TWI386223B - Duck hepatitis vaccine and its preparation method - Google Patents

Description

Duck hepatitis vaccine and preparation method thereof

The invention relates to a duck hepatitis vaccine and a preparation method thereof, in particular to a duck hepatitis vaccine prepared based on a sub-unit vaccine technology and combined with a PreS envelope protein on a duck liver virus and a preparation method thereof.

Ducks are common economic waterfowl animals, but if ducks die during illness or natural disasters, they will cause huge economic losses for duck farmers, and ducks have the same chance of suffering from hepatitis as humans. Duck Hepatitis B virus (hereinafter referred to as DHBV) was isolated by researchers in 1980 (Mason WS et al ., 1980). DHBV is highly contagious in duck populations. It can be transmitted by blood, body fluids, feces or vertical infections. It can continue to cause chronic infection among ducks, but it can lead to lesions in the liver of ducks, which can cause symptoms such as hepatitis and cirrhosis.

In general, most organisms use vaccines to prevent the occurrence of epidemics or infectious diseases. Vaccines can be classified into attenuated vaccines or deadly vaccines depending on the type of antigens contained therein. However, attenuated vaccines may occur. The virulence recovery or viral activity is too low, resulting in insufficient efficacy of the vaccine. In addition, the above vaccines have problems such as difficulty in preparation, high cost, unstable quality, and difficulty in storage.

The vaccine currently used in ducks will be vaccinated with a domesticated live vaccine of the eggs in the breeding ducks or the young ducks of the day or ten days of age, so that the ducks can produce antibodies for the purpose of epidemic prevention. However, this vaccine But it belongs to the aforementioned attenuated vaccine, so it still The domesticated virus is transformed into a virulent pathogen, which forms a loophole in disease control.

In recent years, agricultural product research and development personnel have integrated molecular biology and vaccination technology, and hope to obtain a vaccine that is economical, safe, highly effective, stable and easy to use, among which "subunit vaccine" technology One of the newer technologies, the sub-unit vaccine removes the virion that cannot stimulate the immune response or is harmful to the organism, and retains the effective vaccine components to prepare the vaccine. The vaccine is not a complete pathogen. This unit vaccine is based on the injection of antigenic proteins to elicit a humoral immune response and therefore does not trigger cellular immunity.

However, the "sub-unit vaccine" technology has not been applied to the production of duck hepatitis vaccine. Therefore, in duck farming, duck farmers have to bear the ducks because they do not have a valid vaccine to avoid the disease. Because of the risk of a large number of collective deaths due to the disease, it is still necessary to take into account the research and development of the existing technology in the preparation of the duck hepatitis vaccine, and cooperate with the research and development to provide a duck hepatitis vaccine with the advantages of the "sub-unit vaccine".

The main object of the present invention is to solve the problems of the above prior art, and to provide a duck hepatitis vaccine prepared based on the sub unit vaccine technology and a preparation method thereof.

The preparation method of the duck hepatitis vaccine of the present invention is to clone the DNA sequence of the PreS envelope protein in the duck hepatitis B virus into a vector to form a recombinant plastid, and then transfer the recombinant plastid to a microorganism strain. Through the cultivation of environmental regulation and After protein induction, a large amount of the PreS envelope protein can be produced to prepare a vaccine.

The duck hepatitis vaccine of the present invention comprises at least a PreS envelope protein of duck hepatitis B virus and an additive which is an excipient or an adjuvant by injecting the duck hepatitis virus vaccine or The method of mixing with the feed allows the duck to pass the duck hepatitis virus vaccine only to generate an immune response.

The PreS envelope protein is one of the surface antigens (Duck Hepatitis B surface antigen, DHBsAg) formed on the surface of duck hepatitis B virus to provide protection for the outer layer of the virus, and its presence is associated with the endothelium of the virus. The role of endocytosis and the production of the polymerase enzyme required for replication, so that the PreS envelope protein can be used for vaccine preparation, thereby triggering duck immunity to achieve ducks against duck hepatitis B infection or The possibility of an attack.

In general, the duck hepatitis B vaccine of the present invention has the advantages that the antigen possessed by the subunit vaccine does not proliferate in the living body, is non-infectious, has no side effects, and is simple to develop.

