Fusion protein vaccine for chicken necrotic enteritis and preparation method thereof

A technology of chicken necrotic enteritis and fusion protein, which is applied in the field of biotechnology and biological products, can solve the problems of unclear pathogenic mechanism of necrotic enteritis and non-specific strains of necrotic enteritis, so as to prevent chicken necrotic enteritis and avoid The effect of cumbersome process and biosafety issues, and easy industrial production

Active Publication Date: 2021-11-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenic mechanism of these proteins on necrotic enteritis is not yet

Method used

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  • Fusion protein vaccine for chicken necrotic enteritis and preparation method thereof
  • Fusion protein vaccine for chicken necrotic enteritis and preparation method thereof
  • Fusion protein vaccine for chicken necrotic enteritis and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 fusion protein NAT

[0023] 1. Synthesis of fusion gene NAT

[0024]The present invention designs a fusion gene, selects the truncated fragment of the C-terminus of Tpel toxin and the amino acid sequence of the immunogenic region of the C-terminus of NetB and α-toxin, as shown in SEQ NO.3, SEQ NO.1, and SEQ NO.2. The corresponding coding genes are shown in SEQ NO.7, SEQ NO.5, and SEQ NO.6. At the same time, a linker fragment "A(EAAAK) that does not affect the structure and biological activity of the three toxin proteins was designed 4 A" series, the amino acid sequence is shown in SEQ NO.4, and the nucleotide sequence of the fusion protein encoding gene is shown in SEQ ID NO.8. According to the codon preference of Escherichia coli, the fusion protein gene sequence NetB-ɑ-Tpel (abbreviated as NAT) for codon optimization, the optimized gene sequence is shown in SEQ NO.9, and was synthesized at Sangon Bioengineering (Shanghai) Co., Ltd. Th...

Embodiment 2

[0046] Example 2 Immune Effect Evaluation of Fusion Protein NAT

[0047] 1. Fusion protein vaccine preparation

[0048] The soluble protein and refolding protein purified in Example 1 were adjusted to the required amount with sterile PBS according to the protein concentration for immunization. Mix the fusion protein NAT solution with the propolis adjuvant at a ratio of 1:1 to prepare a suspension vaccine.

[0049] 2. Animal grouping and immunization

[0050] Forty 1-day-old broiler chicks were randomly divided into 5 groups: high-dose refolding protein group, soluble protein group, adjuvant group, challenge group and untreated group. In the first three groups, the first immunization and booster immunization were carried out by injecting 0.3ml / bird in the chest of broiler chickens at 12 and 26 days of age respectively. The adjuvant group and the challenge group were injected with adjuvant or PBS at the same dose and site. 24 hours after inoculation, the spirit, diet and inje...

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Abstract

The invention discloses a fusion protein vaccine for chicken necrotic enteritis and a preparation method thereof. The fusion protein vaccine is prepared by connecting Clostridium perfringens NetB toxin, Tpel toxin with the C tail end and the C end cut off of alpha toxin in series through a joint segment A (EAAAK) 4A and expressing and purifying the toxin in escherichia coli BL21(DE3). The fusion protein and a propolis adjuvant are mixed in an equal volume mode to immunize broiler chickens, and the result shows that the body can be induced to generate a high antibody level, and infection of G-type Clostridium perfringens can be remarkably resisted. The fusion protein vaccine can keep the space structure and immunogenicity of three toxin proteins, can avoid the complex process and biosafety problems of a traditional vaccine, is easy to produce industrially, has the advantages of being low in preparation cost, high in safety and excellent in efficacy, and can well prevent chicken necrotic enteritis clinically.

Description

technical field [0001] The invention belongs to the field of biotechnology and biological products, and in particular relates to a fusion protein vaccine against chicken necrotic enteritis and a preparation method thereof. Background technique [0002] Chicken necrotic enteritis (Necrotic Enteritis, NE) is a common bacterial disease of poultry, which is caused by the proliferation of pathogenic Clostridium perfringens in the gastrointestinal tract of chickens, and is of great economic importance to the global poultry industry. significance. Mainly manifested in clinical and subclinical forms: the clinical form leads to increased mortality in broiler flocks, with losses of about 1% per day for several consecutive days. The subclinical form causes chronic intestinal mucosal damage leading to indigestion, decreased weight gain, and impaired feed conversion. At present, the disease has been found in various poultry such as broiler hens, layer hens, turkeys, and quails. It is m...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C12N1/21A61K39/08A61K39/39A61P31/02C12R1/19
CPCC07K14/33C12N15/70A61K39/08A61K39/39A61P31/02A61K2039/552A61K2039/55588C07K2319/00A61K2300/00Y02A50/30
Inventor 谷长勤赵慧玲谢长清程国富刘晓丽胡薛英张万坡
Owner HUAZHONG AGRI UNIV
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