Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine

A technology of avian leukosis virus and epitope vaccines, applied in the field of immunology, can solve the problems of long purification cycle, severe situation in the prevention and control of subgroup J avian leukemia, virulence recovery, etc., to achieve immune failure avoidance, efficient and safe vaccine protection, specific strong effect

Active Publication Date: 2015-01-28
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In foreign countries, the occurrence of this disease is controlled by purification. Due to the long purification period and high cost, it has not been effectively implemented in my country. We can only carry out large-scale purification by eliminating infected chickens. The infection of ALV-J cannot be controlled, and ALV-J is constantly appearing. The report of large-scale intensive infection, the prevention and control situation of subgroup J avian leukemia in my country is grim
There have been reports at home and abroad on vaccine development methods for subgroup J avian leukemia, mainly traditional formaldehyde inactivated vaccines, attenuated vaccines, SU-based subunit vaccines, and nucleic acid vaccines. protection, but it will cause the possibility of virulence recovery and dispersal of the poison, and the immune effect is unstable and cannot be used clinically

Method used

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  • Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine
  • Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine
  • Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Screening and synthesis of ALV-J multiple epitope genes

[0041] Search for the ALV-J envelope gene sequence published on NCBI, use bioinformatics methods to analyze the 22 envelope epitope, and screen out the epitope gene sequence representing the epitope of the epidemic strains in China in recent years, using encoding glycine, The serine codons are connected in series to obtain a recombinant envelope gene sequence of 998 bp, whose gene sequence is shown in SEQ ID NO.1. The 5'end of the gene sequence contains an Nco Ⅰ restriction site, and the 3'end contains an Xho Ⅰ restriction site.

[0042] The above-mentioned gene sequence was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd., and the synthetic product cENV was further connected to the pUC57 vector using conventional techniques.

[0043] The synthesized product is sequenced, and its gene sequence is shown in SEQ ID NO.1;

[0044] The pET30a and the above-mentioned pUC-57 cloning vector linked to...

Embodiment 2

[0048] Example 2 Expression and purification of recombinant envelope protein

[0049] After sequencing, the positive BL21 bacteria containing the recombinant plasmid were inoculated into LB liquid medium containing kanamycin (final concentration 10μg / ml) (commercially available conventional medium containing 1% (w / v) Tryptone, 0.5 %(W / v) Yeast Extract, 1%(w / v) NaCl, 0.1mg / ml Kanamycin), shake at 37°C (200rpm), culture to OD600 = 1.0, add IPTG at a concentration of 500mMol to the entire medium IPTG The final concentration was 1mmol / L, and the culture was continued at 37°C for 4h. At the same time, the uninduced recombinant plasmid was transformed into BL21 bacteria as a control.

[0050] SDS-PAGE analysis shows that the detection steps are as follows: collect the cultured engineered bacteria solution, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet with standard PBS to obtain a resuspension; follow the ratio of 100ml initial medium to 3ml stan...

Embodiment 3

[0073] Example 3 Activity detection of recombinant protein

[0074] The purified His-cENV protein was used to coat the ELISA plate, and the biological activity of the protein was detected by enzyme-linked immunosorbent assay. Gp85 protein immunized mouse positive serum, HR1 polypeptide, HR2 polypeptide immunized chicken positive serum and IDEXX detection kit test positive chicken serum as positive control, healthy SPF chicken serum as negative control, HRP-labeled goat anti-mouse IgG, goat anti-chicken IgG enzyme-labeled secondary antibody is used according to the instructions. The results showed that the OD ratio of the positive control and the negative control was greater than 2.1, indicating that the protein has good biological activity and antigenicity.

[0075] The specific steps of ELISA are as follows:

[0076] 1) Coating: Take the purified recombinant protein, dilute it with physiological saline to the optimal concentration, and coat the reaction plate with 100μl / well at 4°...

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Abstract

The invention relates to the fields of animal virology and immunology, and provides an epitope vaccine for resisting subgroup J avian leukosis virus infection. The vaccine is prepared from screened and purified high-activity recombinant protein His-cENV after prokaryotic expression with the combination of a freund's adjuvant, wherein the nucleotide sequence of the encoding recombinant protein His-cENV is as shown in SEQ ID NO.1. 7-day breeding poultry chicks by utilizing epitope vaccine immunity can produce neutralizing antibodies of 1 to 128000; in-vitro virus neutralization experiments and animal experiments show that the epitope vaccine can neutralize different ALV-J separation strains, and effectively protect the chicks so as to resist ALV-J strain infection. Env source based multi-epitope antigen gene sequence of the epitope vaccine overcomes the virus variation of ALV-J, develops the ALV-J vaccine new era, provides a new means for resisting ALV-J infection, and provides technical support for ALV-J prevention and control.

Description

Technical field [0001] The present invention relates to the field of immunology, and specifically provides an epitope vaccine against subgroup J avian leukemia virus infection, and a preparation method and application thereof. Background technique [0002] Subgroup J avian leukemia is a tumorous infectious disease caused by subgroup J avian leukemia virus (ALV-J). In clinical practice, it is most common in myeloid leukemia, and appears mainly in immunosuppression, growth inhibition and multiple organs. Tumors are its main characteristics. The occurrence of avian leukemia in subgroup J in my country is quite common. Clinical cases have developed from the initial commercial broiler flocks to commercial layers, local breeder flocks and other poultry, accompanied by increased tumorigenicity and tumor diversity. Studies have shown that my country’s subgroup J avian leukemia virus presents a high infection rate and a high incidence, early infections are common, the age of onset is sig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/21A61P31/14
Inventor 成子强侯敏博张利
Owner SHANDONG AGRICULTURAL UNIVERSITY
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