CN111579783A - Colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection and preparation method thereof - Google Patents
Colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection and preparation method thereof Download PDFInfo
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- CN111579783A CN111579783A CN202010584530.0A CN202010584530A CN111579783A CN 111579783 A CN111579783 A CN 111579783A CN 202010584530 A CN202010584530 A CN 202010584530A CN 111579783 A CN111579783 A CN 111579783A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The invention provides a colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection and a preparation method thereof, and the colloidal gold immunochromatographic test paper comprises: the kit comprises a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad and a bottom plate; the sample pad, the gold label pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate; wherein, the colloidal gold labeled duck plague virus UL2 protein, the colloidal gold labeled duck plague virus gI protein and the colloidal gold labeled goat anti-rabbit IgG are adsorbed on the gold label pad; the cellulose nitrate membrane contains a T1 line, a T2 line and a C line, the T1 line contains duck plague virus UL2 protein, the T2 line contains duck plague virus gI protein, and the C line contains rabbit IgG. The colloidal gold immunochromatographic test paper provided by the invention can effectively identify duck plague commercial attenuated vaccine immunity and wild virus infected duck serum antibodies, and has the advantages of high sensitivity, good specificity, good repeatability and good stability.
Description
Technical Field
The invention belongs to the technical field of colloidal gold immunochromatographic test paper, and particularly relates to colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection and a preparation method thereof.
Background
The colloidal gold immunochromatography technology is that in 1971, salmonella is detected by combining colloidal gold with an antibody for the first time by Faulk and Taylor, thereby introducing immunochemistry. Osikowicz, et al 1990, established the first colloidal gold immunochromatographic assay. Has become one of the practical detection techniques in the detection of animal epidemic diseases at present. The method is a novel immune labeling technology based on antigen-antibody specific binding characteristics, uses colloidal gold as an antigen-antibody tracer label, and can be used for qualitatively or semi-quantitatively detecting pathogens to obtain serum antibodies. The method is simple and convenient to operate, can be operated without using special instruments or special training, and can observe results by naked eyes, so that the clinical convenience is ensured, and the test strip is suitable for clinical and large-batch detection. Colloidal gold is a color indicator, can obtain a result by naked eyes within 10 minutes without adding an additional color reagent, is quick, simple, cheap and intuitive, is suitable for field test, and is widely used for detecting viruses, bacteria, parasites and drug residues at present. There are many techniques for immune colloidal gold test strips, and currently, there are many major technologies for Dot-immune gold filtration assay (DIGFA) and colloidal Gold Immunochromatography (GICA).
Duck plague, also known as duck viral enteritis, is an acute, high temperature, infectious and fatal disease caused by duck plague virus. Duck Plague Virus (DPV) infects ducks and geese and anseriformes. It is characterized by depression, anorexia, fever, feather discoloration, tiredness, hoarseness, nasal obstruction, eyelid and head and neck swelling, lacrimation, diarrhea, white and green feces, malodor, atrophy, congestion, cloacal mucosa edema and green pseudomembrane covering. Severely infected ducks showed dyspnea, ataxia and paralysis. Baudet first reported the disease in 1923. In China, the first outbreak in 1957 was reported by Huang-daoyian et al. The duck plague virus has wide prevalence, the mortality rate of the duck plague virus reaches 100 percent, the egg drop rate reaches 50 percent, and huge economic loss is caused to the duck industry. It has now been demonstrated that DPV attenuated vaccines can effectively induce humoral and cell-mediated immune responses and prevent duck plague infections. The DPV commercial attenuated vaccine is widely used for preventing duck plague and achieves good effect. However, there are cases where duck plague is infected by duck plague that is reported as a commercial attenuated vaccine for duck plague. Therefore, the duck plague is difficult to purify only by means of the attenuated vaccine immunization, and the screening of the infected ducks while the attenuated vaccine immunization has important significance for the purification of the duck plague. Due to the lack of a method capable of distinguishing duck plague virulent virus infection from attenuated vaccine immune duck serum antibody, a scheme for purifying DPV infection through immunization lacks technical support. Currently, there are many conventional methods available for diagnosing duck plague. The method comprises enzyme-linked immunosorbent assay (ELISA), virus separation and identification, Polymerase Chain Reaction (PCR), hemagglutination experiment, neutralization experiment, loop-mediated isothermal amplification and the like. Each of these methods has its advantages and disadvantages. As far as we know, only PCR is used for distinguishing duck plague attenuated vaccine immunity from wild virus infected ducks. However, PCR has some disadvantages that it cannot be used in field detection, such as the need of special equipment, complicated operation, long time consumption, unsuitability for rapid and large-scale clinical detection, and the need of skilled technicians.
