CN116008543A - Lateral chromatography system for detecting canine distemper virus antibody and preparation method thereof - Google Patents
Lateral chromatography system for detecting canine distemper virus antibody and preparation method thereof Download PDFInfo
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Abstract
The invention provides a lateral chromatography system for detecting canine distemper virus antibodies and a preparation method thereof, belonging to the field of biological detection, and being characterized by comprising the following steps: a bond pad and a sense line; the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen is coated on the binding pad; the detection line is coated with an artificial binding antigen; the amino acid sequence of the artificial binding antigen is shown as the sequence ID No.2, and the test strip provided by the invention has the advantages of high sensitivity, rapidness, simplicity, convenience, short time consumption and the like, is suitable for rapid detection on site, and provides a simple serological detection method for on-site immune canine antibody monitoring.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a lateral chromatography system for detecting a canine distemper virus antibody and a preparation method thereof.
Background
Canine distemper (canine distemper) is a highly contagious disease that is caused by infection with canine distemper virus (canine distemper virus, CDV) in multiple animals, which is co-morbid, multi-system infectious. With the increase of population and expansion of wild animal habitat, the inter-species relationship is also increasing, and the host range of CDV is expanding, and the traditional canine, ferret and raccoon families are expanded to the primates monkey, artiodactyla, and carnivorous families, and the like, so that huge losses are caused for the protection of the breeding industry and wild animals. The prevention of CDV is very important for protecting wild animals and reducing loss of farmers. Dogs and economic animals are primarily protected against CDV with attenuated vaccines. Monitoring puppy or immunized dog CDV antibody levels will help determine the timing of the first immunization of a newborn puppy and evaluate the immune effect. The serum neutralization test (SN) is time-consuming and labor-consuming in detecting CDV antibodies, and is complex to operate; although enzyme-linked immunosorbent assay (ELISA) is sensitive and specific, special instruments are needed, and the ELISA is not suitable for detecting small amounts or single serum samples of pets clinically at any time.
Although the domestic pet hospital has an inlet kit for detecting CDV antibodies, the detection cost is high, the monitoring of the CDV antibodies of dogs before immunization is restricted, the immunization programs of most dogs are still determined empirically, and the detection basis of the antibodies is lacking.
The immunochromatography analysis method has the advantages of simple operation, low cost, suitability for a large number of samples and the like, and has wide application in the field of on-site rapid testing (POCT) in recent years. The colloidal gold has the advantages of good biocompatibility, different particle sizes and colors, easy improvement of signal strength through modification, and the like, and becomes a most common marking material in an immunochromatography analysis method. However, the colloidal gold has high price, large batch-to-batch variation, high requirements on materials such as antibodies, coating materials and the like, and weak signals, so that the application of the colloidal gold is limited.
As a substitute for colloidal gold, colloidal carbon is a nanomaterial with good biocompatibility after special activation treatment. As a marker, the method is superior to colloidal gold in stability, detection linear range, effective period, tolerance to whole blood samples, environmental protection, low cost and the like.
In theory, any protein can be non-covalently coupled with colloidal gold or colloidal carbon to prepare a usable test strip, but in the development process of actual products, due to the restriction of specific protein structure/conformation, concentration ratio, coating concentration, spraying amount and other factors of non-covalent coupling between colloidal carbon particles and proteins, no usable effective test strip product for detecting CDV antibodies exists in the market at present.
Therefore, development of a CDV antibody detecting colloidal carbon test strip with good detection effect is needed.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the prior art and providing a test strip for detecting canine distemper virus antibodies and a preparation method thereof. The technology for rapidly detecting the canine distemper virus antibody by using the colloidal carbon immunochromatography is a technology based on the chromatography technology of specific combination of colloidal carbon and antigen-antibody.
The invention selects a prototype in which hemagglutinin protein H-namely canine distemper virus protein H is taken as a binding antigen based on structural analysis of 6 structural proteins coded by CDV genome, namely N-P-M-F-H-L proteins, and selects a truncated fragment as an artificial binding antigen according to the special conformation of the canine distemper virus protein H to prepare optimal proportioning concentration of non-covalent coupling of the binding antigen and colloidal carbon particles, dilution concentration of non-covalent coupling conjugate, coating concentration and spraying amount. Provides a simple serological detection method for on-site immune canine antibody monitoring.
