CN111704656A

CN111704656A - Duck adenovirus I type Penton protein and preparation method and application thereof

Info

Publication number: CN111704656A
Application number: CN202010630390.6A
Authority: CN
Inventors: 王雪平; 张孝俊; 梁秀丽
Original assignee: Anyang Institute of Technology
Current assignee: Anyang Institute of Technology
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2020-09-25

Abstract

The invention belongs to the field of protein engineering, and particularly relates to duck adenovirus type I Penton protein and a preparation method and application thereof. The inventors analyze and find that the Penton protein is hydrophilic, has no transmembrane structural domain, has no advantage selection such as signal peptide prediction and the like to carry out full-length expression. The Penton target segment is obtained by artificial synthesis after sequence optimization, and the expression mode of the protein is cell-free protein expression. Compared with the traditional Penton protein antigen dominant epitope truncated expression, the accuracy of the artificially synthesized and expressed Penton recombinant protein is high, the neutralization of the protein is ensured, the trouble of high mutation rate is avoided, and the development of the antibody detection technology is facilitated. In addition, the expression system has unique advantages, including time savings and increased overall yield of soluble, full-length proteins. In addition, the expression system has low cost, large expression quantity and strong reaction property of Western Blot detection.

Description

Duck adenovirus I type Penton protein and preparation method and application thereof

Technical Field

The invention belongs to the field of protein engineering, and particularly relates to duck adenovirus type I Penton protein and a preparation method and application thereof.

Background

Duck adenovirus type I virus (Duck adenovirus type 1, DAV 1) is a type of adenovirus including Egg Drop Syndrome Virus (EDSV) or EDSV-like, and is a member of the genus Thoracivirus (Atadenvirus) of the family adenoviridae. The representative virus is Egg Drop Syndrome (EDSV), which causes viral infectious diseases that decrease the laying rate of laying hens or breeding hens. After the laying hens are infected with the EDS virus, the egg yield of a chicken group cannot reach a peak period or is greatly reduced, the egg yield can be reduced by 20-30%, the development of the laying hen industry is seriously influenced, huge economic loss is brought to vast farmers and breeding enterprises, and the harmfulness is very serious.

DAV1 has the distribution in the world at present, has brought serious economic loss for the poultry industry of many countries, and ELLSA has advantages such as simple and convenient, swift, sensitive, specific when detecting the antibody, obtains extensive application in the production field, but present kit mainly regards whole virus as the envelope antigen, and the process of purification virion is complicated, and has the risk of dispersing the poison, is not suitable for a series of difficult problems of popularization and application such as monitoring of a large amount of chicken crowd antibody levels and epidemiological investigation.

In view of the severity of the hazards of DAV1 to the poultry industry, serological diagnosis of poultry using reliable and rapid serodiagnostic techniques is the basis for effective prevention and control. Therefore, a kit for accurately diagnosing the DAV1 by a serological diagnosis method is established, and the kit has important significance for preventing and treating the disease.

Disclosure of Invention

The invention provides application of duck adenovirus type I Penton protein, which constructs recombinant plasmid of duck adenovirus type I Penton protein, then adds the plasmid into E.coli dialysis cell-free recombinant protein system and further establishes a kit by taking the purified protein as coating antigen.

The technical scheme of the invention is realized as follows:

a duck adenovirus type I protein, wherein the amino acid sequence of the Penton protein is shown as SEQ ID No. 1.

The preparation method of the duck adenovirus I type Penton protein comprises the following steps:

(1) synthesizing a Penton gene, and connecting the Penton gene between NdeI and BamHI of a pET-11a vector to obtain a recombinant plasmid pET-11 a-Penton;

(2) adding the recombinant plasmid pET-11a-Penton with correct sequencing in the step (1) as a template into an E.coli dialysis cell-free recombinant protein system, and standing for 16 hours at 25 ℃;

(3) and (3) collecting, centrifuging and collecting precipitates in the step (2), and performing affinity purification on the precipitates through a Ni column to finally obtain the duck adenovirus I type Penton protein.

The application of the Penton recombinant protein in preparing an indirect ELISA kit for detecting duck adenovirus type I antibody comprises an expression protein ELISA plate, negative control serum, positive control serum, an enzyme-labeled secondary antibody diluent, a buffer solution, a confining solution, a diluent, a washing solution, a developing solution and a stop solution.

