CN112029792A

CN112029792A - Duck viral hepatitis egg yolk antibody with anti-inflammatory effect and preparation method and application thereof

Info

Publication number: CN112029792A
Application number: CN202010666445.9A
Authority: CN
Inventors: 刘金龙; 丁树新; 滕军; 庄晓峰; 赵学军
Original assignee: Shandong Bairui Kailai Biotechnology Co ltd
Current assignee: Shandong Bairui Kailai Biotechnology Co ltd; Shandong Best Care Biotechnology Co ltd
Priority date: 2020-07-10
Filing date: 2020-07-10
Publication date: 2020-12-04
Anticipated expiration: 2040-07-10
Also published as: CN112029792B

Abstract

The invention relates to the field of genetic engineering, and provides a duck virus hepatitis egg yolk antibody with an anti-inflammatory effect and a preparation method and application thereof, wherein a type I duck hepatitis virus and a duck spleen are taken as templates, VP1 and IL-6 genes are respectively obtained through RT-PCR and are connected to a PMD18-T vector for storage and standby; obtaining a VP1-IL6 fusion gene by utilizing an overlap extension PCR technology, connecting the fusion gene to an expression plasmid pPIC9 after double enzyme digestion, converting a connection product into a receptor bacterium, and finally identifying a positive transposon to obtain a pPIC9-VP1-IL6 positive strain; adding LPS into the expression product, emulsifying with a white oil adjuvant, preparing a vaccine, and immunizing the laying hens to prepare a yolk antibody; the invention successfully clones VP1 of DHV-I and duck IL-6 genes, constructs the two genes into fusion expression plasmid, realizes the high-efficiency expression of recombinant plasmid pichia pastoris, has good yolk antibody protection effect prepared by fusion protein, and has double functions of antivirus and antiphlogosis.

Description

Duck viral hepatitis egg yolk antibody with anti-inflammatory effect and preparation method and application thereof

Technical Field

The invention relates to the field of poultry epidemic disease immune control, relates to a preparation method and application of a duck viral hepatitis egg yolk antibody with an anti-inflammatory effect, and more particularly relates to a pPIC9-VP1-IL6 recombinant plasmid, a construction method, expression in pichia pastoris and application thereof.

Background

Duck viral hepatitis is a highly lethal infectious disease of duckling caused by Duck Hepatitis Virus (DHV). Clinically, the duck is characterized by obvious nervous symptoms, liver swelling and spot bleeding on the surface, mainly attacks ducklings within 6 weeks of age, particularly ducklings from 2 days to 3 weeks, are most susceptible, and adult ducks have resistance. With the rapid development of the duck breeding industry in China in recent years, the disease becomes one of the most serious epidemic diseases harming the duck breeding industry in China, and huge economic losses are brought to the duck breeding industry. The duck hepatitis virus comprises 4 viruses of 3 virus families of picornaviridae, astrovirus and hepatotropic virus, wherein the duck hepatitis virus type I (DHV-I) of the picornaviridae is distributed worldwide, and has the strongest toxicity, the highest infection rate and the most serious harm. The duck hepatitis virus type I occurs and spreads rapidly, and duckling infection is mainly manifested by listlessness, necking down, wing droop and anorexia. The affected duck has nervous symptoms and opisthotonus. Massive death often occurs within 3-5 days. The autopsy lesion is mainly concentrated on the liver, and is characterized by the phenomena of hepatomegaly, brittle and fragile liver and the like, dark or yellow liver color and mottled surface; in addition, gallbladder, spleen, pancreas and other organs also have pathological changes.

The disease mainly damages ducklings, but the immune system of the ducklings within 20 days of age is not completely developed, and the vaccine immune effect on the ducklings is not ideal generally, so the most common prevention and control method for the disease is to use the vaccine to immunize the breeding ducks so that the offspring ducklings obtain high-level maternal antibodies, thereby preventing the disease. However, the clinical morbidity is high due to the reasons that the immunity of the breeding ducks is not timely and standard, or the maternal antibody level of the ducklings is not high or the neutralizing effect is poor caused by virus variation. At present, commercial meat-substitute ducks are generally inoculated with egg yolk antibodies at 3-4 days of age to prevent the diseases or are inoculated with the egg yolk antibodies for treatment in an emergency at the early stage of the diseases. However, the clinical response often shows that the effect is not ideal when the refined yolk antibody is injected for treatment. Researches find that duck hepatitis virus is infected rapidly and rapidly, and the infection for 0.5-1d can induce a large amount of inflammatory cytokine expression to cause 'cytokine storm', which is an important reason for mass death of ducklings. However, the egg yolk antibody mainly contains a specific antibody aiming at duck hepatitis virus, even if the virus can be neutralized, the inflammation is not eliminated, and the duck severe ducklings in the duck group in the peak disease stage still die rapidly, which is a main reason for failure of clinically treating duck viral hepatitis. Therefore, how to effectively reduce the inflammatory reaction in the duckling body is an effective solution for improving the therapeutic effect of the antibody.

Inflammatory cytokines are a class of endogenous polypeptides produced primarily by cells of the immune system with many powerful biological effects that mediate a variety of immune responses. The overexpression of inflammatory cells in vivo can cause symptoms of multiple organ failure. Researches show that the duck hepatitis virus can induce the expression level of various cytokines to be increased after infecting ducklings, wherein the cytokines comprise IFN-alpha, IL-1 beta, IL-2, IL-6, IL-10, TNF-alpha and the like, and the IL-1 beta, the IL-2 and the IL-6 are proinflammatory cytokines and can induce serious inflammatory reaction. Whether inhibition of the activity of these cytokines can reduce the intensity of the inflammatory response in the duck is not relevant, and it is unclear in what manner the inhibition is carried out, and the therapeutic effect of inhibiting which cytokine activity is the best.

Therefore, whether to provide a technical scheme for solving the problem that the duck hepatitis virus antibody level is not high and effectively reducing the inflammatory reaction in the duckling body becomes a technical problem in the field.

Disclosure of Invention

The invention mainly aims to overcome the defects of the prior art and provide a preparation method and application of a duck virus hepatitis egg yolk antibody with an anti-inflammatory effect, in particular to a pPIC9-VP1-IL6 recombinant plasmid, a construction method, expression in pichia pastoris and application thereof, wherein a VP1 gene and an IL-6 gene are respectively obtained by taking a duck hepatitis virus type I (DHV-I) and a duck spleen as templates through RT-PCR and are stored for later use after being connected to a PMD18-T vector; redesigning primers for amplifying VP1 and IL-6 genes, adding a Linker sequence and an enzyme digestion site in the primers, and obtaining a VP1-Linker-IL-6 (abbreviated as VP1-IL6) fusion gene by using an overlap extension PCR technology. The fusion gene is connected into an expression plasmid pPIC9 after double enzyme digestion, the connection product is transformed into a receptor bacterium, and finally a positive transposon is identified to obtain a pPIC9-VP1-IL6 positive strain; LPS is added into the expression product, and the expression product is emulsified with a white oil adjuvant to prepare a vaccine, and then the vaccine is used for immunizing the laying hens to prepare the egg yolk antibody. The invention successfully clones VP1 of DHV-I and duck IL-6 genes, constructs the two genes into fusion expression plasmid, realizes the high-efficiency expression of recombinant plasmid pichia pastoris, has good yolk antibody protection effect prepared by fusion protein, and has double functions of antivirus and antiphlogosis.

