CN111875677A - Canine parvovirus VP2 protein recombinant antigen, encoding gene thereof, expression and application thereof - Google Patents
Canine parvovirus VP2 protein recombinant antigen, encoding gene thereof, expression and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a canine parvovirus VP2 protein recombinant antigen, an encoding gene thereof, and expression and application thereof, and the canine parvovirus VP2 protein recombinant antigen comprises a canine parvovirus VP2 protein recombinant antigen gene, a canine parvovirus VP2 protein recombinant antigen encoded by the gene, a method for expressing the canine parvovirus VP2 protein recombinant antigen, and application thereof in preparing colloidal gold. The VP2 protein is expressed in an escherichia coli expression system in a recombinant mode, and the method has the advantages of short production period, high yield, low cost and good specificity; the His label on the recombinant protein is beneficial to one-step purification to achieve higher purity, and can show better activity without enzyme digestion; the recombinant antigen can be used as a part of a canine parvovirus colloidal gold detection reagent strip, has high sensitivity, good specificity, good stability and simple and convenient operation, and is suitable for being used as a conventional detection means.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a canine parvovirus VP2 protein recombinant antigen, an encoding gene thereof, and expression and application thereof.
Background
Canine parvovirus disease is an acute infectious disease caused by infection of puppies with canine parvovirus. Clinically, there are two manifestations, hemorrhagic enteritis is characterized by severe vomiting, hemorrhagic enteritis and marked leukopenia, and myocarditis is characterized by sudden death. No matter the clinical manifestations of the type, the disease incidence is high, the mortality is high, and the infectivity is strong, and the method is one of the most serious infectious diseases harming the canine industry, and can cause serious economy, so the timely detection and diagnosis of canine parvovirus diseases are very important.
Since canine parvovirus disease has clinical symptoms similar to other canine diseases, it is difficult to confirm CPV infection by medical clinical observation, and it is necessary to confirm viral infection by molecular-level technical means. The detection methods established at present include ELISA, PCR, HA, HI and the like, ELISA can not distinguish wild bacteria infection and vaccine immune effect, PCR sensitivity is high, but requirements on equipment are high, HA and HI need to be matched for use and are inconvenient, for example, the publication No. CN105803112A discloses that 'primers, probes, kits and detection methods for detecting canine parvovirus' include: the upstream detection primer CPV-F comprises a nucleotide sequence; the downstream detection primer CPV-R comprises another nucleotide sequence. The probe includes a third nucleotide sequence. The kit comprises the primer and the probe. Although the invention has high sensitivity, the requirement on equipment is high, the detection time is long, the detection result can be detected within hours, the labeling stability is poor, and the invention is not suitable for conventional detection means.
Disclosure of Invention
The invention aims to overcome the defects of high requirement on equipment, long detection time and unsuitability for conventional detection of the existing detection method, provides the canine parvovirus VP2 protein recombinant antigen, the encoding gene thereof, the expression and the application thereof, can be used for preparing a colloidal gold detection reagent strip by taking the antigen as a labeled antigen, has high sensitivity of a labeling technology, good specificity and good stability, is simple and convenient to operate, and is suitable for being used as a conventional detection means of the canine parvovirus.
In order to achieve the purpose, the invention adopts the following technical scheme:
a canine parvovirus VP2 protein recombinant antigen gene has a sequence shown in SEQ ID NO. 2.
The canine parvovirus VP2 protein recombinant antigen coded by the gene is characterized in that the amino acid sequence of the antigen is shown as SEQ ID NO. 1.
The invention selects VP2 protein as VP2 protein of canine parvovirus as marker antigen in colloidal gold technology, the neutralizing antigen site of CPV is located on VP2, VP2 is immunogenic protein of CPV, and main antigenic determinant of CPV is coded, which can induce organism to generate neutralizing antibody.
A recombinant vector comprising the recombinant antigen gene.
Preferably, the vector is an escherichia coli BL21 expression vector. The recombinant protein gene is artificially synthesized, the used vector is pET30a which is kanamycin resistance, a histidine tag is expressed in a fusion mode, and the protein is positioned in the periplasm of cells.
A recombinant strain comprising the above antigen gene.