The method for preparing a duck hepatitis vaccine of the present invention comprises the steps of: (1) preparing a pre S DNA, chimerizing the DNA into a vector to form a recombinant plastid; and (2) transferring the recombinant plastid to a microorganism. a cell line; (3) using a medium to culture the microbial cell strain to produce a PreS envelope protein; (4) extracting and purifying the PreS envelope protein; and (5) performing the PreS envelope protein with an additive Mix evenly.

The following is a detailed description of the embodiments.

Example

1. Pre S DNA preparation

(1) Preparation of pVAC-EGFP- preS plastid: Polymerase chain reaction (PCR) was used to amplify the preS gene from the blood of ducks infected with hepatitis B, and the gene was transferred to a pVAC vector to form a pVAC- preS The plastid, the pVAC- preS plastid was recombined with a commonly used pEGFP-C3 vector to form a pVAC-EGFP- preS plastid with both the green fluorescent protein gene and the pre gene.

(2) pGEM-T Easy- preS plastid preparation: Polymerase chain reaction was used to amplify the preS gene from the blood of ducks infected with hepatitis B, and the gene was transferred to a commercially available pGEM-T Easy vector kit. That is, the plastid pVAC-EGFP- preS with the pre gene is formed.

2. Construction of duck hepatitis B virus preS recombinant plastid and E. coli expression system

(1) Expression plastid: (1-1) pQE-trisystem-EGFP- preS recombinant plastid: cleavage of plastid pVAC-EGFP- preS with restriction enzymes Kpn I and Nco I, and colonization to quality The pQE-trisystem (QIAGEN) constructs a plastid pQE-trisystem-EGFP- preS which produces an EGFP-PreS fusion protein with a molecular weight of 51.4 kDa and a protein sequence end with 8 consecutive His tag (SEQ ID NO: 2); (1-2) pQE-trisystem- preS recombinant plasmid: pGEM-T Easy- preS with preS gene of duck hepatitis B virus The cleavage of the restriction enzymes Eco RI and Kpn I was carried out and cloned into the pQE-trisystem plastid to construct the expression plastid pQE-trisystem- preS which produced the PreS envelope protein. The molecular weight of the PreS envelope protein was 25.4 KDa. Its protein sequence ends have 8 consecutive histidine tags (SEQ ID NO: 1).

(2) The plastids of the above (1-1) and (1-2) were separately eucroporated into a reactive cell to obtain EGFP-PreS and PreS transgenic cell lines. In the present embodiment, the reaction potential cell line is selected from the E. coli XL-blue-1 cell line.

(3) The transgenic cell line was cultured in an LB medium containing 100 μg/ml ampicillin, and then expanded.

3. Preparation of duck hepatitis B virus vaccine

The cultured transgenic cell line is subjected to protein extraction and purification, and the culture medium for culturing the transgenic cell line is cultured in an LB medium supplemented with 100 μg (μg/ml) of ampicillin per ml, and the culture temperature is 37 ° C. When the concentration of the transgenic cell line is 0.5 in the spectrophotometer, the transfected cell line can be collected for protein extraction and purification.

Protein extraction can be obtained by disrupting the cell wall of the transgenic cell line by chemical or physical means and removing the residual DNA, and then obtaining the protein. In this embodiment, the protein is transformed by an ultrasonic cell crusher. The cells are broken.

The protein purification system can purify the recombinant protein by using a general protein purification method or a rapid protein purification kit. In this embodiment, the extracted recombinant protein is purified by purifying the kit KTA ^TM prime plus and HisTrap ^FF 1 ml column. .

The purified protein is uniformly mixed with an additive, wherein the additive is an adjuvant, such as a saponins adjuvant, a complete adjuvant or an incomplete adjuvant, or is biocompatible. Excipients, by injecting or mixing with the duck hepatitis virus vaccine, the duck can only generate an immune reaction via the duck hepatitis virus vaccine. In this embodiment, the Freund's complete use is used. Freund's adjuvant was uniformly mixed with the protein. In this example, 0.5 microgram (μg) of PreS envelope protein was added to 1 microliter (μl) of Freund's complete adjuvant. The dose was 100 μl/head.