At present, the colloidal gold immunochromatographic assay is used for detecting duck plague serum antibodies, but can only simply detect the duck plague serum antibodies and cannot be used for distinguishing duck plague attenuated vaccine immunized ducks from wild virus infected ducks. Therefore, the development of the colloidal gold immunochromatographic test strip which can be used for field diagnosis quickly, simply and conveniently is very important.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the colloidal gold immunochromatographic test paper for identifying the commercial attenuated vaccine immunity and the wild virus infection of the duck plague and the preparation method thereof, and the test paper can quickly and conveniently distinguish the commercial attenuated immune duck of the duck plague from the wild virus infected duck.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection comprises: the sample pad, the gold label pad, the nitrocellulose membrane, the water absorption pad and the bottom plate are sequentially overlapped and adhered to the bottom plate, and the overlapping of joints is ensured, so that the chromatography can be smoothly carried out; wherein, the colloidal gold labeled duck plague virus UL2 protein, the colloidal gold labeled duck plague virus gI protein and the colloidal gold labeled goat anti-rabbit IgG are adsorbed on the gold label pad; the cellulose nitrate membrane contains a T1 line, a T2 line and a C line, the T1 line contains duck plague virus UL2 protein, the T2 line contains duck plague virus gI protein, and the C line contains rabbit IgG.
Further, the coating concentration of the duck plague virus UL2 protein on the nitrocellulose membrane is 0.5-2.5mg/ml, and preferably 1.5 mg/ml.
Further, the concentration of the coating of the duck plague virus gI protein on the nitrocellulose membrane is 0.5-2.5mg/ml, preferably 2.5 mg/ml.
Further, the concentration of the colloidal gold labeled duck plague virus UL2 protein is 50-250ug/ml, preferably 200 ug/ml; the concentration of the colloidal gold labeled duck plague virus gI protein is 50-250ug/ml, preferably 250 ug/ml; the concentration of colloidal gold-labeled goat anti-rabbit IgG is 20-40. mu.g/ml, preferably 30. mu.g/ml.
Further, the colloidal gold labeled duck plague virus UL2 protein is prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, adding the duck plague virus UL2 protein, and uniformly mixing to obtain the final product; among them, the alkali solution is preferably K having a concentration of 0.1mol/l2CO3And (3) solution.
Further, the colloidal gold-labeled duck plague virus gI protein is prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, adding the duck plague virus gI protein, and uniformly mixing to obtain the composition; among them, the alkali solution is preferably K having a concentration of 0.1mol/l2CO3And (3) solution.
Further, the colloidal gold-labeled goat anti-rabbit IgG was prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, adding goat anti-rabbit IgG, and mixing uniformly to obtain the composition; among them, the alkali solution is preferably K having a concentration of 0.1mol/l2CO3And (3) solution.
Further, the gold-labeled pad is prepared by the following method: soaking the glass cellulose membrane in a mixed solution containing 50-250 mu g/ml of colloidal gold-labeled duck plague virus UL2 protein, 50-250 mu g/ml of colloidal gold-labeled duck plague virus gI protein and 20-40 mu g/ml of colloidal gold-labeled goat anti-rabbit IgG, and drying at 37 ℃ overnight to obtain the product.
The preparation method of the colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection comprises the following steps:
(1) respectively scribing duck plague virus UL2 protein, gI protein and goat anti-rabbit IgG on a nitrocellulose membrane to form a T1 line, a T2 line and a C line, and then placing the nitrocellulose membrane at 37 ℃ for drying for 2 h;
(2) and (2) sequentially adhering the sample pad, the gold-labeled pad, the nitrocellulose membrane treated in the step (1) and the water absorption pad on a bottom plate, wherein a T1 line on the nitrocellulose membrane is close to the gold-labeled pad, a T2 line on the nitrocellulose membrane is close to water absorption paper, a C line is positioned between the detection lines T1 and T2, and then cutting the nitrocellulose membrane into test strips.
The method for identifying duck plague attenuated vaccine immunity and wild virus infection by adopting the test strip comprises the following steps: dripping a serum sample on the sample pad through the sample adding hole, advancing the sample on the chromatographic paper strip, reacting with the colloidal gold labeled antigen on the gold labeled pad, developing color, and judging that the serum sample is from duck plague attenuated vaccine immunized ducks or wild virus infected ducks according to the color development result;
when the T1 line, the T2 line and the C line are all developed, the result is positive, which indicates that the serum sample contains the antibody against the UL2 protein and indicates that the serum sample is from the wild virus infected duck;
when only the T2 line and the C line are developed, the serum sample does not contain anti-UL 2 protein antibodies but contains serum antibodies of duck plague virus, and the serum sample is from duck plague commercial attenuated vaccine immunized ducks;
when only C line is developed, the result is negative, which indicates that the serum sample does not contain the antibody against the duck plague virus, and the sample is from a healthy duck;
if the C line does not develop color, the test strip is invalid.
The colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection and the preparation method thereof provided by the invention have the following beneficial effects:
the sizes of UL2 genes of the attenuated vaccine strain and the wild strain are 474bp and 1002bp respectively, namely compared with the wild strain, the UL2 gene of the attenuated vaccine strain is lack of 528bp (76-240 aa at the N end), so that only the protein expressed by shortening of 76-240 amino acids at the UL2N end of the wild strain is expressed according to the UL2 gene, therefore, the antibody of the protein can be detected in the serum of the wild strain infected duck, and the antibody of the protein can not be detected in the serum of the commercial attenuated vaccine immune duck against duck plague. Therefore, the colloidal gold immunochromatographic test paper provided by the invention can effectively identify the serum antibodies of the duck plague commercial attenuated vaccine immunized ducks and wild virus infected ducks, and has the advantages of high sensitivity, good specificity, good repeatability and good stability.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold test strip for identifying duck plague attenuated vaccine immunity and wild virus infection.