A method for expressing a canine fine VP2 protein recombinant antigen comprises the following steps:
(1) transforming the recombinant vector into escherichia coli BL21 cells to obtain a recombinant strain;
(2) the recombinant strain is cultured in an LB culture medium at the temperature of 35-37 ℃ in a shaking way until OD =0.5-0.7, 0.8-1.2mM IPTG is added, and the induction expression is carried out for 3-5 hours at the temperature of 35-37 ℃ and at the speed of 200-250 rpm;
(3) and after induction, centrifugally recovering, breaking bacteria, taking supernatant and purifying the expressed recombinant antigen of the canine parvovirus VP2 protein.
The expression system of the invention is an escherichia coli BL21 expression system, and has the characteristics of short period, low cost, large expression quantity and the like; in order to better maintain the active site of the recombinant protein, the invention selects a relatively mild culture and induction condition, the induction temperature is 35-37 ℃ during expression, the induction speed is 200-250rpm, and the concentration of induced IPTG is 0.8-1.2mM, so that the recombinant protein has slower expression and sufficient time for spatial conformation formation.
Preferably, the bacteria breaking is carried out by using an ultrasonic breaking mode, wherein the conditions are 350-450w, 2-4s of ultrasonic treatment and 4-8s of interval, and the total time is 150-200 times; the purification method is affinity chromatography, the buffer system is 40-60mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, and 0.0.4-0.6M Imidazole is added into the eluent; after the purification, the mixture was dialyzed 3 to 5 times in the same buffer system as that used for the purification and sterile-filtered, and the concentration was measured by BCA method. The His tag is added into the recombinant protein, the high purity can be achieved through one-step purification, the high activity can be shown without enzyme digestion after the high-purity recombinant protein is obtained, and compared with the recombinant protein which can only generate the activity through enzyme digestion, the method has the advantages that the steps are reduced, and the cost is reduced.
The protein recombinant antigen is applied to the preparation of a colloidal gold detection reagent strip.
Preferably, the preparation method of the colloidal gold test reagent strip comprises the following steps:
(1) adding chloroauric acid and trisodium citrate into boiling pure water, continuously heating and boiling for 10-20min, and cooling to room temperature to obtain colloidal gold solution;
(2) adding K into the cooled colloidal gold solution2CO3Adjusting the pH to be 6.8-7.2, adding the recombinant antigen of the canine parvovirus VP2 protein, stirring for 20-40min, adding a BSA solution, stirring for 20-40min, centrifuging to obtain a precipitate, diluting the precipitate with a gold-labeled diluent, soaking glass fibers, and freeze-drying to obtain a gold-labeled pad;
(3) diluting proteinA to 0.8-1.2mg/mL with a dot-film diluent, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4-0.6mg/mL with the same diluent, streaking the two diluted solutions onto a nitrocellulose membrane, and drying at 35-37 ℃ overnight, wherein the dot-film diluent is 10-20mM PBS (pH 7.2-7.5) and 0.8-1.2wt% sucrose solution;
(4) and assembling the gold label pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the colloidal gold detection reagent strip.
The colloidal gold detection reagent strip prepared by the invention is an indirect colloidal gold detection reagent strip, and compared with other detection modes, the indirect colloidal gold detection reagent strip has the advantages of convenience, rapidness, specificity, sensitivity, strong stability, no need of special equipment and reagents, intuitive result judgment and the like, and is suitable for being used as a conventional detection means.
Preferably, in the preparation method: the gold-labeled diluent is 10-20mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, 0.5-1.5wt% BSA, 0.1-0.2wt% TritonX-100 and 0.01-0.02wt% Proclin300 solution; the addition amount of the chloroauric acid is 0.01-0.02wt%, and the addition amount of the trisodium citrate is 0.01-0.02 wt%.
In conclusion, the invention has the following beneficial effects: (1) the VP2 protein is expressed in an escherichia coli expression system in a recombinant mode, and the method has the advantages of short production period, high yield, low cost and good specificity; (2) the His label on the recombinant protein is beneficial to one-step purification to achieve higher purity, and can show better activity without enzyme digestion; (3) the recombinant antigen can be used as a part of a canine parvovirus colloidal gold detection kit, has high sensitivity, good specificity, good stability and simple and convenient operation, and is suitable for being used as a conventional detection means.
