Salmonella choleraesuis, vaccine and application thereof

A technology of Salmonella and cholera, applied in the field of genetic engineering, can solve the problems of inhibiting reproductive system function, animals not completely losing reproductive ability, and difficult to control the dosage, and achieve the effect of convenient use, various immunization methods, and alleviating restlessness

Active Publication Date: 2020-12-08
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that the regulation of GnRH is dual. Appropriate doses of GnRH can promote the secretion and release of LH and FSH, but when given large doses of exogenous GnRH or GnRH analogs, it inhibits the reproductive system of the body.
The research on GnRH immunization vaccin

Method used

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  • Salmonella choleraesuis, vaccine and application thereof
  • Salmonella choleraesuis, vaccine and application thereof
  • Salmonella choleraesuis, vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Construction of eukaryotic single-expression gonadotropin-releasing hormone plasmid pVAX-S / GnRH-asd.

[0060] 1. Synthesis: Query the GnRH gene (Gene ID: 101111690) from NCBI, connect the HBsAg S gene upstream of the GnRH gene, add NheI and HindIII restriction sites at both ends, and send it to Wuhan Qingke Bio for synthesis. The length of S / GnRH is 993bp, and the sequence is shown in SEQ ID NO.1. The synthesized fragment is inserted into the pUC 57 plasmid and stored as pUC-S / GnRH-Amp;

[0061] 2. Plasmid extraction: take the Salmonella C500 (pVAX-asd) containing the pVAX-asd plasmid and the glycerol bacteria containing pUC-S / GnRH-Amp preserved in the laboratory, and culture them after activation by streaking. Quantitative extraction kit (for the operation steps, refer to the instructions of the plasmid extraction kit of Tiangen Biochemical Technology Co., Ltd.) for plasmid extraction;

[0062] 3. Digestion: Digest the plasmids pVAX-asd and pUC-S / GnRH-Amp wi...

Embodiment 2

[0068] Example 2 Construction of eukaryotic dual expression of gonadotropin-releasing hormone and kissing plasmid pVAX-S / GnRH-2a / KISS1-asd.

[0069] 1. Synthesis: Query the KISS1 gene (Gene ID: 3814) from NCBI, connect the 2A peptide gene upstream of the KISS1 gene, add HindIII and XhoI restriction sites at both ends, and send it to Wuhan Qingke Bio for synthesis. The synthesized 2a The length of / KISS1 is 504bp, and the insertion is stored in the pUC57 plasmid, which is designated as pUC-2a / KISS1-Amp. Wherein the KISS1 sequence is shown in SEQ ID NO.2, and the 2A sequence is shown in SEQ ID NO.3;

[0070] 2. Plasmid extraction: Take the x6097 (pVAX-S / GnRH-asd) constructed in Example 1 and the bacterial strain containing the pUC-2a / KISS1-Amp plasmid, culture them after activation by streaking, and use a small amount of plasmid extraction for the cultured bacterial liquid Kit (for the operation steps, refer to the instructions of the plasmid extraction kit of Tiangen Biochemic...

Embodiment 3

[0076] Example 3 Preparation of eukaryotic C500 competent engineering bacteria expressing anti-gonadotropin-releasing hormone and kissin.

[0077] 1. Preparation of Competent Salmonella C500: Take the frozen C500 strain (preserved by the Animal Genetics, Breeding and Reproduction Laboratory of Huazhong Agricultural University), pick it up with an inoculation loop, add it to the LB agar plate containing 50 μg / ml DAP, and draw a line For culture, the LB agar plate without DAP was streaked as a control. After overnight culture at 37°C, a single colony was picked and placed in 30ml LB liquid medium containing 50μg / ml DAP, and cultured with shaking at 37°C. Inoculate into 300ml LB liquid medium containing DAP at a ratio of 1:100, culture with shaking at 37°C for 4-6h, OD 600 When the value reaches about 0.5, pour the bacterial liquid into a pre-cooled centrifuge bottle, and after 30 minutes in an ice bath, centrifuge at 5000 rpm / min at 4°C for 10 minutes, resuspend the precipitate ...

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Abstract

The invention discloses salmonella choleraesuis C500 (pVAX-S/GnRH-2a/KISS1-asd) which is preserved in the China Center for Type Culture Collection and has the preservation number of CCTCC NO:M 2020055. The strain is applied to preparation of drugs or vaccines for controlling animal oestrus performance. The invention further discloses two plasmids, i.e., a pVAX-S/GnRH-asd plasmid and a pVAX-S/GnRH-2a/KISS1-asd plasmid. The two plasmids can also be applied to preparation of the drugs or the vaccines for controlling animal oestrus performance. The invention also discloses a gene vaccine for controlling animal oestrus performance and a preparation method thereof. After animals are immunized by the vaccine, antibodies of a gonadotrophin releasing hormone and KISS1 are generated, animal endogenous hormones are neutralized, aggressivity of the animals is reduced, and the problems of dysphoria, anorexia and abnormal excitation of the animals in the oestrus period are relieved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Salmonella choleraesuis, a vaccine and applications thereof. Background technique [0002] Under natural circumstances, the reproductive behavior of animals is regulated by multiple factors such as hormone levels and the environment, but human interference and regulation of animal behavior and traits are often required in production and life. In animal husbandry production, especially in the estrus period of large livestock, due to sudden changes in hormone levels, their temperaments often undergo dramatic changes, such phenomena as agitation, roaring, biting, demonstrations, and fighting caused by estrus, It is a risk to the animal itself, its herd, or people who come into direct contact with animals in heat, and can even lead to injury or death. It is not uncommon for pets or animals to hurt people every year. In addition, the fights of group breeding animals oft...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/16A61K39/00A61P5/24C12R1/42
CPCC07K7/23C07K14/575C12N15/74A61K39/0006A61P5/24A61K2039/523A61K2039/53A61K2039/552Y02A50/30
Inventor 熊家军许学林黎平贵陈建国杨艾平杨波荆焕松胡映红李可可游思
Owner HUAZHONG AGRI UNIV
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