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Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof

A live carrier vaccine and chicken coccidiosis technology, applied in the field of live carrier vaccine preparation, can solve problems such as unfavorable transportation and storage, cancer-causing DNA antibodies and gene drift, complex mechanism of action, etc., achieve simple fermentation and production technology, and be popularized and used The effect of value, prevention and treatment

Inactive Publication Date: 2010-12-15
HANGZHOU BAODELI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of attenuated vaccines has the risk of strong virulence, and the antibody does not last long. Some vaccines have certain side effects and are not conducive to transportation and storage due to incomplete attenuation. DNA vaccines have safety problems, including possible carcinogenicity and DNA production. Antibodies and gene drift, etc.; recombinant proteins have disadvantages such as complex production process, high cost, and vaccines need to be stored at low temperature; and Chinese herbal medicines have defects such as high production cost and complicated mechanism of action

Method used

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  • Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof
  • Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof
  • Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the construction that contains antigenic gene 3-1E recombinant plasmid

[0040] 1. Cloning of antigen 3-1E gene

[0041] 1. Cloning of 3-1E gene

[0042] Purify sporulated oocysts from the feces of broiler chickens infected with E.tenella according to conventional techniques, and refer to the total RNA in pure sporulated oocysts, reverse transcribe the RNA into cDNA with a reverse transcription kit, and reverse The recorded cDNA was used as a template, and the upstream primer (SEQ ID NO: 1) was used:

[0043] 5P 31E-BS 5'-TCTAGAAATGGGTGAAGAGGCTGATACT-3' and

[0044] Downstream primer (SEQ ID NO: 2):

[0045] 3P 31E-BS 5'-CTGCAGTTA GTGATGGTGATGGTGATG GAAGCCGCCCTGGTACAGGT-3' was used for PCR to obtain the target gene (3-1E gene).

[0046] The PCR reaction system is:

[0047] cDNA 1.0μl

[0048] Primer 5P 31E-BS (10mM) 1.0μl

[0049] Primer 3P 31E-BS (10mM) 1.0μl

[0050] Taq enzyme (5U / μl) 0.5μl

[0051] 10×PCR buffer (with Mg 2+ ) 5.0μl

...

Embodiment 2

[0080] Embodiment 2, the preparation method of recombinant bacillus subtilis (the live carrier vaccine of chicken coccidiosis prevention):

[0081] Transform the recombinant expression vector (recombinant plasmid) pBES-3-1E constructed in Example 1 above into Bacillus subtilis 1A751 (derived from the American Bacillus Collection Center BGSC) (any Bacillus subtilis strain can be transformed):

[0082] Bacillus subtilis 1A751 was cultured overnight at 30°C in 4ml of GMI medium (test tube), 100r / min (slow shaking) 10% (volume ratio) was transferred to 6ml of GMI medium (erlenmeyer flask), and cultivated at 37°C for 3.5 h, 200r / min (quick shaking); 10% was transferred to 20ml GMII medium (erlenmeyer flask), cultured at 37°C for 90min, 100r / min (slow shaking); the bacteria were collected by centrifugation (1 / 10 volume supernatant Suspended bacteria): Divide 20ml of bacterial liquid into four 10ml centrifuge tubes (5ml each), collect the bacterial cells by centrifugation, and resusp...

Embodiment 3

[0085] Embodiment 3, the preparation of the oral biological preparation of prevention chicken coccidiosis

[0086] Mix 25 parts by weight of zeolite powder with 75 parts by weight of cornstarch, and both the zeolite powder and cornstarch can pass through a 100-mesh sieve; the carrier is obtained.

[0087] The bacteria powder obtained in Example 2 is mixed with the above-mentioned carrier in a weight ratio of 1:9, and simply stirred and mixed to obtain an oral biological agent for preventing and treating chicken coccidiosis; each gram of the oral biological agent contains 10 9 cfu live vector vaccine.

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Abstract

The invention discloses a preparation method of a live vector vaccine for controlling chicken coccidiosis, comprising the following steps of obtaining a recombinant expression vector by connecting a mature peptide of an eimeria tenella sporozoite antigen gene 3-1E gene and an expression vector of bacillus subtilis, obtaining a recombinant strain by transferring the recombinant expression vector into a bacillus subtilis host, and culturing the recombinant strain to finally obtain the live vector vaccine for controlling the chicken coccidiosis by. The live vector vaccine can be used for controlling and / or treating the chicken coccidiosis. The invention also discloses an oral biological preparation for controlling the chicken coccidiosis, prepared by utilizing the live vector vaccine. The oral biological preparation comprises the live vector vaccine and a vector. The oral biological preparation per gram contains the live vector vaccine with a living bacteria number of 108-109cfu.

Description

technical field [0001] The invention provides a preparation method and application of a live carrier vaccine capable of preventing and treating chicken coccidiosis. Background technique [0002] Chicken coccidiosis is a protozoan disease that is very harmful to the chicken industry. Intensive chicken farms are the most suitable places for coccidiosis outbreaks. 78.7% of intensive chicken farms have different degrees of infection. The world economic loss due to chicken coccidiosis is about 2 billion pounds per year (Lee, 2010). Chicken coccidiosis is mainly caused by the infection of one or several Eimeria coccidiosis, a parasitic protozoan disease mainly caused by Eimeria coccidia parasitizing in chicken intestinal epithelial cells; Chickens under the age of 15, especially those aged 15 to 50 days are most susceptible to infection; it occurs frequently in warm and humid months, and the incidence is less in winter. Suffering from acute coccidiosis, the course of the disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/012C12N15/30C12N15/75C12N1/21A61P33/02
Inventor 李卫芬林志伟余东游邓斌毛翔飞
Owner HANGZHOU BAODELI BIOTECH
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