The specific technical scheme of the invention is as follows:

(1) using duck hepatitis virus type I standard strain (strain preservation number: CVCC AV1540, obtained directly from the preservation organization) as template, designing primers for amplifying VP1 gene, specifically primers 1 and 2 (shown as SEQ ID NO: 6 and 7); specific primers are designed according to a predicted sequence (sequence number: XM-027450925.1) of duck IL-6 published by NCBI, primers 5 and 6 (shown as SEQ ID NO: 10 and 11) are designed, and the full-length duck IL-6 gene is amplified. Comparing the sequences of VP1 and IL-6 with the spectrogram information of pPIC9 plasmid to determine double enzyme cutting sites Xho I and Not I;

(2) the construction of the recombinant plasmid pPIC9-VP1-IL6 comprises the following steps:

PCR amplification of I type duck hepatitis virus VP1 gene:

extracting RNA of a duck hepatitis virus type I standard strain (a strain preservation number: CVCC AV1540, directly obtained from a preservation organization), performing reverse transcription on the RNA to obtain cDNA, taking the cDNA as a template, performing PCR amplification on a VP1 gene by using primers 1 and 2 (shown as SEQ ID NO: 6 and 7), recovering PCR product gel, wherein the VP1 gene sequence is shown as SEQ ID NO:1 is shown in the specification; connecting to a PMD18-T vector plasmid to obtain PMD18-T-VP1, and storing for later use;

secondly, designing primers Primer 3 and Primer 4 (shown as SEQ ID NO: 8 and 9) by taking the plasmid constructed in the step I as a template, introducing an Xho I enzyme cutting site sequence and an HIS label sequence into the upstream Primer 3, introducing a Linker sequence into the downstream Primer 4, and performing PCR amplification to obtain VP1-Linker, wherein the Primer sequence is shown as SEQ ID NO: 2 is shown in the specification;

PCR amplification of duck IL-6 gene: extracting duck spleen RNA, performing reverse transcription to obtain cDNA, performing PCR amplification on duck IL-6 gene by using primers 5 and 6 (shown as SEQ ID NO: 10 and 11); after PCR reaction amplification duck IL-6 gene and glue recovery, connecting to PMD18-T vector plasmid to obtain PMD18-T-IL6 recombinant plasmid, and storing for later use; the coding region (CDs) sequence of the duck IL-6 gene intercepted according to the sequencing result is shown as SEQ ID NO:3 is shown in the specification;

and (3) designing primers Pr imer 7 and Pr imer 8 (shown as SEQ ID NO: 12 and 13) by taking the plasmid constructed in the third step as a template, wherein the starting point of an upstream Primer 7 is an IL-6 sequence from which a signal peptide is removed, a linker is introduced into the Primer, a Not I enzyme cutting site sequence is introduced into a downstream Primer 8, and a duck IL-6-linker gene is obtained by PCR amplification, and is shown as SEQ ID NO: 4 is shown in the specification;

respectively recovering VP1-linker and IL-6-linker gene PCR amplification products as templates, and performing SOE-PCR amplification by using Primer 3 (shown as SEQ ID NO: 8) and Primer 8 (shown as SEQ ID NO: 13) as specific primers to obtain VP1-IL6 fusion gene (shown as SEQ ID NO: 5);

since the VP1-linker and the IL-6-linker are embedded with linker linkers, the two can be fused together in the subsequent amplification;

fourthly, connecting the fusion gene obtained by glue recovery to PMD18-T vector plasmid at 16 ℃ overnight, and obtaining PMD18-T-VP1-IL6 recombinant plasmid through transformation, plasmid enzyme digestion and sequencing;

fifthly, the recombinant plasmid PMD18-T-VP1-IL6 is cut by Xho I and Not I restriction enzymes, cut VP1-IL6 fusion gene glue is recovered and is connected with pPIC9 expression plasmid cut by the same restriction enzyme at 16 ℃ overnight, the connection is verified by transformation and enzyme digestion, and then the inserted exogenous gene sequence is verified to be correct by plasmid sequencing, wherein the plasmid is named as: pPIC9-VP1-IL 6.

Wherein, the primers Primer 5 and 6 (shown as SEQ ID NO: 10 and 11) used in the amplification of the duck IL-6 gene are designed from the 5 'non-coding region and the 3' non-coding region of the prediction sequence according to the duck IL-6 gene prediction sequence published by NCBI, the duck IL-6 gene is firstly amplified in the field, complete coding region (CDs) sequence information is obtained, the amplified duck IL-6 gene is compared with the duck IL-6 gene prediction sequence published by NCBI, and the sequences of the two are completely consistent, which indicates that the invention obtains the sequence from a living animal for the first time.

The PCR amplification components of the duck hepatitis virus type I VP1 are as follows: ddH₂O17.3. mu.L, 10 XBuffer (containing Mg)² ⁺)2.5 mu L, 1.0 mu L of upstream primer, 1.0 mu L, dNTP 2.0.0 mu L, cDNA downstream primer template, 1.0 mu L of high fidelity Taq enzyme, and 0.2 mu L of total volume of 25 mu L;

the PCR reaction conditions are as follows: pre-denaturation at 95.0 deg.C for 5 min; 35 cycles of denaturation, annealing and extension I, wherein the denaturation condition is 95.0 ℃ for 30s, the annealing condition is 56.0 ℃ for 45s, and the extension I condition is 72.0 ℃ for 1min for 30 s; extension II at 72.0 ℃ for 10 min;

a connection reaction system of the PMD18-T-VP1 recombinant plasmid: 4.5 mu L of VP1 gene, 0.5 mu L of PMD18-T plasmid and 5.0 mu L of Solution I, all the components are mixed uniformly and connected in water bath at 16 ℃ for 2 h.

Wherein the PCR amplification components of the I type duck hepatitis virus VP1-linker are as follows: ddH₂O17.3. mu.L, 10 XBuffer (containing Mg)²⁺)2.5 mu L, 1.0 mu L of upstream primer, 1.0 mu L, dNTP 2.0.0 mu L, PMD18-T-VP1 recombinant plasmid 1.0 mu L of downstream primer, 0.2 mu L of high fidelity Taq enzyme, and 25 mu L of total volume;

the PCR reaction conditions are as follows: pre-denaturation at 95.0 ℃ for 5 min; 35 cycles of denaturation, annealing and extension I, wherein the denaturation condition is 95.0 ℃ for 30s, the annealing condition is 56.0 ℃ for 45s, and the extension I condition is 72.0 ℃ for 1min for 30 s; extension II at 72.0 ℃ for 10 min.