The specific scheme is as follows:
the invention provides a lateral chromatography system for detecting canine distemper virus antibodies, which is characterized by comprising the following components: a bond pad and a sense line;
the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen is coated on the binding pad;
the detection line is coated with an artificial binding antigen;
the amino acid sequence of the artificial binding antigen is shown as Seq ID No. 2.
Preferably, the lateral chromatography system is characterized in that the mass ratio of the colloidal carbon particles to the artificial binding antigen in the mixture of the colloidal carbon and the artificial binding antigen in the mixture is 1:2-5.
Preferably, the lateral chromatography system is characterized in that the mass ratio of the colloidal carbon particles to the artificial binding antigen is 1:5.
Preferably, the lateral chromatography system further comprises a control line; the control line is positioned at the downstream position of the detection line according to the lateral chromatography direction of the liquid; the control line is coated with rabbit anti-canine distemper virus H protein antibody.
Preferably, any one of the lateral chromatography systems is characterized by sequentially comprising a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially arranged on a bottom liner layer by layer from bottom to top; the detection line and the control line are arranged on the NC film;
preferably, the lateral chromatography system further comprises a clamping shell, wherein the bottom lining, the sample pad, the bonding pad, the NC membrane and the absorbent pad which are arranged on the bottom lining are integrally placed in the clamping shell, and a transparent observation window is arranged on the clamping shell at a position corresponding to the detection line and the contrast line.
In still another aspect, the present invention provides a method for preparing the lateral chromatography system described in any one of the above, which is characterized by comprising the following steps:
spraying a conjugate solution formed by non-covalent coupling and mixing of colloidal carbon and the artificial binding antigen on glass fibers, and drying to prepare a binding pad;
drawing lines on an NC film by using a solution of a rabbit anti-canine distemper virus H protein antibody and drying to prepare a control line;
and scribing and drying the NC film by using the solution for manually binding the antigen to obtain a detection line.
Preferably, the preparation method is characterized by further comprising the steps of preparing the artificial binding antigen, comprising the following steps:
(1) Extracting CDV virus RNA, and reversely transcribing the CDV virus RNA into cDNA;
(2) cDNA amplification is carried out by adopting the following primers to obtain a truncated fragment Seq ID No.3 for encoding the H protein of the canine distemper virus
The sequence of the upstream primer sH-F is: 5 'GGATCCCACCACACAGATGCAGT-3', underlined is BamHI cleavage site;
the sequence of the downstream primer s H-R is: 5 'GAGCTCTTACACCTCTCTCTCAATGCATCCATAG-3', with the SacI cleavage site underlined;
(3) Amplifying to obtain a fragment Seq ID No.3, and carrying out enzyme digestion to obtain a gene fragment Seq ID No.4 for encoding the artificial antigen protein, wherein the enzyme digestion product is connected and loaded into an insect baculovirus expression system; preferably, the insect baculovirus expression system is pFast Bac, and a recombinant expression vector p Fast BacI-H is obtained;
(4) Converting p Fast BacI-H into competent cells to obtain a positive recombinant plasmid Bacmid-H; preferably, wherein the competent cell is DH10Bac;
(5) And (3) carrying out propagation and recovery on the positive recombinant plasmid to obtain a canine distemper virus H protein truncated fragment and an artificial antigen protein shown as a Seq ID No. 2.
In a specific exemplary embodiment of the present invention, steps (3) to (5) are specifically as follows:
the correct PCR product and p Fast Bac plasmid are respectively connected after double enzyme digestion (BamHI+SacI), recombinant expression plasmid p Fast BacI-H is constructed, p Fast BacI-H plasmid is transformed into DH10Bac competent cells, the cells are inoculated on an agar plate containing gentamycin and tetracycline resistance, blue white spot colony screening is carried out, and positive recombinant plasmid is extracted and named as Bacmid-H; the recombinant plasmid Bacmid-H transfects Sf9 cells, the Sf9 culture solution after the second generation inoculation is changed into Grace's without serum, the supernatant and the precipitate are collected after 80 to 90 percent of lesions, protein content is measured after SDS-PAGE separation and recovery, and the protein is preserved at the temperature of minus 70 ℃ for standby.