The expression protein ELISA plate takes Penton recombinant protein as a coating antigen, each hole is coated with 0.4 mu g of the recombinant protein Penton, positive control is duck adenovirus I-resistant chicken serum, and negative control is SPF chicken serum.

The diluent or the washing solution is PBST containing 0.05 percent of Tween-20, and the stop solution is 2M H₂SO₄The solution is 100mL of TMB developing solution.

The invention has the following beneficial effects:

1. the inventors analyze and find that the Penton protein is hydrophilic, has no transmembrane structural domain, has no advantage selection such as signal peptide prediction and the like to carry out full-length expression. The Penton target segment is obtained by artificial synthesis after sequence optimization, and the expression mode of the protein is cell-free protein expression.

2. Compared with the traditional Penton protein antigen dominant epitope truncated expression, the accuracy of the artificially synthesized and expressed Penton recombinant protein is high, the neutralization of the protein is ensured, the trouble of high mutation rate is avoided, and the development of the antibody detection technology is facilitated. On the other hand, we have adopted a cell-free protein expression system that has unique advantages, including time savings and increased overall yield of soluble, full-length protein. In addition, the expression system has low cost, large expression quantity and strong reaction property of Western Blot detection. The indirect ELLSA method established based on the Penton protein as the coating antigen has good specificity. The detection result of clinical samples shows that the method can be used as a method for detecting the duck adenovirus type I antibody, and is favorable for popularization and application in most farmers at the basic level. The detection method of the invention also has the advantages of capability of simultaneously detecting a large number of samples and rapidness and convenience in detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram showing the accuracy of the restriction analysis and identification of the prokaryotic expression plasmid pET-11a-Penton in example 1 of the present invention.

FIG. 2 is a graph showing the result of IPTG-induced expression of the Penton protein, which is identified by SDS-PAGE analysis in example 1 of the present invention.

FIG. 3 is a graph showing the results of SDS-PAGE analysis to identify Ni column affinity purification induced expression in example 1 of the present invention.

FIG. 4 is a Western blot result chart according to example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

1. Amplification of a Penton protein sequence and construction of a prokaryotic expression plasmid pET-11 a-Penton:

the method comprises the steps of synthesizing a Penton protein gene by using a whole gene, wherein the amino acid sequence table of the Penton protein is SEQ.ID.No.1, the nucleotide sequence table of the recombinant Penton protein gene is SEQ.ID.No.2, inserting the recombinant Penton protein gene into a pET-11a expression vector through restriction enzyme sites NdeI and BamHI, respectively adding 3 mu L of the recombinant pET-11a-Penton plasmid, 0.25 mu L of endonuclease I, 0.25 mu L of endonuclease and 1.0 mu L of 10 xBuffer into a 25 mu L centrifugal tube, fully mixing the mixture, placing the mixture into a low-temperature connector at the temperature of 16 ℃ for carrying out a connection reaction for 30min to obtain a connection product, carrying out double enzyme digestion and sequencing on the connection product through restriction enzyme sites Nde I and BamHI to confirm the accuracy of a final expression vector, and obtaining the prokaryotic expression plasmid pET-11 a-Penton. FIG. 1 is a diagram showing the results of enzyme digestion identification of a prokaryotic expression plasmid pET-11a-Penton, in which M is 1Kb DNA Marker; lane 1 is pre-enzyme cut plasmid; lane 2 is the plasmid after digestion, and it can be seen from FIG. 1 that the Penton protein gene fragment matches the expected molecular weight, and sequencing shows that the construction of the Penton protein expression vector is successful.

2. Coli dialysis cell-free protein expression and purification

Recovering and massively culturing the bacterial strain containing the pET-11a-Penton plasmid, and performing PCR amplification on the target gene by using DNA primers at two sides of the gene sequence inserted into the pET-11a-Penton plasmid. Specifically, a forward primer (5'-GATCCCGCGAAATTA-3') upstream of the T7 promoter sequence and a reverse primer (5'-AAAAACCCCTCAAGA-3') downstream of the T7 terminator sequence were used. The DNA template was amplified using Z1 or Z2 DNA polymerase for PCR amplification. FIG. 2 is a graph showing the results of PCR amplification, in which M is 1Kb DNA Marker in FIG. 2;

lanes

1 and 2 are PCR amplification products, the amplified gene fragments correspond to the expected molecular weights. Adding the PCR amplification product into an E.coli dialysis cell-free protein expression system, standing the translation reaction at 25 ℃ for 16 hours, and identifying the sample by SDS-PAGE. FIG. 3 is a graph showing the results, in which M is 10-180Kb Protein Marker; lane 1 is the target protein expression profile; lane 2 is a control expression profile; as can be seen, the Penton protein was successfully expressed.