Drawings
FIG. 1 shows the results of the test using the test strip of the present invention, in which the upper three strips are positive and the lower three strips are negative.
Detailed Description
Example 1: construction of expression vector containing canine parvovirus VP2 protein gene
The canine parvovirus VP2 protein recombinant antigen is designed according to the protein Sequence of VP2 (NCBI Sequence ID: BAB 21028.2).
According to the amino acid sequence, the codon of the escherichia coli is used for reverse translation into a nucleotide sequence, the obtained nucleotide sequence is synthesized into a recombinant gene sequence SEQ ID NO.2 by Shanghai Czejust bioengineering, Inc., and the vector is pET30 a.
Example 2: expression of recombinant antigen containing canine parvovirus VP2 protein
The canine parvovirus VP2 protein plasmid was transformed into Escherichia coli BL21, spread on LB plates containing 50ug/mL kanamycin (Shanghai Biotech, cat # K0408), cultured overnight at 37 ℃, single colonies were picked, cultured at 37 ℃ in300 mL LB medium containing the same concentration of kanamycin to OD600 of about 0.6, and induced to express using IPTG (Shanghai Biotech, cat # IB0168) at a final concentration of 1mM under the following conditions: at 37 ℃ and at a rotation speed of 200rpm for 4 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 3: purification and renaturation of recombinant antigen containing canine parvovirus VP2 protein
The cells were disrupted with 50mL Binding Buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and then sonicated at 400w for 3s with 6s intervals for 180 times, and finally centrifuged at 12000rpm for 30min at 4 ℃ to collect the supernatant, which was the target protein. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 50mM Tris, 0.2M NaCl, 0.5M imidazole, pH = 8.0. The purified recombinant protein was dialyzed against dialysis buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and the dialysate was changed every 12h for 3 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 4: indirect gold labeling method for detecting canine parvovirus antibody
4.1 preparation of test strip by indirect gold labeling method
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 15min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO3Adjusting 1.5mL to gold water pH =7.0, adding a certain amount of VP2 canine parvovirus recombinant antigen after stirring, stirring at room temperature for 30min, adding 10mL of 10wt% BSA solution, stirring at room temperature for 30min, centrifuging at 12000rpm for 30min, and adding the aboveCarefully sucking out the clear solution and discarding, wherein the precipitate is subjected to volume fixing to 1mL by using a gold-labeled diluent with the composition of 20mM Tris, 0.2M NaCl, 1wt% BSA, 0.1wt% Triton X-100, 0.01wt% Proclin300 and pH =8.0, and the volume is the labeled canine parvoantigen colloidal gold complex;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA to 1.0mg/mL with spot membrane diluent of composition 10mM PBS, 1wt% sucrose, pH =7.4, diluting goat anti-OspC-VlsE polyclonal antibody to 0.5mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying at 37 ℃ overnight;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the reagent strip for detecting the canine parvoantibody by the indirect method.
4.2 detection of the test strip by Indirect gold labeling
Adding 50ul of samples (serum and plasma) to be detected to a sample pad, standing at room temperature for 10min, and determining the result according to the following result determination standards:
firstly, only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;
two strips appear, wherein one strip is positioned in the quality control area, and the other strip is positioned in the test area and is positive;
and thirdly, the quality control line has no strip, which indicates that the test strip is damaged, and the test strip should be replaced with a new test strip for retesting.
The test results are shown in FIG. 1, wherein the upper three are positive and the lower three are negative, and it can be clearly seen from the figure that only one band appears on the quality control line of the sample with the negative result, and no band appears in the test area; and positive results are shown in two bands, wherein one band is positioned in the quality control area, and the other band is positioned in the test area.
4.3 Indirect gold labeling method for detecting canine parvoantibody
The indirect gold labeling method is used for detecting 103 positive canine parvovirus disease serum and 254 normal canine serum together, wherein 101 positive sera and 2 negative sera are detected in the 103 positive sera, 4 false positives appear in the 254 negative sera, and the sensitivity and specificity are respectively 98.1% and 98.4%, and the result data can show that the canine parvovirus antigen has very good sensitivity and specificity, is greatly increased compared with the sensitivity and specificity of the existing colloidal gold detection reagent strip which is about 92%, and is very suitable for being used as a raw material for manufacturing the canine parvoantibody detection test strip; the prepared colloidal gold detection strip has the advantages of high sensitivity, good specificity, short detection time, good stability, simple and convenient operation and the like, can judge a result only by placing the strip at room temperature for 10 minutes, and is very suitable for being used as a conventional detection method.