Preferably, the preparation method is characterized in that the solvent of the conjugate solution is PB buffer solution with the concentration of 0.01mol/L and containing 3% of sucrose, 1% of BSA and 0.5% of polyvinyl alcohol in mass-volume ratio;
the concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is 2-4 mg/mL, wherein the mass ratio of the colloidal carbon to the artificial binding antigen is 1-2:5; the spraying amount is 4-8 mug/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration of the rabbit anti-canine distemper virus H protein antibody solution is 3-7 mg/mL, preferably 5mg/mL, and the coating amount is 3-8 mug/cm 2 ；
On the detection line, the coating concentration of the artificial antigen protein is 3-8 mg/mL, preferably 4.9mg/mL, and the dosage is 2-5 mug/cm 2 。
Preferably, the preparation method is characterized in that,
the concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is 3mg/mL, wherein the mass ratio of the colloidal carbon to the artificial binding antigen is 1:5, a step of; the spraying amount was 6. Mu.g/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration of the rabbit anti-canine distemper virus H protein antibody solution is 5mg/m L, and the coating amount is 5.5/cm 2 ；
On the detection line, the coating concentration of the artificial antigen protein is 4.9mg/mL, and the dosage is 3.5/cm 2 。
The invention has the technical advantages that:
(1) CDV is a member of the measles virus genus of Paramyxoviridae, the genome is single-stranded negative strand RNA, the full length 15690nt, and 6 structural proteins, namely N-P-M-F-H-L proteins, are encoded sequentially from the 3 'end to the 5' end. Wherein hemagglutinin (H) protein can mediate the binding of virus to cell surface, play a decisive role in virus invasion, and induce the body to produce anti-CDV neutralizing antibodies. The lateral chromatography system provided by the invention is based on structural analysis of 6 structural proteins coded by CDV genome, namely N-P-M-F-H-L proteins, selects hemagglutinin protein H, namely canine distemper virus protein H (NCBI accession number NP-047206.1 or Seq ID No. 1) as a prototype of a combined antigen, and selects a truncated fragment between 184 th and 334 th positions (shown as Seq ID No. 2) as an artificial antigen after analyzing the special conformation of the canine distemper virus protein H, and after being prepared by the method, the optimal proportioning concentration of non-covalent coupling of protein and colloidal carbon particles, the dilution concentration of non-covalent coupling conjugate, the coating concentration and the spraying amount are prepared.
(2) The invention also provides a preparation method of the test strip for detecting the canine distemper virus antibody. In the invention, an insect baculovirus expression system is adopted to express CDV-H protein; the insect baculovirus expression system is eukaryotic system expression, expressed as glycosylated protein, has protein conformation similar to that of natural protein and high affinity to antibody in serum. On the basis, various reaction conditions are perfected, and test results show that the method for rapidly detecting the warm virus antibodies in the canine serum by using the upper colloidal carbon immunochromatography established by the recombinant protein has good specificity and does not have cross reaction with the CPV, CHV, CPIV, CBb and other 4 canine common infectious disease positive serum; the variation of the repeated tests of the reagent in and among batches is small, and the reagent shows good repeatability; the CDV antibody detection reagent assembled by the research provides an effective technical means for monitoring the CDV antibodies of the immunized canine population and researching the CDVD epidemiology.
(3) The invention develops a rapid immunochromatography method using a colloidal carbon labeled material, and constructs a novel technology for rapid detection on site. Compared with colloidal gold, the advantages of colloidal carbon include higher color intensity, namely higher sensitivity and marking efficiency, low cost, simple manufacturing process, mass production and environmental protection.
CDV-H protein as referred to herein refers to an artificial antigen protein having an amino acid sequence as shown in Seq ID No. 2.
Drawings
FIG. 1 is a schematic diagram of a lateral flow chromatography system according to the invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. The invention is further described below in connection with the drawings and the specific preferred embodiments, but the scope of protection of the invention is not limited thereby.
Consumable and equipment related to experimental example of the invention
SB4 type carbon black: available from Degussa, germany
Dissolving SB4 type carbon black in a binder solvent to obtain a colloidal carbon dispersion, wherein the concentration of the colloidal carbon is as follows: 0.5mg/mL
Binder solvent: PB buffer solution with the mass-volume ratio of 3% of sucrose, 1% of BSA and 0.5% of polyvinyl alcohol and the concentration of 0.01mol/L is prepared in the laboratory.