3. Ni column affinity purification and result analysis of fusion protein

The inclusion body solution was loaded to Ni-IDA Binding-Buffer pre-equilibrated at a flow rate of 0.5 ml/min using a low pressure chromatography system

Ni-IDA-Sepharose CL-6B affinity chromatography column; flushing with Ni-IDA Binding-Buffer at a flow rate of 0.5 ml/min until the effluent OD280 value reaches the baseline; washing with Ni-IDA Washing-Buffer (20M Tris-HCl, 20M M imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min until the effluent OD280 value reaches baseline; eluting the target protein with Ni-IDA Elution-Buffer (20M M Tris-HCl, 250 mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min, and collecting the eluate; adding the collected protein solution into a dialysis bag, and dialyzing overnight with PBS (pH 7.4); 10% SDS-PAGE analysis showed that at a relative molecular mass of about 54 kDa, 1 specific Protein band was observed, which was consistent with the expected size, and the results are shown in FIG. 4, where M is 10-180Kb Protein Marker; lane 1 is the protein of interest;

4. immunoblot assay verification result chart

The result is shown in figure 4, the purified Penton protein is verified by immunoblotting (Western blot), and the purified antibody can perform specific reaction with duck adenovirus type I protein, which shows that the prepared primary antibody has better reactivity. In the figure, M is 10-180Kb Protein Marker, lane 1 is Penton recombinant Protein, and the result shows that a band is obvious at the expected size, which proves that the purified Penton Protein has good reactogenicity.

Example 2

The embodiment is an application of the pentan protein described in embodiment 1, the pentan protein is used for an indirect ELISA kit for detecting duck adenovirus type i antibodies, the indirect ELISA kit comprises an expression protein ELISA plate, negative control serum, positive control serum, an ELISA secondary antibody diluent, a buffer solution, a confining solution, a diluent, a washing solution, a developing solution and a stop solution, and the coated ELISA plate takes the pentan protein as a coating antigen.

1. Preparation of enzyme-labeled secondary antibody: rabbit anti-duck IgG (produced by Beijing Baiolai Boke technology, Inc.) was diluted 100-fold with HRP conjugate stabilizer/diluent I (produced by English, Huzhou, Inc.).

2. Preparing a coated enzyme-labeled secondary antibody diluent, a buffer solution, a confining solution, a sample diluent and a washing solution:

the enzyme-labeled secondary antibody diluent is 0.01M phosphate buffer saline solution b with the pH value of 7.2-7.4, and the mass fraction of chicken serum in the phosphate buffer saline solution b is 5%;

the coating buffer solution is 0.05M carbonate buffer solution with the pH value of 9.6, and the preparation method of the carbonate buffer solution comprises the following steps: taking Na₂CO₃、1.59g、NaHCO₃2.93g, adding 800mL of sterilized double distilled water for dissolving, adjusting the pH to 9.6, and adding the sterilized double distilled water to 1000m L;

the confining liquid is 0.01M phosphate buffer salt solution a with the pH value of 7.2-7.4, the mass fraction of chicken serum in the phosphate buffer salt solution a is 5%, and the mass fraction of casein is 0.25%;

the sample diluent is 0.01M phosphate buffer salt solution d with the pH value of 7.2-7.4, the mass fraction of the chicken serum albumin in the phosphate buffer salt solution d is 0.1%, and the mass fraction of the sucrose in the phosphate buffer salt solution d is 2%.

3. Preparing a color developing solution and a stop solution:

the color development liquid comprises a color development liquid A and a color development liquid B; the preparation method of the color developing liquid A comprises the following steps: dissolving 200mg of tetramethylbenzidine in 100m L absolute ethyl alcohol, and diluting the solution to 1000m L by double distilled water; the preparation method of the color developing solution B comprises the following steps: weighing 21g of citric acid and 28.2g of anhydrous sodium phosphate, adding 6.4m L of 0.75% urea hydrogen peroxide solution, metering the volume of double distilled water to 1000m L, and adjusting the pH value to 4.5-5.0; the stop solution is 2M H₂SO₄And (3) solution.