FIG. 2 is an explanatory diagram of the results of identifying the serum antibodies of the duck plague attenuated vaccine immunized duck and the wild virus infected duck with the colloidal gold test strip.
FIG. 3 is a diagram showing the expression and purification of duck plague virus UL2 protein and Western blotting identification results.
FIG. 4 is a graph showing the results of the expression and purification of duck plague virus gI protein and Western blotting identification.
FIG. 5 is a graph showing the optimized result of the colloidal gold labeled UL2 resuspension concentration for identifying duck plague attenuated vaccine immunity and wild virus infection colloidal gold immunochromatography test paper.
FIG. 6 is a graph showing the results of the optimization of the resuspension concentration of the colloidal gold labeled gI in the colloidal gold immunochromatographic test paper for identifying the immunization of duck plague attenuated vaccines and the wild virus infection.
FIG. 7 is a graph of the concentration optimization result of the colloidal gold immunochromatographic test paper detection line T1 for identifying duck plague attenuated vaccine immunization and wild virus infection.
FIG. 8 is a diagram of the optimized result of the colloidal gold immunochromatographic test paper detection line T2 for identifying duck plague attenuated vaccine immunization and wild virus infection.
FIG. 9 is a graph showing the sensitivity of the colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunization and wild virus infection.
FIG. 10 is a result chart of the specificity of the colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection.
Detailed Description
The experimental materials and main reagents used in the present invention are as follows:
1. experimental Material
Strain, strain and serum: coli BL21(DE3) provided by the university of agriculture in sichuan, aviary disease research center, containing pET32a-gI plasmid; non-immune duck negative serum (negative for DPV antibody detection), DPV positive serum and 123 parts of duck serum to be detected (collected from duck farms in Sichuan province) are provided by the poultry disease research center of Sichuan agricultural university.
2. Primary reagent
Bovine Serum Albumin (BSA) was purchased from Amresco; rabbit IgG, goat anti-rabbit IgG were purchased from Shanghai Bintian Biotech Co., Ltd, and the base plate, the absorbent paper, the nitrocellulose membrane, the glass cellulose membrane and the sample pad were purchased from Shanghai Jie Biotech Co., Ltd.
3 solutions for experiments and their preparation
10% BSA: weighing 10g BSA, adding 100ml purified water, mixing well, and storing at-20 deg.C for use.
Gold labeling solution: 20mmol/l Tris contained 1% BSA and 3% sucrose, 5% trehalose (these are all by volume, e.g. 1% BSA to 1g BSA in 100ml RO water).
PB buffer: weighing Na2HPO41.192g，NaH2PO4Adding 1L purified water into 0.19g, subpackaging, and storing at-20 deg.C for use.
PBS: 800ml of ultrapure water dissolved 8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4And 0.24g KH2PO4Adjusting the pH value to 7.4, and adding deionized water to reach the constant volume of 1000 ml.
20mM Tris buffer: 1.2114g Tris was diluted to 500ml with water.
8M of urea: 48g of urea is added with deionized water to be 100 ml.
LB culture solution: 1g of sodium chloride, 1g of peptone and 0.5g of yeast powder are added with deionized water to be constant volume of 100 ml.
Loading buffer: 1.25ml of 1M Tris-HCl (pH6.8), 0.5g of SDS, 25mg of bromophenol blue, 2.5ml of glycerol and deionized water to a volume of 5ml, wherein the volume is determined according to the following steps of 20: adding beta mercaptoethanol in the volume ratio of 1.
Tris-Glycine electrophoresis buffer: adding 800ml of deionized water into 5g of Tris, 1g of SDS and 18.8g of glycine, dissolving, metering to 1L, and storing at room temperature.
0.2mol/l K2CO3：2.67g K2CO3Deionized water was added to 100 ml.
Coating liquid: 1.59g Na2CO3，2.92g NaHCO3And adding deionized water to the solution to make the volume of the solution constant to 1L.
Stopping liquid: 21.7ml 98% H2SO4178.3ml of double distilled water were added.
Sealing liquid: 5g BSA was added to 100ml deionized water.
LB liquid medium: weighing 10g of Tryptone, 5g of Yeast Extract and 10g of sodium chloride, dissolving in 800mL of deionized water, fully stirring, fixing the volume to lL, and sterilizing at high temperature and high pressure.
The colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the sample pad, the gold-labeled pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate, and the overlapping pressure at a joint is ensured, so that the chromatography can be smoothly carried out; wherein, the colloidal gold labeled duck plague virus UL2 protein, the colloidal gold labeled duck plague virus gI protein and the colloidal gold labeled goat anti-rabbit IgG are adsorbed on the gold label pad; the cellulose nitrate membrane contains a T1 line, a T2 line and a C line, the T1 line contains duck plague virus UL2 protein, the T2 line contains duck plague virus gI protein, and the C line contains rabbit IgG.