NC membrane nitrocellulose membrane, available from Millipore corporation, usa;
the glass fiber, the absorbent paper, the adhesive base liner and the test strip plastic shell are manufactured by Shanghai gold mark biological limited company;
biochemical reagents such as calf serum albumin (BSA), polyvinyl alcohol (PVA), sucrose, etc., available from Sigma company in the united states;
p Fast Bac expression vector, DH10Bac competent cells, sf9 cells, available from full gold biotechnology Co., ltd;
other reagent consumables are commercially available unless specified, and reagents are all analytically pure commonly used in the laboratory.
The brand of the high-speed centrifuge is Hunan instrument; HM3030 three-dimensional reciprocating film-scribing instrument and ZQ2002 microcomputer automatic chopper are manufactured by Shanghai gold mark biotechnology Co.
Sources of biological materials
CDV standard positive and negative serum, positive serum and parvovirus (CPV), canine Paramyxovirus (CPIV), canine Botrytis cinerea (CBb), canine Parvovirus (CPV) and canine infectious hepatitis virus (CHV) positive serum are provided by a central institute (full name: china food and drug inspection institute);
rabbit anti-CDV-H protein antibodies were prepared by the present laboratory;
example 1 preparation of Artificial antigen protein (CDV-H protein truncated fragment)
The viral RNA is used for extraction and reverse transcription into cDNA.
Designing PCR primers for amplifying the CDV-H gene truncated fragment sh: the sequence of the upstream primer sH-F is: 5'-GGATCCCCACCACACAGATGCAGT-3', a BamHI cleavage site is underlined; the sequence of the downstream primer sH-R is: 5'-GAGCTCTTACACTTCCTCAATGTGATCCATAG-3', the Sac I cleavage site is underlined.
Wherein the CDV-H gene has GeneID of 1489792 and total length of 1815bp in ncbi, and the truncated fragment sh of the CDV-H gene amplified by the primer designed by the invention is shown as the sequence ID No.3
The correct PCR product and pFast Bac plasmid are respectively connected after double enzyme digestion (BamHI+SacI), and recombinant expression plasmid pFast Bac I-H is constructed;
transforming pFast Bac I-H plasmid into DH10Bac competent cells, inoculating to an agar plate containing gentamicin and tetracycline resistance, and screening blue white spot colonies to extract positive recombinant Bacmid-H;
the recombinant Bacmid-H transfects Sf9 cells, the Sf9 culture solution after the second generation virus inoculation is changed into Graces without adding serum, the supernatant and the precipitate are collected after 80 to 90 percent of lesions, the protein content is measured to be 4.9mg/mL after SDS-PAGE separation and recovery of protein, and the artificial antigen protein is preserved at the temperature of minus 70 ℃ for standby.
EXAMPLE 2 preparation of Rabbit anti-CDV-H protein antibodies
Taking the artificial antigen protein prepared in the example 1, injecting the artificial antigen protein into New Zealand white rabbits subcutaneously at the neck and back of the neck at a dose of 500 mug, emulsifying the antigen protein by using equivalent Freund's complete adjuvant (Sigma) for the first time,
the second and third phase were emulsified with equal amounts of Freund's incomplete adjuvant (Sigma) and each immunization was performed at 2 weeks intervals. Blood is collected after 1 week of three-phase, and serum is collected for standby.
Example 3 development of test strips
The carbon black is dissolved in a binder solvent to obtain a colloidal carbon dispersion, wherein the concentration of the colloidal carbon is as follows: 0.5mg/mL, to which the artificial antigen protein prepared in example 1 was added to obtain a conjugate diluent;
spraying colloidal carbon particles and the artificial antigen protein prepared in the embodiment 1 on glass fibers to serve as a bonding pad by using a conjugate diluent, rapidly drying at 37 ℃ for 3 hours for standby CDV-H protein and a rabbit anti-CDV-H protein antibody, marking by using a film marking instrument, respectively serving as a T line and a C line, and drying at 37 ℃ for 4 hours for standby;
sequentially adhering a sample pad, a bonding pad, an NC film and a water absorption pad to a bottom lining, cutting into test strips with the width of 4mm by a strip cutting machine, and putting the test strips into a detection card shell for later use;
determination of antigen coating amount, optimal conjugate spray amount and serum dilution to be tested:
the recombinant protein CDV-H is coated on NC film to form detection line, the colloidal carbon particles and the non-covalent coupling conjugate of CDV-H protein are sprayed on glass fiber as binding pad with different spraying amount, CDV standard negative serum is diluted 1:50, 1:100, 1:200, 1:400 and 1:800 times respectively to carry out matrix test, and the result judges that negative serum C line is developed, T line is not developed, positive serum C line is developed and T line is developed.