Example 5: expression of recombinant antigen containing canine parvovirus VP2 protein
The canine parvovirus VP2 protein plasmid was transformed into Escherichia coli BL21, spread on LB plates containing 50ug/mL kanamycin (Shanghai Producer, cat # K0408), cultured overnight at 35 ℃, single colonies were picked, cultured at 35 ℃ in300 mL LB medium containing the same concentration of kanamycin until OD600 reached about 0.7, and induced to express using IPTG (Shanghai Producer, cat # IB0168) at a final concentration of 1.2mM under the following conditions: at 35 ℃ and at 250rpm for 3 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 6: purification and renaturation of recombinant antigen containing canine parvovirus VP2 protein
The cells were disrupted with 50mL Binding Buffer consisting of 40mM Tris, 0.15M NaCl, pH =8.2, and then sonicated under conditions of 450w, sonication for 2s, 4s intervals, 200 times total, and finally, at 12000rpm, 30min, the supernatant was collected by centrifugation at 4 ℃ and the target protein was in the supernatant. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 40mM Tris, 0.15M NaCl, 0.4M imidazole, pH = 8.2. The purified recombinant protein was dialyzed against dialysis buffer consisting of 40mM Tris, 0.15M NaCl, pH =8.2, and the dialysate was changed every 12h for 4 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 7: preparation of indirect gold marking method detection strip
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 10min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO31.5mL of the mixture is adjusted to the pH =7.2 of gold water, a certain amount of VP2 canine parvovirus recombinant antigen is added after stirring, the mixture is stirred for 20min at room temperature, 10mL of 10wt% BSA solution is added, after stirring for 20min at room temperature, the mixture is centrifuged at 12000rpm for 30min, the supernatant is carefully sucked out and discarded, and the precipitate is subjected to volume fixing to 1mL by using a gold-labeled diluent consisting of 10mM Tris, 0.15M NaCl, 1.5wt% BSA, 0.15wt% TritonX-100, 0.015wt% Proclin300 and the pH =8.2, so that the labeled canine parvovirus antigen colloidal gold complex is obtained;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA a to 0.8mg/mL with spot membrane diluent consisting of 10mM PBS, 0.8wt% sucrose, pH =7.2, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying overnight at 35 ℃;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the reagent strip for detecting the canine parvoantibody by the indirect method.
Example 8: expression of recombinant antigen containing canine parvovirus VP2 protein
The canine parvovirus VP2 protein plasmid was transformed into Escherichia coli BL21, spread on LB plate containing 50ug/mL kanamycin (Shanghai Producer, cat # K0408), cultured overnight at 36 ℃, picked up as a single colony, cultured with 300mL LB medium containing kanamycin at the same concentration at 36 ℃ until OD600 reached about 0.5, and induced to express with IPTG (Shanghai Producer, cat # IB0168) at a final concentration of 0.8mM under the following induction conditions: at 36 deg.C, 230rpm, 5 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 9: purification and renaturation of recombinant antigen containing canine parvovirus VP2 protein
The cells were disrupted with 50mL Binding Buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and then sonicated at 350w for 4s with 8s intervals for 150 times, and finally centrifuged at 12000rpm for 30min at 4 ℃ to collect the supernatant, which was the target protein. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 60mM Tris, 0.25M NaCl, 0.5M imidazole, pH = 7.8. The purified recombinant protein was dialyzed against a dialysis buffer consisting of 60mM Tris, 0.25M NaCl, pH =7.8, and the dialysate was changed every 12h for 5 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 10: preparation of indirect gold marking method detection strip
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 20min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO31.5mL of the mixture is adjusted to the pH =6.8 of gold water, a certain amount of VP2 canine parvovirus recombinant antigen is added after stirring, the mixture is stirred for 30min at room temperature, 10mL of 10wt% BSA solution is added, after stirring for 30min at room temperature, centrifugation is carried out at 12000rpm for 30min, the supernatant is carefully sucked out and discarded, and the precipitate is subjected to volume fixing to 1mL by using a gold-labeled diluent consisting of 15mM Tris, 0.25M NaCl, 0.5wt% BSA, 0.2wt% TritonX-100, 0.02wt% Proclin300 and the pH =7.8, so that the labeled canine parvovirus antigen colloidal gold complex is obtained;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA a to 1.2mg/mL with spot membrane diluent of composition 10mM PBS, 1.2wt% sucrose, pH =7.5, diluting goat anti-OspC-VlsE polyclonal antibody to 0.6mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying at 36 ℃ overnight;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the reagent strip for detecting the canine parvoantibody by the indirect method.