The preparation and detection of the colloidal gold immunochromatographic test paper are concretely shown in the following examples and experimental examples:
example 1 induced expression of Duck plague virus UL2 protein and purification of the product
(1) The expression strain BL21(DE3) containing the plasmid pET28a-UL2 was inoculated into liquid LB containing 50. mu.g/ml Kan resistance. Culturing at 37 ℃ overnight, taking the bacterial liquid the next day according to the proportion of 1: a ratio of 100 was inoculated in 500ml of LB liquid medium (containing Kan 50. mu.g/ml), and cultured with vigorous shaking to OD600When the concentration is 0.5, IPTG is added to the final concentration of 0.2mmo/l, 1ml of culture solution is collected after 2h of induction, centrifugation is carried out for 10min at 12000rpm at 4 ℃, the supernatant is discarded, 80 mu l of ultrapure water and 20 mu l of 5 × loading buffer are added into the precipitate, heating and denaturation are carried out in 100 ℃ water bath for 5-10min, 15% SDS-PAGE gel electrophoresis is carried out, and the expression result is observed and is shown in figure 3.
And (4) analyzing results: the result of induction expression and non-induction with IPTG of expression host bacterium BL21(DE3) containing recombinant plasmid pET28a-UL2 by IPTG shows that no specific protein band appears in the non-induction with IPTG; recombinant expression plasmid pET28a-UL2 expressed recombinant protein at 19 kDa.
(2) Solubility analysis of the expression product of the recombinant plasmid pET28a-UL 2: respectively treating 500ml of bacterial liquid with induced expression and 500ml of bacterial liquid without induced expression according to the following steps: centrifuging at 4 deg.C and 5000rpm/min for 30min, and suspending thallus precipitate with 50ml20mmol Tris-HCl (pH8.0); after being placed at-20 ℃ overnight, the thalli are intermittently crushed by ultrasonic waves (ice bath) (600w 30 s/time, 3 times), centrifuged for 10min at 4 ℃ and 1000rpm/min, and the supernatant is taken for later use (I); the precipitate was dissolved in 10ml PBS and stored at low temperature for further use. Mu.l of the supernatant (first) and 80. mu.l of the precipitate (second) dissolved in PBS were added to 20. mu.l of 5 XSDS-PAGE sample buffer, boiled in 100 ℃ water bath for 5-10min, subjected to 15% SDS-PAGE gel electrophoresis, stained with Coomassie brilliant blue, and the results were observed. And scattering, scanning and analyzing the relative percentage content of the recombinant protein in the induced bacteria liquid in the coating slurry (supernatant (first) and the soluble) and the sediment (second) and the inclusion body form) by a full-automatic gel imaging analysis system.
And (4) analyzing results: after the 500ml bacterial liquid of induced expression is processed by solubility analysis, electrophoresis results show that the expressed protein is mainly present in the precipitate, which indicates that the recombinant expressed protein is largely present in insoluble inclusion body form in the thallus.
(3) And (3) carrying out a large-scale gel cutting purification on inclusion bodies of duck plague UL2 protein: taking out the inclusion body stored at the temperature of-20 ℃, thawing, and then mixing according to the weight ratio of 5: 1 into 5 XSDS-PAGE loading buffer (0.313M Tris HCl pH6.8, 50% glycerol, 10% SDS and 0.05% bromophenol blue, lysed with 100mM DTT) for 10 min. The glass plates were mounted as described and an appropriate volume of 8% release glue was dispensed. The glass was filled and sealed with absolute ethanol. After the gel was solidified, the absolute ethanol was poured out and blotted dry with absorbent paper. Without inserting a comb, 5% volume of the concentrated gel was added to the glass plate, leaving room for the sample. Electrophoresis was carried out at 80V for 30min and 120V for 2 h. After completion of the electrophoresis, the PAGE gel was removed and immersed in pre-chilled 0.3mol/l KCl at 4 ℃. The strips are cut after white stripes appear. The bands were cut and placed in dialysis bags, and then the appropriate amount of PBS was added. The dialysis bag was electroeluted in Gly buffer. After electroelution at 120V2h, the target protein was collected and dialyzed in PBS buffer overnight. Stored at-20 ℃. The BCA protein assay kit determines protein concentration.
And (4) analyzing results: the inclusion bodies are subjected to gel cutting and purification to obtain a large amount of purified UL2 protein, SDS-PAGE analysis shows that the purified recombinant UL2 protein has high purity, Western blotting analysis shows that the recombinant protein can have strong immunoreaction with duck DPV-resistant positive serum (see figure 3), and the recombinant protein can be used as an envelope antigen for identifying duck plague commercial attenuated vaccine immunity and wild virus infection colloidal gold immunochromatographic test paper for detecting DPV antibodies.
Example 2 Induction expression of Duck plague Virus gI protein and purification of the product
(1) The expression strain containing pET32a-gI plasmid was inoculated into liquid LB containing 50. mu.g/ml Amp resistance. Culturing at 37 ℃ overnight, taking the bacterial liquid the next day according to the proportion of 1: 100 in the ratio of 500ml LB liquid medium (containing Amp 50u g/ml), intense shaking culture to OD600When the concentration is 0.5, IPTG is added to the final concentration of 0.2mmo/l, 1ml of culture solution is collected after 2h of induction, centrifugation is carried out for 10min at 12000rpm at 4 ℃, the supernatant is discarded, 80 mu l of ultrapure water and 20 mu l of 5 × loading buffer are added into the precipitate, heating and denaturation are carried out in 100 ℃ water bath for 5-10min, 15% SDS-PAGE gel electrophoresis is carried out, and the expression result is observed.