Experimental example 1
Performance verification of the test strip for detecting antibodies to canine distemper prepared in example 3 of the present invention was used for the following experiments:
1. determination of test strip preparation conditions
Specific cross-reaction assay: CPIV, CBb, CPV, CHV positive serum was tested, 4 replicates of each serum were run, negative and positive controls were set, and the procedure was followed to determine if the recombinant antigen cross-reacted with other canine positive serum.
Sensitivity test: CPV positive serum is selected to be diluted in a serial multiple ratio on the basis of dilution of 1:100, and is measured by a program, and the highest dilution capable of detecting positive results is calculated.
Repeatability test:
in-batch reproducibility test the test strips prepared in the same batch were used to obtain 4 parts of positive serum and 4 parts of negative serum, the test strips were tested according to the program, 8 serum replicates were used for each serum, and the results were analyzed statistically.
The batch-to-batch reproducibility test uses 3 test strips prepared in batches, 4 parts of positive serum and 4 parts of negative serum are taken and measured by a program, and the results are subjected to statistical analysis.
Through test result analysis, the preferred conditions for preparing the test strip are as follows:
the mass ratio of the colloidal carbon to the artificial binding antigen in the conjugate diluent is 1:2 to 5; the final concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is in the range of 2-4 mg/mL, and the spraying amount is in the range of 4-8 mug/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration range of the rabbit anti-canine distemper virus H protein antibody solution is 3-7 mg/mL, and the coating amount range is 3-8 mug/cm 2 ；
On the detection line, the coating concentration range of the artificial antigen protein is 3-8 mg/mL, and the dosage range is 2-5 mug/cm 2 。
The optimal conditions for preparing the test strip are determined as follows:
the mass ratio of the colloidal carbon to the artificial binding antigen in the conjugate diluent is 1:5;
the final concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is 3mg/mL, and the spraying amount is 6 mug/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration range of the rabbit anti-canine distemper virus H protein antibody solution is 5mg/mL, and the coating amount range is 5.5 mug/cm 2 ；
On the detection line, the coating concentration range of the artificial antigen protein is 4.9mg/mL, and the dosage range is 3.5 mug/cm 2 。
2. Determination of test strip decision criteria
50 parts of test strips which are manufactured under the optimal conditions and within the optimal condition range determined in the step 1 are adopted to be randomly matched with 30 parts of CDV negative serum detection and 20 parts of CDV positive serum detection; the results show that:
in 30 CDV negative serum samples, the line C develops color, and the line T does not develop color;
in 20 CDV positive serum samples, the C line developed and the T line developed.
The test strip of the invention has the detection accuracy up to 100%.
3. Specific cross-reaction test
Positive serum of CPIV, CBb, CPV, CHV canine diseases is detected, and no color is developed on the T line; as a result, the test strip prepared in the step 1 under the optimal conditions and within the optimal condition can detect positive serum of 4 epidemic diseases without cross reaction, which indicates that the recombinant protein selected and prepared by the invention brings stable and good specificity to the detection method.
4. Sensitivity test
CDV positive serum was detected.
The test strip under the preferred preparation condition has positive detection result when the serum is diluted to 50-800 times;
under the optimal preparation condition, when the serum dilution is continued to 1600-3200 times (the concentration of the antibody is 40-80 ng/mL), the detection result is still positive, and the established method is proved to have better sensitivity.
5. Repeatability test
Batch repeatability test: 8 parts of serum are used for carrying out 8 times of repeatability test results, and the results show that the negative and positive results are consistent, thus showing good repeatability.
Batch-to-batch repeatability test: 8 serum is subjected to repeatability tests by using test strips of different batches, negative positives are consistent, and the same sample has smaller variation degree in the tests of different batches and has better repeatability.
The above examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the above examples. All technical schemes belonging to the concept of the invention belong to the protection scope of the invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (10)
1. A lateral chromatography system for detecting antibodies to canine distemper virus comprising: a bond pad and a sense line;
the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen is coated on the binding pad;
the detection line is coated with an artificial binding antigen;
the amino acid sequence of the artificial binding antigen is shown as Seq ID No. 2.
2. The lateral flow chromatography system of claim 1, wherein the mass ratio of colloidal carbon particles to artificial binding antigen in the non-covalently coupled mixture of colloidal carbon and artificial binding antigen is 1:2-5.