The PCR amplification components of the duck IL-6 gene are as follows: ddH₂O17.3. mu.L, 10 XBuffer (containing Mg)²⁺)2.5 mu L, 1.0 mu L of upstream primer, 1.0 mu L, dNTP 2.0.0 mu L, cDNA downstream primer, 1.0 mu L of template, 0.2 mu L of high fidelity Taq enzyme and 25 mu L of total volume;

the PCR reaction conditions are as follows: pre-denaturation at 95.0 deg.C for 5 min; 35 cycles of denaturation, annealing and extension I, wherein the denaturation conditions are 95.0 ℃ for 30s, the annealing conditions are 58.0 ℃ for 45s, and the extension I conditions are 72.0 ℃ for 90 s; extension II at 72.0 ℃ for 10 min;

a connection reaction system of the PMD18-T-IL-6 recombinant plasmid: IL-6 gene 4.5 μ L, PMD18-T plasmid 0.5 μ L, Solution I Solution 5.0 μ L, mixing the components, connecting in water bath at 16 deg.C for 2 h.

Wherein the PCR amplification components of the duck IL-6-linker are as follows: ddH₂O17.3. mu.L, 10 XBuffer (containing Mg)²⁺)2.5 mu L, 1.0 mu L of upstream primer, 1.0 mu L, dNTP 2.0.0 mu L, PMD18-T-IL-6 recombinant plasmid 1.0 mu L of downstream primer, 0.2 mu L of high fidelity Taq enzyme and 25 mu L of total volume;

the Linker adopted above is a flexible Linker peptide segment Linker, the Linker is 10 amino acids, and the amino acid sequence is as follows: GGGGSGGGGS.

Wherein, the components of PCR amplification of the fusion gene are as follows: ddH₂O17.3. mu.L, 10 XBuffer (containing Mg)²⁺)2.5 mu L, 1.0 mu L of upstream primer, 1.0 mu L, dNTP 2.0, 2.0 mu L, IL-6-linker template 0.5 mu L, VP1-linker template 0.5 mu L of high fidelity Taq enzyme, and the total volume is 25 mu L; a

The PCR reaction conditions are as follows: pre-denaturation at 95.0 deg.C for 5 min; 35 cycles of denaturation, annealing and extension I, wherein the denaturation conditions are 95.0 ℃ for 30s, the annealing conditions are 58.0 ℃ for 45s, and the extension I conditions are 72.0 ℃ for 90 s; extension II at 72.0 ℃ for 10 min.

Wherein, the connection reaction system of the PMD18-T-VP1-IL6 recombinant plasmid comprises: 4.5 mu L of VP1-IL6 gene, 0.5 mu L of PMD18-T plasmid and 5.0 mu L of Solution I, all the components are mixed uniformly and connected for 2h in a water bath at 16 ℃;

double-restriction reaction system of PMD18-T-VP1-IL 6: 1.0 mu g of recombinant plasmid PMD18-T-VP1-IL6, 0.5 mu L of Xho I endonuclease, 0.5 mu L of Not I endonuclease and 2.0 mu L of 10 Xbuffer, supplementing sterilized ultrapure water to 20 mu L, uniformly mixing the components, and placing the components in a water bath at 37 ℃ for 2 hours; the double digestion products were electrophoresed through agarose gel to obtain a band of 2692bp and a band of 1373bp, indicating successful construction of the cloned plasmid.

pPIC9-VP1-IL6 recombinant plasmid ligation reaction system: 10 Xbuffer 1.0 μ L, VP1-IL 62.0 μ L, pPIC96.0 μ L after enzyme digestion, and T4DNA ligase 1.0 μ L, mixing the components uniformly, and connecting in water bath at 16 ℃ overnight;

the reaction system of the double-enzyme digestion pPIC9-VP1-IL6 is as follows: 1.0 mu g of pPIC9-VP1-IL6 recombinant plasmid, 0.5 mu L of Xhol I endonuclease, 0.5 mu L of Not I endonuclease and 2.0 mu L of 10 Xbuffer, and sterilized ultrapure water is supplemented to 20 mu L, and the components are mixed uniformly and then placed in a water bath at 37 ℃ for 2 h; and (3) carrying out agarose gel electrophoresis on the double digestion product, and carrying out digestion to obtain a strip with the size of 8000bp and 1373bp, which indicates that the fusion gene is successfully connected to an expression plasmid.

The invention also provides a method for expressing the pPIC9-VP1-IL6 recombinant plasmid in pichia pastoris, which comprises the following specific steps:

(1) expression of VP1-IL6 fusion protein in Pichia pastoris:

firstly, carrying out linearization on the constructed recombinant plasmid by Sal I incision enzyme digestion, carrying out dephosphorylation treatment, extracting and recovering phenol/chloroform, and dissolving in a TE buffer solution; at the same time, the pPIC9 empty vector plasmid is linearized by the same enzyme digestion, after dephosphorylation treatment, phenol/chloroform extraction recovery is carried out, and the recovered phenol/chloroform extraction is dissolved in TE buffer solution to be used as negative control;

respectively transforming the two plasmids into GS115 competent cells by a LiCl method, coating an RDB plate, and obtaining a recombinant plasmid positive transformant and an empty plasmid negative transformant;

secondly, extracting genomes of the two transformants as templates, amplifying Aox1 gene by PCR, identifying by agarose gel electrophoresis, generating a 2200bp and 493bp band on a negative transformant, and generating a 2200bp and 1816bp band on a positive transformant, which shows that the pPIC9-VP1-IL6 recombinant plasmid is successfully transformed;

thirdly, inoculating the screened positive transformants and negative transformants into 20mL BMGY medium, performing shake culture at 30 ℃ and 250rpm until OD is reached₆₀₀2-6, and centrifugally collecting thalli; then, adding an equal volume of BMMY culture medium for resuspension of the positive transformants, and adding 1/5 volume of BMMY culture medium for resuspension of the negative transformants; carrying out shake culture on the negative and positive transformants at 30 ℃ and 250rpm for 96h, supplementing methanol to 1% every 24h, taking 1.0mL of culture medium to analyze the expression level of the exogenous gene when the methanol is respectively 0, 12, 24, 36, 48, 60, 72, 84 and 96h, taking the negative transformants as a control, and finally determining the optimal bacteria receiving time to be 96 h;

(2) identification of VP1-IL6 fusion protein:

extracting DNA of the induced yeast, and extracting the DNA of the induced yeast by using Primer 3 with a gene sequence shown as SEQ ID NO: 8, and Primer 8 has a gene sequence shown as SEQ ID NO: 13, carrying out PCR reaction for the primers;

after the recombinants are induced by methanol, collecting the culture medium of the recombinants, and adding a sample buffer solution to carry out SDS-PAGE electrophoresis;

western-blotting is used for identifying the expression of the I type duck hepatitis virus VP1 and duck IL-6 fusion protein in the yeast recombinant.