4. Determination of ELISA reaction conditions:

determination of optimal coating concentration optimal dilution factor of sample: respectively diluting the antigen (Penton protein) by a multiple ratio according to (1 mu g/mL, 2 mu g/mL, 4 mu g/mL, 8 mu g/mL, 16 mu g/mL, 32 mu g/mL and 64 mu g/mL) by adopting a matrix method, sequentially coating the antigen (Penton protein) in an enzyme label plate, repeating 4 holes for each gradient, coating the antigen in each hole at 4 ℃ by 100 mu L, diluting the negative control serum and the positive serum by sample diluent of 1: 25, 1: 50, 1: 100 and 1: 200 times respectively, performing indirect ELISA, finally determining the optimal antigen coating amount to be 4 mu g/mL and 100 mu L/hole according to a P/N value (positive sample OD value/negative sample OD value), and determining the optimal dilution of the sample to be 1: 50 The enzyme-labeled secondary antibody action time, the substrate color development time and the like are optimized one by one, and finally the optimal reaction conditions of the indirect ELISA of the duck adenovirus type I antibody are determined as follows: the optimal amount of coating antigen is 5 mug/mL, 100 mug/hole, coating for 16h at 4 ℃, and sealing for 2h at 37 ℃; diluting the serum to be detected by 50 times, and incubating for 0.5h at 37 ℃; enzyme-labeled secondary antibody dilution is 1: 10000, 37 ℃ for 0.5 h; the substrate color development condition is that TMB (3,3',5,5' -tetramethyl benzidine) substrate is developed for 10min at room temperature in dark.

5. Determination of negative and positive cut-off values for indirect ELISA

Under the optimal conditions of indirect ELISA, 46 negative serum samples from non-infected duck adenovirus type I are used to determine the Cut-Off value (Cut-Off) of the indirect ELISA, the calculation method is to add three times of standard deviation (S.D.) to the average value of 46 negative serum OD450, and the test sample OD450nm is determined to be positive when the value is greater than or equal to the Cut-Off value, otherwise, the test sample is determined to be negative.

The cutoff value of the indirect ELISA method established by pentan was calculated to be 0.425 according to the cutoff calculation formula (Cut-Off ═ negative sample OD450 mean value +3 × s.d.). According to the method, when a duck serum sample is detected by using an indirect ELISA method established by Penton, the duck adenovirus type I antibody is determined to be positive when the OD450 value is more than or equal to 0.534; on the contrary, when the OD450 value is less than 0.534, the duck adenovirus type I antibody is determined to be negative.

6. Intra-and inter-batch reproducibility of indirect ELISA detection methods

9 duck adenovirus type I positive sera were selected to determine the intra-and inter-batch reproducibility of the indirect ELISA. Respectively detecting 9 positive serum samples in the ELISA plates coated in the same batch and 5 different batches, making 3 repeated holes on the same ELISA plate for each sample, determining OD450 values, respectively calculating the OD450 average value and Standard Deviation (SD) of each serum sample, and determining the in-batch and inter-batch repeatability of the established indirect ELISA method by calculating the coefficient of variation. The result shows that the intra-batch repeated variation coefficient of the indirect ELISA method established based on Penton is between 1% and 4.57%, the inter-batch repeated variation coefficient is between 1.58% and 5.25%, and all the variation coefficients are within 10%, which indicates that the indirect ELISA method established based on Penton has good repeatability and reliable result.

7. Specificity analysis of Indirect ELISA method

The OD values of positive sera of duck adenovirus type I (DAdV-1), Avian Influenza Virus (AIV), newcastle disease virus, avian adenovirus type 4 (FAdV-4), avian adenovirus type 8 (FAdV-8) and avian Reovirus (REO) chickens are simultaneously determined by the ELISA method established above, and the result shows that only the detection result of the serum of the duck adenovirus type 1 is positive, and the detection results of the positive sera of other pathogens are negative.

8. The duck adenovirus type I indirect ELISA detection kit comprises:

(1) the coated plate is coated with an enzyme label plate (coated with Penton protein) 6;

(2) negative control serum 3m L;

(3) positive control serum 3m L;

(4) enzyme-labeled secondary antibody 0.8m L;

(5) enzyme-labeled secondary antibody diluent 80m L;

(6) sample diluent 150m L;

(7) color developing liquid a 50m L;

(8) color developing solution B50 m L;

(9) stop solution 50m L;

(10) concentrated washings (20 × washings) 150m L;

(11) 8 cover plate films;

(12) 8 serum dilution plates;

(13) 1 part of a specification.