3. The lateral flow chromatography system of claim 1, wherein the mass ratio of colloidal carbon particles to artificial binding antigen is 1:5.
4. The lateral chromatography system for detecting canine distemper virus antibody of claim 1, further comprising a control line; the control line is positioned at the downstream position of the detection line according to the lateral chromatography direction of the liquid; the control line is coated with rabbit anti-canine distemper virus H protein antibody.
5. The lateral flow chromatography system according to any one of claims 1 to 4, comprising a sample pad, the conjugate pad, an NC membrane, and a water absorbing pad sequentially arranged layer by layer on a substrate from bottom to top; the detection line and the control line are provided on the NC film.
6. The lateral flow chromatography system of claim 5, further comprising a cartridge housing, wherein the substrate, sample pad, conjugate pad, NC membrane, absorbent pad disposed on the substrate are disposed as a unit within the cartridge housing, and wherein transparent viewing windows are formed in the cartridge housing at positions corresponding to the detection and control lines.
7. A method of preparing a lateral flow chromatography system according to any one of claims 1 to 6, comprising the steps of:
spraying a conjugate solution formed by non-covalent coupling and mixing of colloidal carbon and the artificial binding antigen on glass fibers, and drying to prepare a binding pad;
drawing lines on an NC film by using a solution of a rabbit anti-canine distemper virus H protein antibody and drying to prepare a control line;
and scribing and drying the NC film by using the solution for manually binding the antigen to obtain a detection line.
8. The method of preparing according to claim 7, further comprising preparing the artificial binding antigen comprising the steps of:
(1) Extracting CDV virus RNA, and reversely transcribing the CDV virus RNA into cDNA;
(2) cDNA amplification is carried out by adopting the following primers to obtain a truncated fragment Seq ID No.3 for encoding the H protein of the canine distemper virus
The sequence of the upstream primer sH-F is: 5 'GGATCCCACCACACAGATGCAGT-3', underlined is BamHI cleavage site;
the sequence of the downstream primer s H-R is: 5 'GAGCTCTTACACCTCTCTCTCAATGTG-ATCCATAG-3', with the SacI cleavage site underlined;
(3) Amplifying to obtain a fragment Seq ID No.3, and carrying out enzyme digestion to obtain a gene fragment Seq ID No.4 for encoding the artificial antigen protein, wherein the enzyme digestion product is connected and loaded into an insect baculovirus expression system; preferably, the insect baculovirus expression system is pFast Bac, and a recombinant expression vector p Fast BacI-H is obtained;
(4) Converting p Fast BacI-H into competent cells to obtain a positive recombinant plasmid Bacmid-H; preferably, wherein the competent cell is DH10Bac;
(5) And (3) carrying out propagation and recovery on the positive recombinant plasmid to obtain a canine distemper virus H protein truncated fragment and an artificial antigen protein shown as a Seq ID No. 2.
9. The method according to claim 7 or 8, wherein the solvent of the conjugate solution is a PB buffer containing 3% sucrose, 1% BSA and 0.5% polyvinyl alcohol in a concentration of 0.01mol/L by mass/volume ratio;
the concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is 2-4 mg/mL, wherein the mass ratio of the colloidal carbon to the artificial binding antigen is 1-2:5; the spraying amount is 4-8 mug/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration of the rabbit anti-canine distemper virus H protein antibody solution is 3-7 mg/mL, preferably 5mg/m L, and the coating amount is 3-8 mug/cm 2 ；
On the detection line, the coating concentration of the artificial antigen protein is 3-8 mg/mL, preferably 4.9mg/mL, and the dosage is 2-5 mug/cm 2 。
10. The method according to claim 9, wherein,
the concentration of the non-covalent coupling mixture of the colloidal carbon and the artificial binding antigen in the conjugate diluent is 3mg/mL, wherein the mass ratio of the colloidal carbon to the artificial binding antigen is 1-5; the spraying amount was 6. Mu.g/cm 2 Then drying at 37 ℃ for 3 hours to prepare a bonding pad for standby;
on a control line, the coating concentration of the rabbit anti-canine distemper virus H protein antibody solution is 5mg/m L, and the coating amount is 5.5/cm 2 ；
On the detection line, the coating concentration of the artificial antigen protein is 4.9mg/mL, and the dosage is 3.5/cm 2 。
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