And (4) analyzing results: the result of induction expression of expression host bacterium BL21(DE3) containing recombinant plasmid pET32a-gI by IPTG and induction without IPTG shows that no specific protein band appears in the strain without IPTG induction; recombinant expression plasmid pET32a-gI expressed the recombinant protein at 65kDa (see FIG. 4).
(2) The solubility analysis of the recombinant plasmid pET32a-gI expression product comprises the following steps of respectively treating 500ml of bacterial liquid with induced expression and 500ml of bacterial liquid without induced expression: centrifuging at 4 deg.C and 5000rpm/min for 30min, and suspending thallus precipitate with 50ml20mmol Tris-HCl (pH8.0); after being placed at-20 ℃ overnight, the thalli are intermittently crushed by ultrasonic waves (ice bath) (600w 30 s/time, 3 times), centrifuged for 10min at 4 ℃ and 1000rpm/min, and the supernatant is taken for later use (I); the precipitate was dissolved in 10ml PBS and stored at low temperature for further use. Mu.l of the supernatant (first) and 80. mu.l of the precipitate (second) dissolved in PBS were added to 20. mu.l of 5 XSDS-PAGE sample buffer, boiled in 100 ℃ water bath for 5-10min, subjected to 15% SDS-PAGE gel electrophoresis, stained with Coomassie brilliant blue, and the results were observed. And scattering, scanning and analyzing the relative percentage content of the recombinant protein in the induced bacteria liquid in the coating slurry (supernatant (first) and the soluble) and the sediment (second) and the inclusion body form) by a full-automatic gel imaging analysis system.
And (4) analyzing results: after the 500ml bacterial liquid of induced expression is processed by solubility analysis, electrophoresis results show that the expressed protein is mainly present in the precipitate, which indicates that the recombinant expressed protein is largely present in insoluble inclusion body form in the thallus.
(3) And (3) cutting and purifying a large amount of inclusion bodies of the duck plague gI protein by gel: taking out the inclusion body stored at the temperature of-20 ℃, thawing, and then mixing according to the weight ratio of 5: 1 was added to 5 XSDS-PAGE loading buffer (0.313M Tris HCl pH6.8, 50% glycerol, 10% SDS and 0.05% bromophenol blue, lysed with 100mM DTT) and boiled for 10 min. The glass plates were mounted as described and an appropriate volume of 8% release glue was dispensed. The glass was filled and sealed with absolute ethanol. After the gel was solidified, the absolute ethanol was poured out and blotted dry with absorbent paper. Without inserting a comb, 5% volume of the concentrated gel was added to the glass plate, leaving room for the sample. Electrophoresis was carried out at 80V 30min, 120V2 h. After completion of the electrophoresis, the PAGE gel was removed and immersed in pre-chilled 0.3mol/l KCl at 4 ℃. The strips are cut after white stripes appear. The bands were cut and placed in dialysis bags, and then the appropriate amount of PBS was added. The dialysis bag was electroeluted in Gly buffer. After electroelution at 120V2h, the target protein was collected and dialyzed in PBS buffer overnight. Stored at-20 ℃. The BCA protein assay kit determines protein concentration.
And (4) analyzing results: the inclusion bodies are subjected to gel cutting and purification to obtain a large amount of purified gI proteins, SDS-PAGE analysis shows that the purified recombinant gI proteins have high purity, Western blotting analysis shows that the recombinant proteins can have strong immunoreaction with duck DPV-resistant positive serum (see figure 4), and the recombinant proteins can be used as an envelope antigen for identifying duck plague attenuated vaccine immunity and wild virus infection colloidal gold immunochromatographic test paper for detecting DPV antibodies.
Example 3 pH optimization of protein labeling with colloidal gold solution
And (3) taking 6 Eppendorf tubes, and respectively adding 500 mu l of 20nm colloidal gold solution with the serial number of 1-6.
1, 2, 3, 4, 5 and 6. mu.l of 0.1mol/l K2CO3Add separately to the EP tube with the corresponding number and mix well. To each tube was added 5. mu.g of DPV-UL2 protein. After mixing, the mixture was allowed to stand at room temperature for 30min and then centrifuged at 12,000rpm/min for 30 min. And (4) observing whether the sediment exists at the bottom of the test tube, then flicking the bottom of the test tube, and observing whether the colloidal gold labeled protein sediment is redissolved and whether the redissolution is complete. Colloidal gold labels K added to a soft and easily-redissolved tube of UL2 protein precipitate2CO3The amount of pH adjustment is optimal. The same method is used for optimizing the optimum labeling pH of the gI protein labeled by the colloidal gold solution and the goat anti-rabbit IgG.
Example 4 determination of the coating concentration of purified recombinant UL2 protein and gI protein on nitrocellulose Membrane
The coating concentrations of C-line rabbit IgG and gI protein were fixed, and the purified recombinant UL2 protein was then diluted with PBS to various concentrations of 2.5mg/ml, 2.0mg/ml, 1.5mg/ml, 1.0mg/ml, 0.5 mg/ml. And (3) scribing the test paper on a nitrocellulose membrane by using an XYZ-3000 three-dimensional dot spraying instrument, assembling colloidal gold immunochromatography test paper, and detecting a serum sample. The optimal UL2 working concentration was determined from the color development of the T1 line (fig. 7). The same procedure was performed to optimize the streaking concentration of detection line gI protein on nitrocellulose membrane (fig. 8).
