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Construction of recombinant bacteria expressing avian encephalomyelitis virus VP1 protein

A technology of avian encephalomyelitis and protein recombination, which is applied in the field of constructing recombinant bacteria expressing VP1 protein of avian encephalomyelitis virus, can solve the problem of high safety

Active Publication Date: 2012-07-18
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And do not need inactivation, but high security

Method used

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  • Construction of recombinant bacteria expressing avian encephalomyelitis virus VP1 protein
  • Construction of recombinant bacteria expressing avian encephalomyelitis virus VP1 protein
  • Construction of recombinant bacteria expressing avian encephalomyelitis virus VP1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Propagation of AEV

[0062] Aseptically inoculate the yolk sac of 6-day-old SPF chicken embryos with AEV VR strain, and hatch at 37°C. On the 9th day after inoculation, put the chicken embryos into a 4°C refrigerator to collect the brain, pancreas, gastrointestinal tract and allantoic fluid of chicken embryos with typical lesions ; The collected allantoic fluid was used as a diluent to treat the disease material, after grinding, repeated freezing and thawing 3 times, and centrifugation at 7000r / min for 10min, the virus suspension was obtained and stored at -80°C.

[0063] 2. PCR amplification of VP1 gene, cloning and sequence determination (see figure 1 , figure 2 , image 3 )

[0064] A pair of primer P1 (sequence 3) containing an ECORI restriction site and primer P2 (sequence 4) containing a SalI restriction site were designed and synthesized according to the published sequence. The purified virus suspension was cracked by adding Trizol, extracted with chloro...

Embodiment 2

[0072] 1. Fermentation and induced expression

[0073] The recombinant Escherichia coli Rossetta / pET-32a-VP1 was inoculated on the LB plate of ampicillin + chloramphenicol, and cultured at 37°C for 10-12 hours. Pick 10 larger smooth colonies and inoculate them in 500 mL of LB culture medium with ampicillin + chloramphenicol. Incubate overnight at 37°C with shaking. Transfer the saturated bacterial solution to 20,000 mL of Escherichia coli culture medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride) at a volume ratio of 2%. Cultivate at 37°C and 30% oxygen saturation until the OD value of the culture medium is 0.6-0.8, add 0.2M lactose to a final concentration of 0.02M, and continue culturing for 5-7 hours. Bacteria were collected by centrifugation. Add 4 parts of normal saline according to the wet weight of 1 part of bacteria. Resuspend the bacteria and store at -20°C.

[0074] 2. Purification process of expressed protein: thaw the frozen bacterial cell suspen...

Embodiment 3

[0076] Vaccine preparation:

[0077] The expression product of the recombinant strain CGMCC No.5581 obtained in Example 2 was used as the antigen for preparing subunit vaccine, and the vaccine was prepared according to the conventional mineral oil adjuvant inactivated vaccine method.

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Abstract

The invention relates to construction of a recombinant bacteria expressing avian encephalomyelitis virus VP1 protein. According to the invention, through synthesizing a primer of an avian encephalomyelitis virus VP1 gene, the recombinant bacteria with an efficient expression vector of the gene is constructed, efficient expression of the avian encephalomyelitis virus VP1 protein in Escherichia Coli is realized, and the amount of the expressed soluble protein accounts for about 60 percent of the total protein of the bacteria. The expression product is soluble protein in cytosol and has the antigen activity of natural VP1 protein, and avian encephalomyelitis VP1 subunit vaccine prepared from the expression product can effectively protect birds against the attack of avian encephalomyelitis virus. Therefore, a firm basis is established for industrially producing the avian encephalomyelitis subunit inactivated vaccine.

Description

technical field [0001] The invention relates to the construction of recombinant bacteria expressing VP1 protein of avian encephalomyelitis virus, which belongs to the field of molecular biology and also belongs to the field of veterinary biological products. Background technique [0002] Avian encephalomyelitis, also known as epidemic tremor, is an infectious disease that mainly affects chicks caused by avian encephalomyelitis virus (AEV). Its characteristic pathological change is non-suppurative encephalomyelitis. Typical symptoms It is movement disorder, head and neck tremor and progressive paralysis (Zhao Zhenhua, Yu Wangsheng, Hao Xianpu, etc. Occurrence and diagnosis of avian encephalomyelitis. Inner Mongolia Animal Husbandry Science. 1994, 3: 34-37). The disease has occurred in almost all areas where commercial chickens are raised in the world. Since the 1980s, the disease has broken out in various places in my country, which has brought great economic losses to the po...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/41C12N15/63A61K39/125A61P31/14C12R1/19
Inventor 郭伟伟范根成左青山刘新文胡潇
Owner YEBIO BIOENG OF QINGDAO
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