In addition, in order to be conveniently applied, the invention also provides a preparation method of the yolk antibody with anti-inflammatory effect for duck viral hepatitis, in particular to a preparation method for preparing the yolk antibody by a pichia pastoris expression product pPIC9-VP1-IL6, which is characterized in that: the method comprises the following specific steps:

(1) preparation of the vaccine: inducing and expressing a large amount of pPIC9-VP1-IL6 recombinant pichia pastoris, and harvesting culture medium supernatant after culturing for 96 h; measuring the concentration of VP1-IL6 protein by using a spectrophotometer, and storing at-20 ℃; adjusting the final protein concentration of VP1-IL6 to 100 mug/mL by using sterile water, adding 5.0 mug/mL LPS (lipopolysaccharide) into the solution as a polyclonal antibody activator, mixing the prepared protein solution serving as a water phase with an equal volume of white oil adjuvant, and fully emulsifying for 20min by using a homogenizer to prepare the vaccine;

(2) immunizing the laying hens which are about 1 month before the laying by using the prepared vaccine for 4 times, wherein the immunization interval is 2 weeks each time; after 4 times of immunization in the early stage, the subsequent immunization is carried out every 1-2 months, and the immunization dose is 1.0-2.0 mL/mouse;

(3) collecting the immunized eggs two weeks after the 4 th immunization, and keeping the immunization once every 1-2 months in the continuous egg collecting process; separating egg yolk from egg white by using an egg yolk separator, mixing the egg yolk with purified water preheated to 30-37 ℃ according to a volume ratio of 3-5:1 to dilute egg yolk liquid, uniformly stirring to obtain egg yolk diluted liquid, and preheating for 1h at 30-37 ℃;

(4) adding PEG6000 into the yolk diluent, wherein the mass fraction of the mixed PEG6000 is 3-4%, mixing with the yolk, fully stirring uniformly, standing for 4-6h, and fully reacting;

(5) extracting the reacted supernatant, and coarsely filtering with a 100-200-mesh filter screen, wherein the supernatant is firstly filtered with 100 meshes and then filtered with 200 meshes;

(6) putting the filtered supernatant into a sterile barrel for secondary precipitation for 24-48 h;

(7) pumping out the supernatant of the secondary precipitation, coarsely filtering once by using a 200-mesh filter screen, and then filtering and sterilizing by using a 0.22-micron filter membrane; adding formaldehyde with volume fraction of one thousandth into the sterilized supernatant to inactivate unknown viruses and using the inactivated unknown viruses as a preservative, namely a refined egg yolk antibody, wherein the detected agar expansion test titer of the anti-type I duck virus hepatitis virus and the duck IL-6 antibody is not lower than 1: 64;

(8) and sub-packaging the filtrate in sterile vaccine bottles under sterile conditions, covering a rubber plug, rolling an aluminum cover, and labeling to obtain a finished egg yolk antibody product, and preserving at 4-8 ℃.

Preferably, after the previous 4 immunizations in step (1) are completed, the subsequent immunizations are performed every 1.5 months;

the dilution volume ratio in the step (2) is 3.5:1, and the temperature is 35 ℃;

the addition amount of PEG6000 in the step (3) is 3.5 percent; wherein PEG6000 can be dissolved in small amount of warm water

Compared with the prior art, the invention has the beneficial effects that:

the invention successfully clones the duck IL-6 gene containing the complete CDs sequence in the field for the first time, fills the blank of the field, successfully clones the VP1 gene of the duck hepatitis virus type I, constructs the two genes into a fusion expression plasmid, realizes the high-efficiency expression of the recombinant plasmid Pichia pastoris, and has the constructed expression plasmid taking methanol as an inducer, low cost and convenient operation;

the prepared refined egg yolk antibody has good protection effect, especially has anti-inflammatory and anti-inflammatory effects, has overall effect superior to that of the conventional duck viral hepatitis egg yolk antibody sold in the market, provides a new treatment idea for the treatment of duck hepatitis I, and can remarkably promote the development of poultry industry.

The invention provides a sequence table of related sequences, wherein the information of each sequence in the sequence table is as follows:

SEQ ID NO: 1: a gene sequence of duck hepatitis virus I VP 1;

SEQ ID NO: 2: a gene sequence of type I duck hepatitis virus VP 1-linker;

SEQ ID NO: 3: the gene sequence of the coding region (CDs) of the duck IL-6 gene;

SEQ ID NO: 4: duck IL-6-linker gene sequence;

SEQ ID NO: 5: fusion of the VP1-IL6 gene sequence;

SEQ ID NO: 6: an upstream Primer 1 of a type I duck hepatitis virus VP1 gene;

SEQ ID NO: 7: a Primer 2 at the downstream of a VP1 gene of the I-type duck hepatitis virus;

SEQ ID NO: 8: i type duck hepatitis virus VP1-linker gene upstream Primer 3;

SEQ ID NO: 9: a duck hepatitis virus type I VP1-linker gene downstream Primer 4;

SEQ ID NO: 10: an upstream Primer 5 of the duck IL-6 gene;

SEQ ID NO: 11: a duck IL-6 gene downstream Primer 6;

SEQ ID NO: 12: an upstream Primer 7 of the duck IL-6-linker gene;

SEQ ID NO: 13: duck IL-6-linker gene downstream Primer 8.

Drawings

FIG. 1 shows the PCR amplification result of the VP1 gene of duck hepatitis virus type I:

in the figure, M is DL2000 Marker, and Lane 1-2 is duck hepatitis I virus VP1 gene DNA. bp is a base pair unit.

FIG. 2 is a graph showing the PCR amplification result of the gene VP1-linker of duck hepatitis virus type I.

FIG. 3 is a diagram showing the result of PCR amplification of the IL-6 gene of duck:

in the figure, M is DL2000 Marker, and lanes 1-2 are DNA of duck IL-6 gene. bp is a base pair unit.

FIG. 4 is a graph showing the result of PCR amplification of the duck IL-6-linker gene (signal peptide removed).

FIG. 5 is a graph showing the PCR amplification result of the fusion gene of type I duck hepatitis virus VP1-IL 6:

in the figure, M is DL5000 Marker, and lanes 1-4 are duck hepatitis virus I VP1-IL6 DNA. bp is a base pair unit.

FIG. 6 shows the cleavage of the PMD-18-T-VP1-IL6 plasmid:

in the figure, M is DL2000 Marker, and lanes 1-2 are recombinant clone plasmid PMD-18-T-VP1-IL6 after enzyme digestion. bp is a base pair unit.

FIG. 7 shows the cleavage of pPIC9-VP1-IL6 plasmid:

in the figure, M is DL8000 Marker, and 1 is recombinant expression plasmid pPIC9-VP1-IL6 after enzyme digestion. bp is a base pair unit.

FIG. 8 is a graph showing the PCR amplification result of Pichia positive transposon:

in the figure, M is DL10000 Marker, lane 1-2 shows a positive image, and lane 3 shows a negative image. bp is a base pair unit.

FIG. 9 is a diagram showing the SWISS-MODEL software analysis of the fusion protein spatial folding state prediction.

FIG. 10 is a diagram showing SDS-PAGE:

in the figure, M is Blue Plus Protein Marker, lanes 1-4 are supernatant samples after induction at 96h, 72h, 48h and 24h, respectively, and lane 5 is an empty plasmid negative control. KDa is the protein molecular mass unit.

Fig. 11 is a protein purification diagram:

in the figure, M is Blue Plus Protein Marker, lane 1 is a negative control, and lane 2 is a purified Protein sample. KDa is the protein molecular mass unit.

FIG. 12 is a Western-blotting reaction chart:

in the figure, M is Blue Plus Protein Marker, lane 1 is a negative control, and lane 2 is a Protein sample. KDa is the protein molecular mass unit.