Example 5 determination of dilution concentration of colloidal gold-labeled recombinant protein
Fixing the concentration of the colloidal gold labeled gI protein and the colloidal gold labeled goat anti-rabbit immunoglobulin. The colloidal gold labeled UL2 protein was resuspended in different amounts of resuspension buffer to obtain different concentrations of colloidal gold labeled UL 250. mu.g/ml, 100. mu.g/ml, 150. mu.g/ml, 200. mu.g/ml, 250. mu.g/ml. Mixing with colloidal gold labeled gI protein and colloidal gold labeled goat anti-rabbit IgG according to the ratio of 1: 1: 1, fixing the mixture on a gold label pad by using a gold spraying instrument to assemble the colloidal gold immunochromatographic test paper. And (3) detecting a serum sample, comparing the color development condition, and determining the optimal colloidal gold labeled UL2 protein concentration as the optimal working concentration (figure 5). The same procedure was used to optimize the concentration of colloidal gold-labeled gI protein (FIG. 6).
EXAMPLE 6 preparation of the colloidal gold immunochromatographic test strip
Through the optimization and screening, the optimal conditions and the preparation process for identifying the duck plague attenuated vaccine immunity and the wild virus infection colloidal gold immunochromatographic test paper are as follows:
(1) an XYZ-3000 three-dimensional dot spraying instrument is used for marking the recombinant UL2 protein and the gI protein with the protein concentrations of 1.5mg/ml and 2.5mg/ml on a nitrocellulose membrane to form T1 and T2 lines, goat anti-rabbit IgG with the concentration of 30 mu g/ml is fixed on a quality control line C, and the nitrocellulose membrane is dried at 37 ℃ for 2 hours and then is stored at 4 ℃ in a sealing way for later use.
(2) Soaking the glass cellulose membrane in a mixed solution of 200 mu g/ml colloidal gold labeled recombinant UL2 protein, 250 mu g/ml colloidal gold labeled gI protein and colloidal gold labeled goat anti-rabbit IgG, and drying at 37 ℃ overnight to prepare a gold-labeled pad.
(3) Respectively adhering a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad (absorbent paper) on a white plastic back plate in sequence, wherein a detection line T1 on the nitrocellulose membrane is close to the gold-labeled pad, a detection line T2 is close to the absorbent paper, a line C is positioned between the detection lines T1 and T2, then assembling the test strips into test strips, cutting the test strips into test strips with the width of 0.4cm by using an LN-5000 cutting machine, putting the test strips into a card shell, sealing and storing the test strips at 4 ℃.
Examples of the experiments
1. Colloidal gold test paper strip detection sample result explanation
After the serum sample was diluted with the PB buffer solution, 80. mu.L of the diluted serum sample was dropped onto the sample pad as shown in FIG. 2. If the detection sample is negative serum, the serum sample cannot be combined with the gI protein and the UL2 protein marked by the colloidal gold at the gold-marked pad, and two red detection line strips cannot be formed; however, the colloidal gold labeled goat anti-rabbit IgG still reaches the quality control line due to chromatography and is combined with the rabbit IgG. If the sample to be detected is commercial attenuated vaccine immune duck serum, the sample is combined with the colloidal gold labeled gI protein on the gold labeled pad to form a colloidal gold labeled gI protein-serum antibody complex. And then combining with the gI protein at the detection line T2 on a nitrocellulose membrane to form a colloidal gold labeled gI protein-serum antibody-gI protein complex, thereby forming a red band, and because the anti-UL 2 antibody does not exist in the duck serum after duck plague attenuated immunization, the complex cannot be combined with the UL2 protein of the T1 line to form the red band. If the detection sample is duck serum infected by duck plague virus wild virus, the serum antibody is combined with the gI protein marked by colloidal gold and the UL2 protein marked by colloidal gold at the gold-marked pad, and then is combined with detection lines T1 and T2 on the nitrocellulose membrane to form two red strips.
2. Sensitivity test
DPV positive sera were taken as 1: 20. 1: 40. 1: 60. 1: 80. 1: 100. 1: 120 and 1: diluting by 140 times, and detecting by using the established colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection. Results when positive sera were diluted to 1: at 100, three clear red bands are still visible (fig. 9). This method was shown to be able to detect 1: DPV positive serum diluted by 100 times has stronger sensitivity.
3. Specificity test
The established colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection is used for respectively detecting DPV positive serum, duck plague Riemerella positive serum, duck salmonella positive serum, duck pasteurella positive serum, duck Tembusu virus positive serum, duck hepatitis I positive serum, duck hepatitis III positive serum, duck influenza virus positive serum and duck goose parvovirus positive serum. The specificity of the method was observed.