Sequence listing
<110> Hangzhou AoRui biomedical science and technology Co., Ltd
<120> canine parvovirus VP2 protein recombinant antigen, encoding gene thereof, expression and application thereof
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<213> Canine Parvovirus VP2 protein (Canine Parvovirus VP2)
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Met Ser Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala Val Arg
1 5 10 15
Asn Glu Arg Ala Thr Gly Ser Gly Asn Gly Ser Gly Gly Gly Gly Gly
20 25 30
Gly Gly Ser Gly Gly Val Gly Ile Ser Thr Gly Thr Phe Asn Asn Gln
35 40 45
Thr Glu Phe Lys Phe Leu Glu Asn Gly Trp Val Glu Ile Thr Ala Asn
50 55 60
Ser Ser Arg Leu Val His Leu Asn Met Pro Glu Ser Glu Asn Tyr Arg
65 70 75 80
Arg Val Val Val Asn Asn Leu Asp Lys Thr Ala Val Asn Gly Asn Met
85 90 95
Ala Leu Asp Asp Thr His Ala Gln Ile Val Thr Pro Trp Ser Leu Val
100 105 110
Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln Leu
115 120 125
Ile Val Asn Thr Met Ser Glu Leu HisLeu Val Ser Phe Glu Gln Glu
130 135 140
Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro
145 150 155 160
Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
165 170 175
Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser
180 185 190
Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp
195 200 205
Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly
210 215 220
Thr Ser Gly Thr Pro Thr Asn Ile Tyr His Gly Thr Asp Pro Asp Asp
225 230 235 240
Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg
245 250 255
Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Phe Phe Asp Cys Lys Pro
260 265 270
Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu Pro
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Pro Phe Leu Asn Ser Leu Pro Gln Ala Glu GlyAsp Thr Asn Phe Gly
290 295 300
Tyr Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly
305 310 315 320
Asn Thr Asn Tyr Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val
325 330 335
Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro
340 345 350
Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp Glu
355 360 365
Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His
370 375 380
Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr Tyr
385 390 395 400
Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile Gln
405 410 415
Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu Pro
420 425 430
Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile Phe
435 440 445
Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val ProPro Val Tyr
450 455 460
Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro
465 470 475 480
Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro Gly
485 490 495
Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp Pro
500 505 510
Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe Trp
515 520 525
Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His Thr
530 535 540
Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe Asn
545 550 555 560
Tyr Val Pro Ser Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys Ser
565 570 575
Gln Leu Ala Pro Arg Lys Leu Tyr
580
<210>2
<211>1755
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60
acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120
tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg atgggtggaa 180
atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataga 240
agagtggttg taaataattt ggataaaact gcagttaacg gaaacatggc tttagatgat 300
actcatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360
tttaatccag gagattggca actaattgtt aatactatga gtgagttgca tttagttagt 420
tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480
ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagcaat 540
aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600
aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660
tctcatactg gaactagtgg cacaccaaca aatatatacc atggtacaga tccagatgat 720
gttcaatttt atactattga aaattctgtg ccagtacact tactaagaac aggtgatgaa 780
tttgctacag gaacattttt ttttgattgt aaaccatgta gactaacaca tacatggcaa 840
acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggagat 900
actaactttg gttatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960
aatacaaact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020
ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080
cgggggggag cgcaaacaga tgaaaatcaa gcagcagatg gtgatccaag atatgcattt 1140
ggtagacaac atggtcaaaa aactaccaca acaggagaaa cacctgagag atttacatat 1200
atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260
aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320
ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380
ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440
agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500
aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560
attgtaactt actcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620
gcctctcata cttggaatcc aattcagcaa atgagtatta atgtagataa ccaatttaac 1680
tatgtaccaa gtaatattgg aggtatgaaa attgtatatg aaaaatctca actagcacct 1740
agaaaattat attaa 1755
Claims (10)
1. A canine parvovirus VP2 protein recombinant antigen gene is characterized in that the recombinant antigen gene sequence is shown as SEQ ID NO. 2.