FIG. 13 shows the expression level of IL-6 in duck serum after duck hepatitis virus infection by ELISA method.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention:

EXAMPLE 1 construction of recombinant plasmid pPIC9-VP1-IL6

The method comprises the following steps:

(1) determination of cleavage sites

According to the sequencing result of finding the gene sequence (SEQ ID NO:1) of the standard strain VP1 of the I-type duck hepatitis virus from NCBI and the duck IL-6 gene sequence (SEQ ID NO:3) obtained by PCR amplification, compared with the plasmid spectrogram information of pPIC9, the double enzyme cutting sites Xho I and Not I are determined.

(2) Construction of recombinant plasmid pPIC9-VP1-IL6

PCR amplification of I type duck hepatitis virus VP1 gene:

the following primers were designed according to GenBank published duck hepatitis virus type I VP 1:

primer 1: 5'-GGTGATTCTAACCAGTTAGG-3', as shown in SEQ ID NO: 6 is shown in the specification;

primer 2: 5'-TTCAATTTCCAGATTGAGTT-3', as shown in SEQ ID NO: shown at 7.

RNA of a duck hepatitis virus type I standard strain (strain preservation number: CVCC AV1540, directly obtained from a preservation organization) is extracted, reverse-transcribed into cDNA and used as a template, and PCR amplification is carried out on the VP1 gene by using Primer 1 and Primer 2 as primers:

the PCR reaction conditions are as follows:

the recovery result of the amplification product is shown in FIG. 1. agarose gel electrophoresis of the recovered VP1 gene gave a 714bp band, which is consistent with the expected result. After cloning the target band into a PMD18-T vector, the sequencing result shows that the target band is consistent with a VP1 sequence published by GenBank. The VP1 gene is successfully cloned, and the recombinant plasmid with correct sequencing is named as PMD18-T-VP1 and is stored for later use.

PCR amplification of type I duck hepatitis virus VP1-linker gene:

for the purpose of fusing the VP1 and IL-6 genes and cloning the fused gene into pPIC9 plasmid, a linker sequence is inserted into the amplified product, so that the primers are designed as follows:

Primer 3:

5′-CCGCTCGAGATGCATCATCATCATCATCATGGTGATTCTAACCAGTTAGG-3', the short-chain part is an Xho I cleavage site, and the long-chain part is a His tag. As shown in a sequence table SEQ ID NO: shown in fig. 8.

Primer 4:

5′-TGAACCTCCACCTCCTGATCCACCTCCACCTTCAATTTCCAGATTGAGTT-3', the underlined part is the linker sequence. As shown in a sequence table SEQ ID NO: shown at 9.

Carrying out PCR amplification on the VP1-linker gene by taking the recombinant plasmid PMD18-T-VP1 as a template and the Primer 3 and the Primer 4 as primers:

the PCR reaction conditions are as follows:

the result is shown in FIG. 2, a 774bp band (containing linker gene, His tag, restriction site and protective base sequence) is obtained by agarose gel electrophoresis of the recovered VP1-linker gene, and is consistent with the expected result.

PCR amplification of duck IL-6 gene:

according to a predicted sequence of a duck IL-6 gene published by GenBank, the following primers are designed:

Primer 5:

5'-ATGAGCTCTCCCGACGGCTG-3', as shown in SEQ ID NO: 10 is shown in the figure;

Primer 6:

5'-TCAGACACTGACACTCCTGG-3', as shown in SEQ ID NO: shown at 11.

Extracting duck spleen RNA, performing reverse transcription to obtain cDNA serving as a template, and performing PCR amplification on a duck IL-6 gene by using Primer 5 and Primer 6 as primers:

the PCR reaction conditions are as follows:

the results are shown in FIG. 3, which shows that agarose gel electrophoresis of the recovered IL-6 gene gave a band of 961bp, consistent with the expected results. After cloning the target band into a PMD18-T vector, a sequencing result shows that the gene sequence comprises a coding region (CDs) part which is consistent with a duck IL-6 gene predicted sequence published by GenBank. The full-length sequence of the duck IL-6 gene CDs region is successfully cloned, and the gene sequence of the duck IL-6 gene coding region (CDs) is intercepted, and is shown as a sequence table SEQ ID NO:3, the sequence fills the blank in the field, and the recombinant plasmid with correct sequencing is named as PMD18-T-IL6 and is stored for later use.

PCR amplification of duck IL-6-linker gene:

based on the three purposes that the IL-6 and VP1 genes are fused, the signal peptide sequence of duck IL-6 needs to be removed from the fused gene, and the fusion expression product needs to be cloned to pPIC9, the following primers are designed:

Primer 7

5′-GGTGGAGGTGGATCAGGAGGTGGAGGTTCAGCGCCGCTGCCCCTGGCCGC-3', the underlined part is the linker sequence. As shown in a sequence table SEQ ID NO: shown at 12.

Primer 8:

5′-TATGCGGCCGCTCAGACACTGACACTCCTGG-3', the underlined part is the NotI cleavage site. As shown in a sequence table SEQ ID NO: shown at 13.

Carrying out PCR amplification on the IL-6-linker gene by taking the recombinant plasmid PMD18-T-IL-6 as a template and the Primer 7 and the Primer 8 as primers:

the PCR reaction conditions are as follows:

the results are shown in FIG. 4: the IL-6-linker gene PCR product was subjected to agarose gel electrophoresis to obtain a band of 633bp in size, which was consistent with the expected results.

PCR amplification of fusion gene:

respectively recovering amplified VP1-linker and IL-6-linker genes, and performing SOE-PCR amplification by using Primer 3(SEQ ID NO: 8) and Primer 8(SEQ ID NO: 13) as specific primers to obtain VP1-IL6 fusion genes;

the PCR reaction conditions are as follows:

the results are shown in FIG. 5: the SOE-PCR product of the fusion gene was subjected to agarose gel electrophoresis to obtain a band of 1379bp in size, which was consistent with the expected results.

Sixthly, the fusion gene obtained by glue recovery is connected overnight at 16 ℃, transformed into escherichia coli DH5 alpha competent cells, single colony is selected to shake bacteria and extract plasmid, and PMD18-T-VP1-IL6 recombinant plasmid is obtained by enzyme digestion and sequencing; the specific operation is as follows:

connecting a reaction system: 4.5 mu L of VP1-IL6 gene, 0.5 mu L of PMD18-T plasmid and 5.0 mu L of Solution I, all the components are mixed uniformly and connected for 2h in a water bath at 16 ℃;

the reaction system of the double-enzyme digestion PMD18-T-VP1-IL6 is as follows: 1.0 mu g of recombinant plasmid PMD18-T-VP1-IL6, 0.5 mu L of Xho I endonuclease, 0.5 mu L of Not I endonuclease and 2.0 mu L of 10 Xbuffer, supplementing sterilized ultrapure water to 20 mu L, uniformly mixing the components, and placing the components in a water bath at 37 ℃ for 2 hours;

the results are shown in FIG. 6: the recombinant plasmid PMD18-T-VP1-IL6 is cut by enzyme to obtain a strip with the size of 2692bp and 1373bp (3 protective bases at two ends are removed), which indicates that the clone plasmid is successfully constructed.