The DPV positive serum, duck Riemerella anatipestifer positive serum, duck salmonella positive serum, duck pasteurella positive serum, duck Tembusu virus positive serum, duck hepatitis I positive serum, duck hepatitis III positive serum, duck influenza virus positive serum and duck goose parvovirus positive serum are respectively detected by the colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection established by the method, the result shows that only three clear red strips can be seen from the DPV positive serum, and only one clear red strip can be seen from other sera at the C line (figure 10), thereby showing that the test paper has good specificity.
4. Repeatability test
In-batch repeatability test: the colloidal gold immunochromatographic test paper established by the method is used for respectively detecting non-immune duck negative serum (1 part) and DPV positive serum (1 part) for 3 times of repetition and observing the result.
Batch to batch repeatability test: and (3) respectively detecting non-immune duck negative serum (1 part) and DPV positive serum (1 part) by using test strips prepared in different batches, and observing results.
The results of the in-batch repeatability tests show that: the 3-time repeated detection results of the non-immune duck negative serum and the DPV positive serum are consistent; the results of the batch to batch repeatability tests show: the test paper strips prepared by 3 different batches are used for respectively detecting the non-immune duck negative serum and the DPV positive serum, and the obtained results are also uniform. The colloidal gold immunochromatographic test paper is proved to have good repeatability in batches and among batches.
5. The colloidal gold immunochromatographic test paper has higher sensitivity than neutralization test
Neutralization experiment: taking 11 parts of serum sample to be detected, removing bacteria on a 0.22 mu m microporous filter membrane to prevent pollution, and inactivating complement in the serum in a 56 ℃ water bath for 30 min; 250 μ L MEM serum samples were serially diluted 2-fold; the DPV CHv virus was diluted to 1000TCID50 in MEM, 250. mu.L of the diluted virus was added to each diluted serum sample, mixed well in a dilution tank, added to a 96-well cell plate, and incubated at 37 ℃ for 3.5 hours. Preparing duck embryo fibroblasts, subpackaging the duck embryo fibroblasts into a 96-well cell plate, adding 100 mu L of cell suspension into each well, culturing the duck embryo fibroblasts in a 37 ℃ 5% carbon dioxide incubator for about 36h until the cells overgrow the cell plate, adding a mixed solution of serum to be detected and viruses into the duck embryo fibroblasts, culturing the duck embryo fibroblasts in a 5% carbon dioxide incubator for about 6d at 100 mu L and 37 ℃ per well, observing cytopathic effect under a microscope, counting the number of pathological changes, and calculating the titer of neutralizing antibodies in the serum to be detected by using a Reed-Muench formula.
Detecting by using colloidal gold immunochromatographic test paper: and (3) diluting 11 serum samples by 2 times, then dropwise adding the diluted serum samples into a sample adding hole of the colloidal gold test strip, and observing the maximum sample dilution which can be detected by the colloidal gold test strip as the sensitivity of the colloidal gold test strip.
The above test results are shown in table 1:
TABLE 1 results of comparing the sensitivity of the colloidal gold immunochromatographic test paper with that of the neutralization test
| Serum.no | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| NT | 0 | 2-2 | 2-4 | 2-4.375 | 2-3.33 | 2-3.16 | 2-3.16 | 2-4.33 | 2-4.33 | 2-4.5 | 2-3.87 |
| CGS | 0 | 1:100 | 1:60 | 1:120 | 1:40 | 1:100 | 1:60 | 1:60 | 1:60 | 1:80 | 1:60 |
As can be seen from Table 1, the sensitivity of the colloidal gold test strip is higher than that of the neutralization test method.
6. Detection of clinical serum sample to be detected
123 serum samples were selected for compliance comparison with the UL2-ELISA method. UL2 protein was added to the plate at a concentration of 62.5 ng/. mu.L, 100. mu.L/well, and the plate was coated overnight at 4 ℃. Discarding the coating solution, patting the liquid in the ELISA plate, washing with PBST buffer solution for 3 times (200 μ L/well), each time for 5min, and patting the liquid for the last time. Add 200. mu.L blocking solution (10% BSA) to each well, incubate for 1h at 37 ℃; after removing the blocking solution, PBST buffer solution was washed 3 times at 200. mu.L/well for 5min with shaking. Diluting the serum to be detected by PBS buffer solution, adding the diluted serum to an ELISA plate, incubating for 2h at the temperature of 37 ℃ at a concentration of 100 mu L/hole; discarding the liquid, washing with 200 μ L/well PBST buffer solution for 3 times, each time for 5min, and drying the liquid for the last time; diluting HRP-labeled goat anti-duck IgG with PBS buffer solution 1:800, adding 100 μ L diluted goat anti-duck IgG into each hole, incubating for 1h at 37 ℃, and washing for 3 times with PBST buffer solution; and (3) adding TMB color development solution after sufficiently spin-drying the washing solution in the 96-well plate, incubating at the temperature of 37 ℃ for 15min, and adding 50 mu L stop solution into each reaction hole to stop reaction. And reading the OD value of 450nm by using a microplate reader. And judging whether the ELISA detected sample is negative or positive according to the OD value. Similarly, the 123 serum samples are diluted 2 times and then dropped into the sample adding holes of the colloidal gold test strips, and the serum samples are judged to be wild virus infection, commercial attenuated vaccine immunity or negative serum according to the color development condition.