2. The canine parvovirus VP2 protein recombinant antigen encoded by the gene of claim 1, wherein the amino acid sequence of the antigen is shown as SEQ ID No. 1.
3. A recombinant vector comprising the recombinant antigen gene according to claim 1.
4. The recombinant vector according to claim 3, wherein the vector is an E.coli BL21 expression vector.
5. A recombinant strain comprising the antigenic gene of claim 1.
6. A method for expressing a canine fine VP2 protein recombinant antigen is characterized by comprising the following steps:
(1) transforming the recombinant vector of claim 4 into Escherichia coli BL21 cells to obtain a recombinant strain;
(2) the recombinant strain is cultured in an LB culture medium at the temperature of 35-37 ℃ in a shaking way until OD =0.5-0.7, 0.8-1.2mM IPTG is added, and the induction expression is carried out for 3-5 hours at the temperature of 35-37 ℃ and at the speed of 200-250 rpm;
(3) and after induction, centrifugally recovering, breaking bacteria, taking supernatant and purifying the expressed recombinant antigen of the canine parvovirus VP2 protein.
7. The method as claimed in claim 6, wherein the disruption is performed by ultrasonic disruption under conditions of 350-450w, 2-4s of ultrasonic treatment, and 4-8s of interval, and for a total of 150-200 times; the purification method is affinity chromatography, the buffer system is 40-60mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, and 0.4-0.6M Imidazole is added into the eluent; after the purification, the mixture was dialyzed 3 to 5 times in the same buffer system as that used for the purification and sterile-filtered, and the concentration was measured by BCA method.
8. The protein recombinant antigen of claim 2 is applied to the preparation of a colloidal gold detection reagent strip.
9. The use of claim 8, wherein the colloidal gold test strip is prepared by the following method:
(1) adding chloroauric acid and trisodium citrate into boiling pure water, continuously heating and boiling for 10-20min, and cooling to room temperature to obtain colloidal gold solution;
(2) adding K into the cooled colloidal gold solution2CO3Adjusting the pH to be 6.8-7.2, adding the recombinant antigen of the canine parvovirus VP2 protein, stirring for 20-40min, adding a BSA solution, stirring for 20-40min, centrifuging to obtain a precipitate, diluting the precipitate with a gold-labeled diluent, soaking glass fibers, and freeze-drying to obtain a gold-labeled pad;
(3) diluting proteinA to 0.8-1.2mg/mL with a dot-film diluent, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4-0.6mg/mL with the same diluent, streaking the two diluted solutions onto a nitrocellulose membrane, and drying at 35-37 ℃ overnight, wherein the dot-film diluent is 10-20mM PBS (pH 7.2-7.5) and 0.8-1.2wt% sucrose solution;
(4) and assembling the gold label pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the colloidal gold detection reagent strip.
10. Use according to claim 9, characterized in that in the preparation method: the gold-labeled diluent is 10-20mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, 0.5-1.5wt% BSA, 0.1-0.2wt% TritonX-100 and 0.01-0.02wt% Proclin300 solution; the addition amount of the chloroauric acid is 0.01-0.02wt%, and the addition amount of the trisodium citrate is 0.01-0.02 wt%.
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| CN114478710A (en) * | 2022-02-21 | 2022-05-13 | 苏州西山生物技术有限公司 | Rat parvovirus RPV-1 specific VP2 gene recombinant antigen, synthetic method and kit |
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| CN114478710A (en) * | 2022-02-21 | 2022-05-13 | 苏州西山生物技术有限公司 | Rat parvovirus RPV-1 specific VP2 gene recombinant antigen, synthetic method and kit |
| CN114478710B (en) * | 2022-02-21 | 2023-09-29 | 苏州西山生物技术有限公司 | Recombinant antigen of rat parvovirus RPV-1 specific VP2 gene, synthetic method and kit |
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