Seventhly, the recombinant plasmid PMD18-T-VP1-IL6 is cut by using Xho I and Not I restriction endonucleases, cut VP1-IL6 fusion gene glue is recovered, the recovered VP1-IL6 fusion gene glue is connected with a pPIC9 expression plasmid cut by using the same restriction endonucleases at 16 ℃ overnight, the obtained product is connected through transformation and enzyme digestion verification, then the inserted exogenous gene sequence is verified to be correct through plasmid sequencing, and the plasmid is named as: pPIC9-VP1-IL 6;

the specific operation is as follows:

connecting a reaction system: 10 Xbuffer 1.0 μ L, VP1-IL6 gene 2.0 μ L, pPIC96.0 μ L after restriction enzyme, and T4DNA ligase 1.0 μ L, mixing the components uniformly, and connecting in water bath at 16 ℃ overnight;

the reaction system of the double-enzyme digestion pPIC9-VP1-IL6 is as follows: pPIC9-VP1-IL6 recombinant plasmid 1.0 μ g, Not I endonuclease 0.5 μ L, Xho I endonuclease 0.5 μ L, 10 XBuffer 2.0 μ L, sterilized ultrapure water to 20 μ L, after mixing the components, they were placed in a 37 ℃ water bath for 2 h.

The results are shown in FIG. 7: the recombinant pPIC9-VP1-IL6 is subjected to enzyme digestion to obtain a band with the size of 8000bp and 1373bp (3 protective bases at two ends are removed), which indicates that the fusion gene is successfully connected into an expression plasmid.

Example 2 pPIC9-VP1-IL6 plasmid Pichia pastoris expression and purification

(1) Expression and identification of VP1-IL6 fusion protein in pichia pastoris

Firstly, carrying out linearization and gel recovery on the constructed recombinant plasmid by using Sal I incision enzyme digestion, simultaneously carrying out linearization and gel recovery on an empty vector by using the same incision enzyme digestion, carrying out dephosphorylation treatment, extracting and recovering phenol/chloroform, and dissolving in a TE buffer solution; the recombinant plasmid was transformed into pichia pastoris GS115 competent cells by the conventional LiCl method, and RDB plates were coated to select positive transformants.

Wherein the enzyme digestion reaction system is as follows: 1.0 mu g of pPIC9-VP1-IL6 recombinant plasmid, 1.0 mu L of Sal I endonuclease, 2.5 mu L of 10 Xbuffer and 20 mu L of sterilized ultrapure water, wherein the components are uniformly mixed and then placed in a water bath at 37 ℃ for 2 hours;

② the identification of positive transformants: extracting the genome of the transformant, and amplifying the Aox1 gene by PCR by taking the genome as a template, wherein the PCR reaction system comprises the following steps:

the PCR reaction conditions are as follows:

the results are shown in FIG. 8: and (3) identifying by agarose gel electrophoresis, wherein a blank plasmid transformant is identified when a 2200bp and 493bp band appears, a positive transformant is identified when a 2200bp and 1816bp band appears, and the 1816bp length represents the sum of the lengths of the 493bp gene fragment and an inserted gene sequence 1323bp (the inserted sequence is the sum of the length of 1373bp of the digested VP1-IL6 fusion gene minus the length of 14bp of two digestion sites on the pPIC9 plasmid and 36bp between the two digestion sites) on the plasmid.

And (4) selecting positive transformants for subsequent protein expression.

After the positive transformants are successfully constructed, the spatial structure of the connected genes is predicted by SWISS-MODEL software:

the results are shown in FIG. 9: according to the analysis result of SWISS-MODEL software, it can be seen that the two genes connected in the test are connected by one peptide segment, and the proteins expressed by the two genes of the peptide segment respectively keep their respective spatial structures and do not interfere with each other. Therefore, the protein expressed by the expression system selected in the test can still respectively reserve the spatial structures of the two genes and keep the respective immunogenicity.

(2) Expression of I-type duck hepatitis virus VP1 and duck IL-6 fusion protein in pichia pastoris

Inducing expression of VP1-IL6 fusion protein in Pichia pastoris:

inoculating the selected positive recombinant into 20mL BMGY medium, culturing at 30 ℃ and 250rpm with shaking to OD₆₀₀2-6, the cells were collected by centrifugation. Adding an equal volume of BMMY culture medium for resuspension if the transformant is positive, and adding 1/5 volumes of BMMY culture medium for resuspension if the transformant is negative; shaking culture at 30 ℃ and 250rpm for 96h, supplementing methanol to 1% every 24h, taking 1.0mL of culture medium at 0, 12, 24, 36, 48, 60, 72, 84 and 96h respectively for analyzing expression level, and finally determining the optimal bacteria harvesting time to be 96 h.

The results are shown in FIG. 10: an 49.98kDa band was obtained in the sampled lanes at different time periods compared to the control group, consistent with the expected results. And with the extension of the induction time, the expression quantity is gradually increased, which accords with the expression rule of pichia pastoris, so that the recombinant expression plasmid can be successfully expressed in a pichia pastoris expression system.

② the identification and purification of VP1-IL6 fusion protein.

The protein extract was taken, added to SDS loading buffer to adjust protein concentration, boiled for 10min, subjected to SDS-PAGE with 40. mu.L loading per well, and transferred to NC membrane for a total of 3 replicate membranes. Blocking with 0.5% skimmed milk powder for 3h, and immersing the blocked 3 NC membranes in 1: a1000-fold dilution of rabbit anti-His tag primary antibody (purchased, formulated with 0.05% PBST), 1:200(v/v) rabbit anti-VP 1 primary antibody (prepared from immunized rabbits), 1:200(v/v) rabbit anti-IL-6 primary antibody (prepared from immunized rabbits), shaking at 37 ℃ for 2.5h, taking out and rinsing 3 times, immersing in a 1:2000(v/v) diluted HRP-labeled goat anti-rabbit secondary antibody (purchased, formulated with PBST) for binding for 1.5h, and performing DEB chemochromic after membrane washing.

The results are shown in FIG. 11: the obtained protein is purified and then subjected to SDS-PAGE electrophoresis to obtain a band with the size of 49.98kDa, and the result is consistent, so that the fusion protein is successfully expressed by pichia pastoris and then is well purified.

As shown in fig. 12: the purified fusion protein is respectively identified by an anti-His tag antibody, a rabbit anti-VP 1 primary antibody, a rabbit anti-IL-6 primary antibody and corresponding secondary antibodies, and bands with the size of 49.98kDa are respectively obtained at target bands, which shows that the proteins expressed by the expression system respectively keep the respective immunogenicity of VP1 and IL-6 proteins.

Example 3 preparation of anti-VP 1-IL6 yolk antibody

Example 4 quality test of yolk antibody

(1) Safety inspection

After 20 cherry valley ducklings of 1 day old are injected into the muscle at multiple points, 2.0mL of the egg yolk antibody prepared in the embodiment 3 is observed for 14 days, the ducklings are susceptible to all healthy survival, and the safety of the egg yolk antibody is good.

(2) Sterility testing

The results of the implementation according to the pharmacopoeia of the people's republic of China (2015 edition) show that the egg yolk antibody of the invention is free from bacterial, mycoplasma and foreign virus contamination.