9. The duck adenovirus type I antibody indirect ELISA kit operation instruction:

(1) coating: taking the purified Penton protein as a coating antigen, diluting the Penton protein with a carbonate buffer solution, adding the diluted Penton protein into an ELISA plastic hole plate with 96 holes, coating each hole with 100 mu L of the solution at 4 ℃ overnight; discarding redundant antigens in the coated wells, adding 200 mu L of PBST into each well, washing for 4 times, and then patting the plate to be dry;

(2) and (3) sealing: adding 200 mu L of sealing liquid into each hole, sealing for 2h at 37 ℃; discarding the confining liquid, washing with PBST for 4 times, and drying after washing;

(3) adding serum to be detected: adding 100 mu L of chicken serum diluted by PBS buffer solution into each hole, and incubating for 1h at 37 ℃; discarding redundant serum in the incubated hole, washing with PBST for 4 times, and drying after washing;

(4) adding a secondary antibody: mu.L of goat anti-chicken IgG enzyme-labeled secondary antibody diluted with PBS buffer was added to each well and incubated at 37 ℃ for 1 h. Then, discarding redundant enzyme-labeled secondary antibodies in the incubated holes, washing for 4 times by using PBST, and then patting dry after washing;

(5) color development: adding 100 μ L of TMB color developing agent into each well, and developing at 37 deg.C in dark for 10 min; and (4) terminating: blue-green color appears in duck type 1 adenovirus positive serum sample hole, and 0.5M HSO is added immediately₄Terminating the reaction;

(6) and (5) judging a result: after the reaction was terminated, the reaction mixture was placed in a microplate reader, and the OD value of each sample was read at 450 nm. Immediately reading on a microplate reader at 450nm, and determining the titer of the sample according to the dilution corresponding to the well with the OD value being greater than 2.1 times of the set negative control OD value.

Example 3

98 parts of chicken serum samples which are clinically checked are detected by using the duck adenovirus type I antibody indirect ELISA method. The chicken serum detects 25 parts of duck type 1 adenovirus positive serum, the positive rate is 25%, which indicates that the duck type 1 adenovirus has higher infection rate in the chicken group to be inspected, and the indirect ELISA method established by taking the purified Penton protein as the coating antigen has the characteristics of good specificity and high sensitivity.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Gly Ala Ala Ala Ala Ala Cys Ala Gly Cys Cys Thr Gly Cys Cys Gly

1235 1240 1245

Gly Gly Thr Gly Thr Thr Cys Ala Ala Ala Gly Ala Ala Thr Ala Ala

1250 1255 1260

Cys Thr Cys Thr Gly Ala Cys Cys Gly Ala Cys Gly Ala Thr Ala Gly

1265 1270 1275 1280

Gly Cys Gly Gly Cys Gly Cys Ala Cys Thr Thr Gly Cys Cys Cys Gly

1285 1290 1295

Thr Ala Cys Ala Thr Cys Thr Ala Cys Ala Ala Ala Thr Cys Thr Thr

1300 1305 1310

Thr Gly Gly Cys Cys Cys Gly Cys Gly Thr Ala Thr Cys Thr Cys Cys

1315 1320 1325

Thr Cys Gly Gly Gly Thr Gly Ala Thr Thr Thr Cys Thr Ala Gly Cys

1330 1335 1340

Gly Cys Thr Ala Cys Thr Thr Thr Gly Cys Ala Ala Thr Ala Ala

1345 1350 1355

Claims

1. Duck adenovirus type I Penton protein, its characterized in that: the amino acid sequence of the recombinant protein Penton is shown as SEQID No. 1.

2. The method for preparing duck adenovirus type I Penton protein according to claim 1, characterized in that the method comprises the following steps:

3. The use of the pentan protein of claim 2 in the preparation of an indirect ELISA kit for detecting duck adenovirus type I antibodies, wherein the kit comprises: the indirect ELISA kit comprises an expression protein ELISA plate, negative control serum, positive control serum, an enzyme-labeled secondary antibody diluent, a washing solution, a developing solution and a stop solution.

4. Use according to claim 3, characterized in that: the expression protein ELISA plate takes Penton as a coating antigen, each hole is coated with 0.5 mu g of the Penton protein, positive control is duck adenovirus I-resistant chicken serum, and negative control is SPF chicken serum.

5. Use according to claim 4, characterized in that: the diluent or the washing solution is PBST containing 0.05 percent of Tween-20, and the stop solution is 2M H₂SO₄The solution is 100mL of TMB developing solution.