The above test results are shown in Table 2:
TABLE 2 results of the test using colloidal gold immunochromatographic strip and UL2-ELISA
As can be seen from table 2, the positive coincidence rates of the two detection methods were 51/(51+4) × 100%: 92.7%, the negative coincidence rates were (38+21)/68 × 100%: 86.7%, and the total coincidence rate was: the result of (51+38+21)/123 × 100% ═ 89.4% indicates that the UL2-ELISA method established in the present study has a high coincidence rate with the detection method using colloidal gold immunochromatographic test paper.
7. Stability test
The test strip is dried, sealed and then placed at 4 ℃ for sealed storage, non-immune duck negative serum (1 part) and DPV positive serum (1 part) are detected respectively in 3 months, 6 months, 9 months and 12 months, the result of detecting the non-immune duck negative serum (1 part) by colloidal gold is negative serum, the result of detecting the positive serum is positive serum, and the colloidal gold test strip has better stability according to the current color development effect.
Claims (10)
1. A colloidal gold immunochromatographic test paper for identifying duck plague attenuated vaccine immunity and wild virus infection is characterized by comprising the following components: the kit comprises a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad and a bottom plate; the sample pad, the gold label pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate; wherein, the colloidal gold labeled duck plague virus UL2 protein, the colloidal gold labeled duck plague virus gI protein and the colloidal gold labeled goat anti-rabbit IgG are adsorbed on the gold label pad; the cellulose nitrate membrane contains a T1 line, a T2 line and a C line, the T1 line contains duck plague virus UL2 protein, the T2 line contains duck plague virus gI protein, and the C line contains rabbit IgG.
2. The colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection according to claim 1, characterized in that the coating concentration of duck plague virus UL2 protein on nitrocellulose membrane is 0.5-2.5 mg/ml.
3. The colloidal gold immunochromatographic strip for use in the discrimination of duck plague attenuated vaccine immunity and wild virus infection according to claim 1, characterized in that the coating concentration of duck plague virus gI protein on nitrocellulose membrane is 0.5-2.5 mg/ml.
4. The colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection according to claim 1, characterized in that the concentration of the colloidal gold labeled duck plague virus UL2 protein is 50-250 μ g/ml; the concentration of the colloidal gold labeled duck plague virus gI protein is 50-250 mug/ml; the concentration of the colloidal gold labeled goat anti-rabbit IgG is 20-40 mug/ml.
5. The colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection according to claim 1 or 4, characterized in that the colloidal gold labeled duck plague virus UL2 protein is prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, adding the duck plague virus UL2 protein, and mixing uniformly to obtain the final product.
6. The colloidal gold immunochromatographic strip for use in the discrimination of duck plague attenuated vaccine immunity and wild virus infection according to claim 1 or 4, characterized in that the colloidal gold labeled duck plague virus gI protein is prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, then adding the duck plague virus gI protein, and uniformly mixing to obtain the product.
7. The colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection according to claim 1 or 4, characterized in that the colloidal gold labeled goat anti-rabbit IgG is prepared by the following method: mixing the colloidal gold solution and the alkaline solution, adjusting the pH value of the colloidal gold solution to the isoelectric point of protein, adding goat anti-rabbit IgG, and mixing uniformly to obtain the product.
8. The colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection according to claim 1, characterized in that the gold pad is prepared by the following method: soaking the glass cellulose membrane in a mixed solution containing 50-250 mu g/ml of colloidal gold-labeled duck plague virus UL2 protein, 50-250 mu g/ml of colloidal gold-labeled duck plague virus gI protein and 20-40 mu g/ml of colloidal gold-labeled goat anti-rabbit IgG, and drying at 37 ℃ overnight to obtain the product.
9. The method for preparing the colloidal gold immunochromatographic strip for discriminating duck plague attenuated vaccine immunity and wild virus infection as claimed in any one of claims 1 to 8, which comprises the following steps:
(1) respectively scribing duck plague virus UL2 protein, gI protein and rabbit IgG on a nitrocellulose membrane to form a T1 line, a T2 line and a C line, and then placing the nitrocellulose membrane at 37 ℃ for drying for 2 h;
(2) and (2) sequentially adhering the sample pad, the gold-labeled pad, the nitrocellulose membrane treated in the step (1) and the water absorption pad on a bottom plate, wherein a T1 line on the nitrocellulose membrane is close to the gold-labeled pad, a T2 line on the nitrocellulose membrane is close to water absorption paper, a C line is positioned between the detection lines T1 and T2, and then cutting the nitrocellulose membrane into test strips.
10. The method for discriminating duck plague attenuated vaccine immunity and wild virus infection using the colloidal gold immunochromatographic strip of any one of claims 1 to 8, comprising: dropwise adding the serum sample on the sample pad through the sample adding hole, and judging that the serum sample is from duck plague attenuated vaccine immunized ducks or wild virus infected ducks according to color development results on lines T1, T2 and C;
when the T1, T2 and C lines are all colored, the serum sample is indicated to be from a wild infected duck;
when only the T2 line and the C line are developed, the serum sample is from duck plague attenuated vaccine immunized ducks;
when only the C line is developed, the serum sample is from a healthy duck;
if the C line does not develop color, the test strip is invalid.
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