0.5M sulfuric acid, 50. Mu.l/well, and OD was measured by using a microplate reader 450nm Values.
The results are shown in Table 1, the clinical serum detection result of the NTD 27-157aa truncated coating antigen of the N protein of the canine coronavirus is close to that of a positive control, and OD 450nm The value is larger than 1.0, the CTD 245-346aa truncated coated antigen has better antigen activity, and the detection result of the CTD 245-346aa truncated coated antigen clinical serum is close to that of a negative control, so that the CTD 245-346aa truncated coated antigen has no antigen activity. Thus, the NTD 27-157aa truncated protein was selected for use as a raw material for a subsequent ELISA coating antigen.
TABLE 1 results of antigen Activity verification
The truncated amino acid sequence of NTD 27-157aa of N protein of canine coronavirus (SEQ ID No. 2):
QNRTNASIPLSWFTSIIDESNGNFTSLMPPSGVPTGMGTAAQQCGYWYRAPTFYQVRRGKRVPLPPVWYFYFLGTGPHSNAAYGTAMDGVFWVKTKNGQIDPKSIKALGVRDSGTDPRRANIPNLPEGLRV。
the truncated nucleotide sequence of NTD 27-157aa of N protein of canine coronavirus (SEQ ID No. 4):
CAAAACAGAACCAATGCTTCAATTCCGTTGTCTTGGTTTACCTCAATCATTGATGAGTCTAATGGAAACTTCACTAGTCTTATGCCTCCTAGTGGTGTACCTACTGGTATGGGCACTGCAGCTCAGCAGTGTGGTTACTGGTATCGTGCGCCAACCTTCTATCAGGTGCGCCGCGGTAAAAGAGTACCTCTACCTCCTGTATGGTACTTCTACTTTTTGGGTACTGGTCCTCATTCTAATGCCGCTTACGGAACAGCAATGGATGGCGTTTTCTGGGTTAAGACGAAAAATGGTCAGATTGACCCTAAGTCTATTAAAGCTCTTGGTGTACGTGACAGTGGTACAGATCCTAGGCGCGCTAACATACCTAACCTTCCTGAAGGGTTGCGTGTG。
example 3 ELISA antibody detection kit based on canine coronavirus N protein NTD 27-157aa truncations as antigen
1. Preparation and detection steps of ELISA antibody detection kit with N protein NTD 27-157aa truncated form of canine coronavirus as antigen coating:
(1) Coating: an indirect ELISA antibody detection kit prepared by taking the N protein NTD 27-157aa truncated form of the canine coronavirus prepared in the example 2 as an antigen is used for detecting an ELISA antibody of the canine coronavirus in canine serum, wherein the concentration of the N protein NTD 27-157aa truncated form of the canine coronavirus which is coated and purified in the detection kit is 1 mug/ml.
(2) Sample adding: taking out the antigen coating plate coated with the canine coronavirus N protein NTD 27-157aa truncated antigen from the detection kit, diluting the serum to be detected (198 mu l of sample diluent and 2 mu l of serum to be detected) on a serum dilution plate by 100 times by using the sample diluent, adding 100 mu l of the mixed solution into an ELISA plate, and simultaneously adding 2 holes into positive serum and negative serum respectively, wherein each hole is 100 mu l.
(3) Incubation resistance: the samples in each well were gently shaken, covered with a plate cover, and incubated at 37℃for 30 minutes.
(4) Washing: 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.
(5) Secondary antibody incubation: 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.
(6) Washing: 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.
(7) Color development: mu.l of substrate solution was added to each well, the coated plate was covered with a sealing plate membrane and incubated at 37℃for 10 minutes.
(8) And (3) terminating: the reaction was stopped by adding 50. Mu.l of stop solution to each well.
(9) Results: determination of the absorbance of the sample and control at 450nm (OD 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 0.45, and the sample to be detected is positive for serum antibody; S/P is less than 0.45, and the sample to be detected is negative for serum antibody.
2. ELISA antibody detection kit specificity test
And detecting canine parvovirus CPV, canine distemper virus CDV, canine infectious hepatitis virus CAV I antibody positive serum and canine coronavirus CCV antibody positive serum by using the prepared kit. The detection results are shown in Table 2, and the S/P value of the canine coronavirus antibody positive serum is obviously more than 0.45, and the S/P value of the rest serum is less than 0.45, so that the detection kit meets the judgment standard of negative serum, and has good specificity.
TABLE 2 specific serum assay results
3. ELISA antibody detection kit sensitivity test
The canine coronavirus antibody positive serum is diluted by 2 times, 4 times, 8 times, 16 times, 32 times, 64 times and 128 times respectively, and the ELISA antibody detection kit prepared by the invention and the commercial canine coronavirus ELISA antibody detection kit are used for simultaneous detection. The results are shown in Table 3, and the results show that the two methods can detect the positive serum of the canine coronavirus antibody diluted by 32 times, and the detection kit has good sensitivity.
TABLE 3 sensitive serum assay results
4. ELISA antibody detection kit coincidence rate test
The ELISA antibody detection kit and the commercial canine coronavirus ELISA antibody detection kit are used for simultaneously detecting 30 canine serum samples, the results are compared, the coincidence rate of detection of the ELISA antibody detection kit and the commercial kit sample is 96.7%, the ELISA antibody detection kit and the commercial kit sample are good in adaptability, and the detection results and the analysis results are shown in tables 4 and 5.
Table 4 coincidence rate detection results
TABLE 5 coincidence rate analysis results
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 (7)
1. The amino acid sequence of the antigen truncated body is shown as SEQ ID No. 2.
2. A canine coronavirus N protein antigen truncate encoding gene of claim 1.
3. The coding gene of the canine coronavirus N protein antigen truncate of claim 2, wherein the coding gene is a DNA molecule shown in SEQ ID No. 4.
4. A biological material of any one of the following (a 1) to (a 4):
(a1) An expression cassette comprising the coding gene of claim 2 or 3;
(a2) A recombinant vector comprising the coding gene of claim 2 or 3;
(a3) A recombinant bacterium comprising the coding gene of claim 2 or 3;
(a4) A transgenic cell line comprising the coding gene of claim 2 or 3.
5. Use of a canine coronavirus N protein antigen truncate according to claim 1 and/or an antigen truncate encoding gene according to claims 2-3 and/or a biomaterial according to claim 4 in the preparation of an antigen.
6. A detection reagent for canine coronavirus antibody, wherein the active ingredient is the canine coronavirus N protein antigen truncate of claim 1.
7. The detection reagent for canine coronavirus antibody according to claim 6, wherein the detection reagent is an ELISA antibody detection reagent or a fluorescent microsphere antibody detection reagent or a colloidal gold antibody detection reagent.
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