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Preparation method of attenuated salmonella transferring chicken gamma interferon

A Salmonella and interferon technology, applied in the biological field, can solve the problems of cumbersome protein purification steps, easy to be damaged by gastric acid, etc., and achieve the effects of easy storage, good antiviral activity, and simple storage methods

Inactive Publication Date: 2011-11-23
山东步步赢生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Our research group has also cloned chIFN-γ into the prokaryotic expression vector pET32a, but the expressed protein has cumbersome purification steps and is easily destroyed by gastric acid in the stomach.

Method used

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  • Preparation method of attenuated salmonella transferring chicken gamma interferon
  • Preparation method of attenuated salmonella transferring chicken gamma interferon
  • Preparation method of attenuated salmonella transferring chicken gamma interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation method of the chicken gamma interferon delivered by the attenuated Salmonella comprises the following steps:

[0035] (1) Amplification of the gene sequence of the mature protein of chicken gamma interferon (chIFN-γ):

[0036] (1-1) Design and synthesis of primers:

[0037] (1-1-1) According to the gene sequence of chIFN-γ published by GenBank (GenBank no. AY501004), design a pair of specific primers for amplifying the gene sequence of mature chIFN-γ protein. Introduced at the 5' end of primer chIFN-γ-Fwd Eco RI restriction site and Kozak sequence were introduced at the 5' end of primer chIFN-γ-Rev xho I restriction site, the primer sequence is as follows:

[0038] chIFN-γ-Fwd: 5’-TATGAATTCGGTACCACCATGGGACATACTGCAAGTAGT-3’

[0039] chIFN-γ-Rev:

[0040] 5'-GAGCTCGAGAAGCTTATTAGCAATTGCATCTC-3'

[0041] The upstream and downstream primers span the chIFN-γ mature protein coding region, and the length of the amplified fragment is expected to be about 4...

Embodiment 2

[0054] Post-challenge protection assay of attenuated Salmonella delivered orally to chickens with chIFN-γ:

[0055] will contain the plasmid pVAX1 ? - The attenuated Salmonella of chIFN-γ was cultured by shaking overnight, then centrifuged, resuspended in PBS, and the cfu was calculated. At the same time, attenuated Salmonella without any plasmid was set up as a control. will contain the plasmid pVAX1 ? -The concentration of the attenuated Salmonella of chIFN-γ and the control attenuated Salmonella were both adjusted to 10 8 cfu / mL. Get 20 20-day-old SPF chickens and divide them into 2 groups at random, and the test group takes 10 orally 8 cfu containing plasmid pVAX1 ? -chIFN-γ attenuated Salmonella, and the control group was orally administered attenuated Salmonella without any plasmid. On the first day and the second day of the test, it was taken orally once each, and on the third day, 50 LD 50 The Newcastle disease virus NDV challenged the virus, observed that all 10...

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Abstract

The invention belongs to the biotechnology field, in particular discloses a preparation method of attenuated salmonella transferring chicken gamma interferon. The preparation method is characterized by comprising the following steps: amplifying a gene sequence of a maturation protein of the chicken gamma interferon (chIFN-gamma); obtaining a recombinant eukaryotic expression plasmid; detecting a chIFN-gamma activity unit expressed by the recombinant eukaryotic plasmid; electrically transferring the recombinant plasmid pVAX1<c>-chIFN-gamma into the attenuated salmonella to obtain a strain. By the adoption of the attenuated salmonella transferring chicken gamma interferon, the problems that the interferon is easy to be degraded by gastric acid after being orally taken by the chicken and the protein purifying step is fussy can be avoided effectively. The chIFN-gamma can be prepared by bacteria fermentation, and is easy to store; the chIFN-gamma has obvious NDV (Newcastle disease virus) protection force for chicken, and can provide guarantee for preventing and treating the viral diseases such as NDV in a scale chicken house.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a preparation method for attenuated Salmonella delivering chicken gamma interferon. (2) Background technology [0003] IFN can be divided into two types (type I and type II). IFN-α and β belong to type I interferon, and IFN-γ belongs to type II interferon, which has antiviral, antitumor and immunomodulatory effects. After Digby and Lowenthal successfully cloned chicken IFN-γ (chIFN-γ) for the first time in 1995, scholars at home and abroad have done a lot of research on chIFN-γ through expression systems such as Escherichia coli, yeast, and baculovirus. Our research group has also cloned chIFN-γ into the prokaryotic expression vector pET32a, but the expressed protein has cumbersome purification steps and is easily destroyed by gastric acid in the stomach. (3) Contents of the invention [0004] In order to make up for the deficiencies in the prior a...

Claims

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Application Information

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IPC IPC(8): C12N15/23C12N15/79C12N1/21C12R1/42
Inventor 张旭东李玲
Owner 山东步步赢生物科技有限公司
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