4. The effect of inducing agent and culture time on protein expression and salt concentration to promote protein precipitation

(1) Effect of the inducing agent on the amount of protein expression: During the cultivation of the microorganism, the production of the protein can be promoted by the addition of the inducing agent, and in the present embodiment, the isopropyl-β-D-thio half is used. isopropyl-beta-D-thiogalactopyranoside (hereinafter referred to as IPTG) is an inducing agent to induce protein expression by adding IPTG to the above-mentioned LB medium supplemented with 100 μg (μg/ml) of ampicillin per ml. The effect of EGFP-PreS and PreS transgenic cell lines on the production of EGFP-PreS envelope protein or PreS envelope protein was detected by anti-histidine (Anti-His) antibody by Western blotting method and Specificity (results are shown in Table 1).

(2) Effect of culture time on protein expression: 1 mM IPTG was added to LB medium supplemented with 100 μg (μg/ml) of ampicillin per ml, and the transfected cell line was cultured at 37 ° C for different culture time. The effect of induced protein expression was determined by Western blotting using anti-His (Anti-His) as an antibody to detect protein expression and specificity (results shown in Table 2).

(3) Effect of salt concentration on promoting protein precipitation: When extracting protein, salt precipitation can be used to precipitate and precipitate proteins. In this embodiment, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) is used for salting out. Add 1 mM IPTG and different concentrations of ammonium sulfate to the LB medium supplemented with 100 μg (μg/ml) of ampicillin per ml. The effect of ammonium sulfate concentration on protein precipitation was anti-His (Anti-His) antibody. The expression and specificity of the protein were determined by Western blotting (the results are shown in Table 3).

5. Immunoassay:

(1) Test duck screening: The following primers were used to detect whether duck blood had DHBV by polymerase chain reaction (PCR) to confirm whether the duck was infected with DHBV: DHBV- preS- 778~805 ( Eco RV)

5'-GATATCAGTCACATCAAGTTCCTGATGGG-3'DHBV- preS -2165~2145 ( Eco RI)

5’-GGAATCGCTACACGTGGCAAAGGTACAG-3’

(2) Grouping and processing as shown in Table 4 using uninfected DHBV ducks as test samples.

Among them, the three-day-old duckling and the ten-day-old duckling (referred to as the experimental group) were injected with duck hepatitis virus on the first day and the eighth day, and on the 35th day, the virus-challenge was applied. On the third and tenth day after the blood collection, the antibody (IgG) was detected by dot blotting, and the presence of the PreS gene of DHBV was detected by PCR. The results are shown in Table 5. As can be seen from Table 5, the experimental group The ducks can indeed avoid DHBV infection after applying the duck hepatitis virus vaccine of the present invention.

The antigenic epitope of the duck hepatitis virus vaccine of the present invention is the main vaccinogen composition, and does not have the toxicity of the virus, so there is no concern of virulence recovery, and the antigen has no ability to replicate. Therefore, it does not proliferate in the living body, and there is no risk of infection. In addition, the preparation method of the duck hepatitis virus vaccine of the present invention is very simple, and it is easy to prepare and produce in large quantities for use in feeding ducks.

Supplemental sequence table

<110> Chiayi University

<120> Duck hepatitis vaccine and preparation method thereof

<140> TW 097143630

<141> 2008-11-12

<160> 2

<210> 1

<211> 236

<212> DNA

<213> Artificial sequence

<400> 1

<210> 2

<211> 457

<212> DNA

<213> Artificial sequence

<400> 2

Claims (10)

