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A kind of epitope vaccine against a/b subgroup avian leukosis virus infection and its preparation method and application

An avian leukemia virus and epitope vaccine technology, applied in the field of immunology, can solve the problems of unstable immune effect, virulence recovery, and long purification cycle, and achieve the effect of efficient and safe vaccine protection, strong specificity, and avoiding immune failure.

Active Publication Date: 2017-03-22
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 cycle and high cost, it has not been effectively implemented in our country. We can only carry out large-scale purification by eliminating infected chickens. The infection of ALV-A / B cannot be controlled, and ALV continues to appear. -Reports of large-scale intensive infection of A / B, the situation of prevention and control of A / B subgroup avian leukemia in my country is grim
There have been reports on the vaccine research and development methods for A / B subgroup avian leukemia at home and abroad, mainly traditional formaldehyde inactivated vaccines, attenuated vaccines, SU-based subunit vaccines, and nucleic acid vaccines. The body provides 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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  • A kind of epitope vaccine against a/b subgroup avian leukosis virus infection and its preparation method and application
  • A kind of epitope vaccine against a/b subgroup avian leukosis virus infection and its preparation method and application
  • A kind of epitope vaccine against a/b subgroup avian leukosis virus infection and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The screening synthesis of embodiment 1ALV-A / B multi-epitope gene

[0042] Search the ALV-A / B envelope gene sequence published on NCBI, and use bioinformatics methods to analyze and obtain 22 segments of the envelope antigen epitope, screen out the envelope epitope gene sequence representing the epidemic strains in China in recent years, and use the code Glycine and serine codons were concatenated to obtain a recombinant envelope gene sequence of 1148 bp, the gene sequence of which is shown in SEQ ID NO.1. The 5' end of the gene sequence contains an NcoI restriction site, and the 3' end contains an XhoI restriction site.

[0043] The above gene sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the synthetic product cENV was further connected to the pUC57 vector by conventional techniques.

[0044] Synthetic product sequencing, its gene sequence is shown in SEQ ID NO.1;

[0045] Digest pET30a and the above-mentioned pUC-57 cloning vector linked ...

Embodiment 2

[0049] Example 2 Expression and purification of recombinant envelope protein

[0050] The positive BL21 bacteria containing the recombinant plasmid after sequencing were inoculated in LB liquid medium containing kanamycin (final concentration of 10 μg / 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 with a concentration of 500mMol to the whole medium IPTG The final concentration was 1mmol / L, and the culture was continued at 37°C for 4h. At the same time, BL21 bacteria transformed with uninduced recombinant plasmids were set as a control.

[0051] SDS-PAGE analysis shows that the detection steps are as follows: collect the cultured engineering bacteria liquid, centrifuge at 5000rpm for 5min, discard the supernatant, and resuspend the precipitate with standard PBS to obtain a resuspension; add 3ml of standard PBS to 100ml of initial me...

Embodiment 3

[0074] Activity detection of embodiment 3 recombinant protein

[0075] The above 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. The chicken serum tested positive by the His-tag antibody and the IDEXX detection kit was used as the positive control, and the healthy SPF chicken serum was used as the negative control. HRP-labeled goat anti-mouse IgG and goat anti-chicken IgG enzyme-labeled secondary antibodies were used according to the instructions. The results showed that the ratio of the OD value of the positive control to the negative control was greater than 2.1, indicating that the protein had good biological activity and antigenicity.

[0076] The specific steps of ELISA are as follows:

[0077] 1) Coating: Take the purified recombinant protein, dilute it to the optimal concentration with physiological saline, and coat the reaction plate, 100 μl / well, overnight at 4°C...

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Abstract

The invention relates to the field of animal virology and immunology, and provides an epitope vaccine for resisting A / B subgroup avian leucosis virus infection. The epitope vaccine is prepared by highly active recombinant protein His-cENV which is obtained through screening and purification after prokaryotic expression and a freund's adjuvant in a united manner, wherein the nucleotide sequence for encoding the recombinant protein His-cENV is shown as SEQ ID NO.1. Through the epitope vaccine, 7-day-old breeding poultry chicks are immune and can produce 1:128000 neutralizing antibodies. Vitro virus neutralization experiments and animal experiments show that the epitope vaccine can neutralize different ALV-A / B isolated strains, so that chicken flocks are effectively protected to resist the infection of ALV-A / B strains. Because the epitope vaccine is based on a multi-epitope antigen gene sequence which is originated form env, the defect of ALV-A / B virus variation is overcome, a new era of the ALV-A / B vaccine is opened, a new way of resisting the ALV-A / B infection is provided, and a technical support for preventing and controlling the ALV-A / B is provided.

Description

technical field [0001] The invention relates to the field of immunology, and specifically provides an epitope vaccine against A / B subgroup avian leukosis virus infection, a preparation method and application thereof. Background technique [0002] Avian leukosis refers to various transmissible neoplastic diseases caused by avian retroviruses of the family Retroviridae, Alpharetroviruses, and the excessive proliferation of certain cellular components in poultry hematopoietic tissues. Avian lymphocytic leukemia and myeloid leukemia are more common, and are widely prevalent in chicken flocks around the world through vertical transmission and horizontal transmission, causing chicken death, emaciation, poor growth and development, and at the same time causing decreased body resistance and immunosuppression. It is one of the main diseases that endanger the poultry industry. According to the antigenic differences of the viral envelope glycoprotein, virus interference experiments, h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/21A61P31/14
Inventor 成子强侯敏博杨治聪马晓倩王永波孟薇李根庄萍萍冯卫国王桂花
Owner SHANDONG AGRICULTURAL UNIVERSITY
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