(3) Efficacy test

3-day-old susceptible duckling (DHV-I maternal antibody)<1:4)80, randomly divided into A, B, C, D4 groups of 20. Each duck is infected by 10LD₅₀Doses of DHV-I virus. 6h after infection with the virus, group A was injected intramuscularly with 1.0mL each of the yolk antibodies prepared in example 4; group B intramuscular injection of 1.0mL of a commercially available refined yolk antibody (product No. 1) for type I duck viral hepatitis; c group intramuscular injection of 1.0mL of a commercially available refined yolk antibody (No. 2 manufacturer) for type I duck viral hepatitis; group D was a control group, and 1.0mL of saline was injected intramuscularly and kept separately. After 8h of injection, 10 animals are killed in each group, and the content of IL-6 in the liver is detected; meanwhile, liver tissues are collected to prepare pathological tissue sections, and the inflammation score condition in the liver is evaluated. 10 remaining per group, 20 days of age were observed and mortality was recorded.

The results show that the IL-6 content in the liver of 10 ducks in the A group (anti-VP 1-IL6 yolk antibody group) is significantly lower than that in the B group and the C group (figure 13); histopathological observations found that group a had a lower pathology score than group B, C, D (table 1); in addition, the remaining 10 ducks in group a were all alive during the observation period, 2 were dead in group B, 3 were dead in group C, and all dead in group D. The results show that the anti-VP 1-IL6 yolk antibody prepared in example 4 has better protective effect than 2 commercial yolk antibodies, and obviously reduces the inflammatory reaction in the liver. The ideal treatment effect can be achieved by injecting 1.0mL of the 3-day-old duckling.

TABLE 1 Duck liver pathology score (mean) after Duck hepatitis Virus infection

Group of	Group A	Group B	Group C	Group D
					Score of	0.8	1.5	1.7	3.6

Note: the scoring was performed as follows, and the scores of each group were represented by means of averages.

0: normal liver histological morphology;

1: mild focal degeneration of hepatocytes;

2: moderate multifocal hepatocyte degeneration, a small amount of bleeding, infiltration of a small amount of inflammatory cells such as lymphocytes and heterophilic granulocytes;

3: diffuse degeneration and necrosis of liver cells, a large amount of inflammatory cell infiltration and obvious blood focus;

4: extensive hepatic cells are severely necrotic, severe bleeding focus, severe liver tissue damage and disappearance of liver tissue structure.

Example 5 comparison of Effect of anti-VP 1-IL6 yolk antibody

The same technology is used for preparing VP1-IL1 beta and VP1-IL2 fusion proteins respectively, yolk antibodies are prepared by the same method, and the protective effects of the VP1-IL1 beta, VP1-IL2 and VP1-IL6 yolk antibodies are compared.

3-day-old susceptible duckling (DHV-I maternal antibody)<1:4)80, randomly divided into A, B, C, D4 groups of 20. Each duck is infected with 100EID₅₀Doses of DHV-I virus. 6h after infection with the virus, group A was injected intramuscularly with 1.0mL each of the anti-VP 1-IL6 yolk antibody prepared in example 4; intramuscular injection of anti-VP 1-IL1 beta yolk antibody to group B, 1.0mL each; intramuscular injection of anti-VP 1-IL2 yolk antibody to group C, 1.0mL each; group D was a control group, and 1.0mL of saline was injected intramuscularly and kept separately. Injecting for 8h, killing 10 per group, collecting liverPathological tissue sections were prepared and the inflammation score in the liver was evaluated. 10 remaining per group, 20 days of age were observed and mortality was recorded.

The results showed that the remaining 10 ducks in group A (anti-VP 1-IL6 yolk antibody group) were all alive during the observation period, 1 dead in group B, 2 dead in group C, and all dead in group D; histopathological observations revealed that the pathological score in group a was lower than that in group B, C, D (table 2). The results show that the anti-inflammatory effect and the protective effect of the anti-VP 1-IL6 yolk antibody prepared in example 4 are better than those of the other two, namely the anti-inflammatory effect of the fusion expressed IL-6 is better than that of the fusion of two cytokines IL-1 beta and IL-2.

TABLE 2 Duck liver pathology score (mean) after Duck hepatitis Virus infection

Group of	Group A	Group B	Group C	Group D
					Score of	0.8	1.2	1.3	3.6

0: normal liver histological morphology;

1: mild focal degeneration of hepatocytes;

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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Claims

A pPIC9-VP1-IL6 recombinant plasmid, characterized in that: the recombinant plasmid contains VP1-IL6 fusion gene, and the sequence of the gene is shown as SEQ ID NO: 5, respectively.
2. The inactivated vaccine for duck circovirus tissue according to claim 1, wherein: the VP1-IL6 fusion gene is obtained by the following method:

respectively extracting duck hepatitis virus I RNA and duck spleen RNA, performing reverse transcription, and amplifying a duck hepatitis virus I VP1 gene sequence and a duck IL-6 gene sequence by using PCR (polymerase chain reaction), wherein the nucleotide sequences are respectively shown as SEQ ID NO:1 and 3, and cloning the two plasmids into a PMD18-T cloning plasmid respectively for later use; redesigning primers aiming at VP1 and duck IL-6 genes, adding protective bases, enzyme cutting sites and Linker sequences into the primers, and amplifying by using a PCR (polymerase chain reaction) mode to obtain VP1-Linker and IL-6-Linker, wherein the gene sequences are respectively shown as SEQ ID NO: 2 and 4; the two sequences were amplified by overlap extension PCR technique into VP1-IL6 fusion gene.
3. The construction method of the recombinant plasmid pPIC9-VP1-IL6 is characterized in that: the method comprises the following steps:

PCR amplification of I type duck hepatitis virus VP1 gene:

extracting RNA of a duck hepatitis virus type I standard strain, performing reverse transcription to obtain cDNA, taking the cDNA as a template, and using primers Primer 1 and Primer 2, wherein the gene sequence of the cDNA is shown as SEQ ID NO: 6 and 7, performing PCR amplification on the VP1 gene, recovering PCR product gel, wherein the VP1 gene sequence is shown as SEQ ID NO:1 is shown in the specification; connecting to a PMD18-T vector plasmid to obtain PMD18-T-VP1, and storing for later use;

secondly, using the plasmid constructed in the step (i) as a template, designing primers Primer 3 and Primer 4, wherein the gene sequences are shown as SEQ ID NO: 8 and 9, introducing an Xho I enzyme cutting site sequence and an HIS label sequence into an upstream Primer 3, introducing a Linker sequence into a downstream Primer 4, and performing PCR amplification to obtain VP1-Linker, wherein the gene sequence of the VP1-Linker is shown as SEQ ID NO: 2 is shown in the specification;