A duck hepatitis vaccine comprising: a duck hepatitis B virus PreS envelope protein having the following amino acid sequence: Met-Gly-Gln-Asn-Pro-Ala-Lys-Ser-Lys-Asp-Val- Arg-Arg-Ile-Glu-Gly-Gly-Glu-Ile-Leu-Leu-Asn-Gln-Leu-Ala-Gly-Arg-Met-Ile-Pro-Lys-Gly-Thr-Val-Thr-Trp- Ser-Gly-Lys-Phe-Pro-Thr-Ile-Asp-His-Val-Leu-Asp-His-Val-Asp-Thr-Met-Glu-Glu-Ile-Asn-Thr-Leu-Gln-Asn- Gln-Gly-Ala-Trp-Pro-Glu-Gly-Ala-Gly-Arg-Arg-Ala-Gly-Leu-Thr-Asn-Pro-Ala-Pro-Gln-Glu-Ile-Pro-Gln-Pro- Lys-Trp-Thr-Pro-Glu-Glu-Asp-Gln-Lys-Ala-Arg-Glu-Ala-Phe-Arg-Arg-Tyr-Gln-Glu-Glu-Arg-Pro-Pro-Glu-Thr- Thr-Thr-Ile-Pro-Pro-Thr-Ser-Pro-Thr-Gln-Trp-Lys-Leu-Gln-Pro-Gly-Asp-Asp-Pro-Leu-Leu-Gly-Asn-Lys-Ser- Leu-Leu-Glu-Thr-His-Pro-Leu-Tyr-Gln-Asn-Pro-Glu-Pro-Ala-Val-Pro-Val-Ile-Lys-Thr-Pro-Pro-Leu-Lys-Lys- Lys-Met-Ser; and an additive, which is an excipient or adjuvant. The vaccine of claim 1, wherein the duck hepatitis B virus PreS envelope protein is more fused to a green fluorescent protein. The vaccine of claim 1, wherein the duck hepatitis B virus PreS envelope protein is further fused to a stretch of 8 consecutive histidine tags. The vaccine of claim 1, wherein the adjuvant is a saponin adjuvant, a complete adjuvant or an incomplete adjuvant. The vaccine of claim 4, wherein the complete adjuvant is Freund's complete adjuvant. A method for preparing a duck hepatitis vaccine comprises the steps of: preparing a polynucleotide encoding the following amino acid sequence: Met-Gly-Gln-Asn-Pro-Ala-Lys-Ser-Lys-Asp-Val -Arg-Arg-Ile-Glu-Gly-Gly-Glu-Ile- Leu-Leu-Asn-Gln-Leu-Ala-Gly-Arg-Met-Ile-Pro-Lys-Gly-Thr-Val-Thr-Trp-Ser-Gly-Lys-Phe-Pro-Thr-Ile-Asp- His-Val-Leu-Asp-His-Val-Asp-Thr-Met-Glu-Glu-Ile-Asn-Thr-Leu-Gln-Asn-Gln-Gly-Ala-Trp-Pro-Glu-Gly-Ala- Gly-Arg-Arg-Ala-Gly-Leu-Thr-Asn-Pro-Ala-Pro-Gln-Glu-Ile-Pro-Gln-Pro-Lys-Trp-Thr-Pro-Glu-Glu-Asp-Gln- Lys-Ala-Arg-Glu-Ala-Phe-Arg-Arg-Tyr-Gln-Glu-Glu-Arg-Pro-Pro-Glu-Thr-Thr-Thr-Ile-Pro-Pro-Thr-Ser-Pro- Thr-Gln-Trp-Lys-Leu-Gln-Pro-Gly-Asp-Asp-Pro-Leu-Leu-Gly-Asn-Lys-Ser-Leu-Leu-Glu-Thr-His-Pro-Leu-Tyr- Gln-Asn-Pro-Glu-Pro-Ala-Val-Pro-Val-Ile-Lys-Thr-Pro-Pro-Leu-Lys-Lys-Lys-Met-Ser; the polynucleotide is chimeric to one The vector forms a recombinant plastid; the recombinant plastid is transferred to a microbial cell strain; the microbial cell strain is cultured in large quantities to produce a recombinant protein having the amino acid sequence encoded by the polynucleotide; Purifying the recombinant protein; and uniformly mixing the recombinant protein with an additive. The method of claim 6, wherein the polynucleotide further encodes a green fluorescent protein. The method of claim 6, wherein the polynucleotide further encodes a stretch of 8 consecutive histidine tags. The method of claim 6, wherein the adjuvant is a saponin adjuvant, a complete adjuvant or an incomplete adjuvant. The method of claim 9, wherein the complete adjuvant is Freund's complete adjuvant.