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Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation

A cystic larval virus and structural protein technology, applied in antiviral immunoglobulins, egg-derived immunoglobulins, introduction of foreign genetic material using vectors, etc. Problems such as not being able to eat well

Inactive Publication Date: 2017-03-22
JINZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, although there are traditional Chinese medicines used for the treatment of bee cystic larvae, the traditional Chinese medicine treatment is mainly through the immune regulation, indirect anti-virus, can not directly kill the virus, relatively slow onset, at the same time, the smell of traditional Chinese medicine often makes the Bees cannot feed well, and even escape, especially for seriously ill bee colonies, the healing effect is poor
In view of this, CN102504023A discloses a method for preparing egg yolk antibody by using the vesicular larvae virus. However, since the vesicle larvae virus has no suitable culture system so far, it cannot be artificially propagated in vitro, which directly limits the production of yolk antibodies. mass production and application of

Method used

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  • Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation
  • Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation
  • Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Based on the VP2 gene of the structural protein of the Chinese bee vesicular larvae virus, according to the codon preference of Escherichia coli, and while ensuring that the amino acid sequence of the expressed protein remains unchanged, the codon modification was carried out, and a rare codon modification was obtained. The VP2 gene is shown in SEQ ID No.1.

Embodiment 2

[0020] 1. Materials and methods

[0021] 1.1 Materials

[0022] The CSBV strain (GenBank accession number: HM237361.1) was isolated from a bee farm in Kuandian, Liaoning Province and preserved by our laboratory;

[0023] The total RNA extraction kit was produced by Promega in the United States; the prokaryotic expression vector pET-28a (gifted by Professor Wang Hong of Jinan University); the low relative molecular weight protein marker, AMV reverse transcriptase, and DNA ligation kit were produced by Dalian Bao Biological Company; plasmids Small extraction kit, DNA purification and recovery kit and BIOTECH, His band protein purification kit were purchased from Novagen, Germany; anti-CSBV egg yolk antibody (preserved by our laboratory); HRP-labeled rabbit anti-chicken IgG was purchased from Beijing Boaosen Biotechnology Co., Ltd. Technology Co., Ltd.; isopropyl-β-D-thiogalactoside (IPTG) was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0024] method

[0025] 1...

Embodiment 3

[0053] 1. Method for preparing egg yolk antibody

[0054] 1.1 Antigen preparation

[0055] The purified VP2 fusion protein obtained in Example 2 was diluted to 0.5 mg / mL with 0.01 M PBS at pH=7.4. Antigen emulsification adopts double syringe mutual pushing method.

[0056] 1.2 Immunization procedure

[0057] 15 Bailaihang hens were randomly divided into three groups, and the prepared VP2 protein, the whole virus inactivated vaccine group and normal saline were used to immunize the abdominal and back muscles at multiple points. The dose of VP2 protein for the first immunization was 0.4mg, 0.8ml per mouse, the immunogen was mixed with Freund's complete adjuvant in equal amounts, and phacoemulsified. The interval between the second immunization and the first one was one month, the dose was halved, and the immunogen was emulsified with incomplete Freund's adjuvant. The interval between the third immunization and the previous immunization is 2 weeks, and the dose of immunizatio...

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Abstract

The invention provides a Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation. A Chinese bee cysticercus virus structure protein VP2 gene transformed through a rare codon obtained according to the preference of escherichia coli to a codon is shown in SEQ ID No.1. The Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression has the advantage that the Chinese bee cysticercus virus structure protein VP2 gene is optimized and transformed. The transformed VP2 gene is subjected to prokaryotic expression and can produce a large number of VP2 proteins, and an egg yolk antibody prepared through a VP2 immunized chicken can be used for preventing a Chinese bee cysticercus disease and is remarkable in curative effect.

Description

technical field [0001] The invention relates to the prokaryotic expression of the VP2 gene of the vesicle virus structural protein used for the preparation of egg yolk antibody. Background technique [0002] Apis chinensis has been included in the national livestock and poultry genetic resources protection list, and it is also one of the top ten livestock and poultry genetic resources protection species in Liaoning Province. It is a precious local bee species resource in my country. Raising bees has also become an effective way for farmers in mountainous areas to make a fortune . Chinese bees play an irreplaceable role in maintaining the plant ecosystem in our country. Studies have shown that once Chinese bees go extinct, 30% of the plants in my country will become extinct. At present, due to the reduction in production of Chinese bees, some national-level secondary protected plants are on the verge of extinction. [0003] In recent years, in order to strengthen the developm...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K16/10C07K16/02
CPCC07K16/10C07K2317/11
Inventor 马鸣潇费东亮李慧张皓淳李永森
Owner JINZHOU MEDICAL UNIV
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