PCR amplification of duck IL-6 gene: extracting duck spleen RNA, performing reverse transcription to obtain cDNA, performing PCR amplification on duck IL-6 genes, wherein the primers are Pr imer 5 and Pr imer 6, and the gene sequences are shown as SEQ ID NO: 10 and 11; after the duck IL-6 gene is amplified by PCR reaction and the glue is recovered, the CDs sequence of the duck IL-6 gene is shown as SEQ ID NO:3 is shown in the specification; connecting to a PMD18-T vector plasmid to obtain PMD18-T-IL6, and storing for later use;

using plasmids constructed in the third step as templates, designing primers Pr imer 7 and Pr imer 8, wherein the gene sequences are shown in SEQ ID NO: 12 and 13, the starting point of the upstream Primer 7 is an IL-6 sequence with signal peptide removed, a linker is introduced into the Primer, a Not I enzyme cutting site sequence is introduced into the downstream Primer 8, and a duck IL-6-linker gene is obtained by PCR amplification, wherein the gene sequence is shown as SEQ ID NO: 4 is shown in the specification;

respectively recovering VP1-linker and IL-6-linker gene PCR amplification products as templates, and taking Primer 3 with a gene sequence shown as SEQ ID NO: 8 and Primer 8, wherein the gene sequence is shown as SEQ ID NO: 13 shows that the specific primer is used for SOE-PCR amplification to obtain VP1-IL6 fusion gene, and the gene sequence of the fusion gene is shown as SEQ ID NO: 5 is shown in the specification;

fourthly, connecting the fusion gene obtained by glue recovery to PMD18-T vector plasmid at 16 ℃ overnight, and obtaining PMD18-T-VP1-IL6 recombinant plasmid through transformation, plasmid enzyme digestion and sequencing;

fifthly, the recombinant plasmid PMD18-T-VP1-IL6 is cut by Xho I and Not I restriction enzymes, cut VP1-IL6 fusion gene glue is recovered and is connected with pPIC9 expression plasmid cut by the same restriction enzyme at 16 ℃ overnight, the connection is verified by transformation and enzyme digestion, and then the inserted exogenous gene sequence is verified to be correct by plasmid sequencing, wherein the plasmid is named as: pPIC9-VP1-IL 6.
The expression method of the pPIC9-VP1-IL6 recombinant plasmid in pichia pastoris is characterized in that: the method comprises the following specific steps:

(1) expression of VP1-IL6 fusion protein in Pichia pastoris:

firstly, carrying out linearization on the constructed recombinant plasmid by Sal I incision enzyme digestion, carrying out dephosphorylation treatment, extracting and recovering phenol/chloroform, and dissolving in a TE buffer solution; at the same time, the pPIC9 empty vector plasmid is linearized by the same enzyme digestion, after dephosphorylation treatment, phenol/chloroform extraction recovery is carried out, and the recovered phenol/chloroform extraction is dissolved in TE buffer solution to be used as negative control;

respectively transforming the two plasmids into GS115 competent cells by a LiCl method, coating an RDB plate, and obtaining a recombinant plasmid positive transformant and an empty plasmid negative transformant;

secondly, extracting genomes of the two transformants as templates, amplifying Aox1 gene by PCR, identifying by agarose gel electrophoresis, generating a 2200bp and 493bp band on a negative transformant, and generating a 2200bp and 1816bp band on a positive transformant, which shows that the pPIC9-VP1-IL6 recombinant plasmid is successfully transformed;

thirdly, inoculating the screened positive transformants and negative transformants into 20mL BMGY medium, performing shake culture at 30 ℃ and 250rpm until OD is reached₆₀₀2-6, and centrifugally collecting thalli; subsequently, positive transformants were resuspended in an equal volume of BMMY medium and negative transformants were resuspended in 1/5 mediumResuspending the volume of BMMY media; carrying out shake culture on the negative and positive transformants at 30 ℃ and 250rpm for 96h, supplementing methanol to 1% every 24h, taking 1.0mL of culture medium to analyze the expression level of the exogenous gene when the methanol is respectively 0, 12, 24, 36, 48, 60, 72, 84 and 96h, taking the negative transformants as a control, and finally determining the optimal bacteria receiving time to be 96 h;

(2) identification of VP1-IL6 fusion protein:

extracting DNA of the induced yeast, and extracting the DNA of the induced yeast by using Primer 3 with a gene sequence shown as SEQ ID NO: 8, and Primer 8 has a gene sequence shown as SEQ ID NO: 13, carrying out PCR reaction for the primers;

after the recombinants are induced by methanol, collecting the culture medium of the recombinants, and adding a sample buffer solution to carry out SDS-PAGE electrophoresis;

western-blotting is used for identifying the expression of the I type duck hepatitis virus VP1 and duck IL-6 fusion protein in the yeast recombinant.
5. A preparation method of a duck virus hepatitis egg yolk antibody with anti-inflammatory effect adopts pPIC9-VP1-IL6 Pichia pastoris expression products to prepare the egg yolk antibody, and is characterized in that: the method comprises the following specific steps:

(1) preparation of the vaccine: inducing and expressing a large amount of pPIC9-VP1-IL6 recombinant pichia pastoris, and harvesting culture medium supernatant after culturing for 96 h; measuring the concentration of VP1-IL6 protein by using a spectrophotometer, and storing at-20 ℃; adjusting the final protein concentration of VP1-IL6 to 100 mug/mL by using sterile water, adding 5.0 mug/mL LPS (lipopolysaccharide) into the solution as a polyclonal antibody activator, mixing the prepared protein solution serving as a water phase with an equal volume of white oil adjuvant, and fully emulsifying for 20min by using a homogenizer to prepare the vaccine;

(2) immunizing the laying hens which are about 1 month before the laying by using the prepared vaccine for 4 times, wherein the immunization interval is 2 weeks each time; after 4 times of immunization in the early stage, the subsequent immunization is carried out every 1-2 months, and the immunization dose is 1.0-2.0 mL/mouse;

(3) collecting the immunized eggs two weeks after the 4 th immunization, and keeping the immunization once every 1-2 months in the continuous egg collecting process; separating egg yolk from egg white by using an egg yolk separator, mixing the egg yolk with purified water preheated to 30-37 ℃ according to a volume ratio of 3-5:1 to dilute egg yolk liquid, uniformly stirring to obtain egg yolk diluted liquid, and preheating for 1h at 30-37 ℃;

(4) adding PEG6000 into the yolk diluent, wherein the mass fraction of the mixed PEG6000 is 3-4%, mixing with the yolk, fully stirring uniformly, standing for 4-6h, and fully reacting;

(5) extracting the reacted supernatant, and coarsely filtering with a 100-200-mesh filter screen, wherein the supernatant is firstly filtered with 100 meshes and then filtered with 200 meshes;

(6) putting the filtered supernatant into a sterile barrel for secondary precipitation for 24-48 h;

(7) pumping out the supernatant of the secondary precipitation, coarsely filtering once by using a 200-mesh filter screen, and then filtering and sterilizing by using a 0.22-micron filter membrane; adding formaldehyde with volume fraction of one thousandth into the sterilized supernatant to inactivate unknown viruses and using the inactivated unknown viruses as a preservative, namely a refined egg yolk antibody, wherein the detected agar expansion test titer of the anti-type I duck virus hepatitis virus and the duck IL-6 antibody is not lower than 1: 64;

(8) and sub-packaging the filtrate in sterile vaccine bottles under sterile conditions, covering a rubber plug, rolling an aluminum cover, and labeling to obtain a finished egg yolk antibody product, and preserving at 4-8 ℃.
6. The method for preparing a yolk antibody against duck viral hepatitis with an anti-inflammatory effect according to claim 5, wherein the yolk antibody is prepared by the following steps:

after 4 immunizations in the early stage in the step (1) are finished, carrying out subsequent immunizations once every 1.5 months;

the dilution volume ratio in the step (2) is 3.5:1, and the temperature is 35 ℃;

the addition amount of PEG6000 in the step (3) is 3.5%.