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hn-vp233-221aa fusion protein and its preparation method and application

A technology of fusion protein and bursal disease, applied in the field of HN-VP233-221aa fusion protein and its preparation, can solve the problems of reduced immune effect of traditional vaccines, increased susceptibility to other diseases, impaired lymphocyte function, etc., and achieve enhanced Humoral and cellular immune response, good immune protection rate, effect of good protection

Active Publication Date: 2021-01-12
HENAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

IBD mainly affects chicks and young chickens aged 3-6 weeks, and can destroy the central organ of humoral immunity in chickens—the bursa of Fabricius, resulting in impaired lymphocyte function and immunosuppression, thereby affecting the immune effects of vaccines such as Newcastle disease and avian influenza. and increased susceptibility to other diseases
At present, due to the prevalence of NDV and IBDV mutant strains, super-virulent strains and new strains, the immune effect of traditional vaccines has been reduced. Therefore, the prevention and control of these two severe infectious diseases is of great significance to the development of the poultry industry. Research and development New vaccines to prevent and control ND and IBD are particularly important

Method used

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  • hn-vp233-221aa fusion protein and its preparation method and application
  • hn-vp233-221aa fusion protein and its preparation method and application
  • hn-vp233-221aa fusion protein and its preparation method and application

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preparation example Construction

[0049] Recombinant HN-VP2 of the present invention 33-221aa The preparation method of fusion protein, its production comprises the following steps:

[0050] (a) HN-VP2 33-221aa and HN gene sequence acquisition

[0051] ①IBDV VP2 33-221aa Obtaining the gene sequence:

[0052] According to the nucleotide sequence of IBDV VP2 in GenBank (No:AF508177), design a pair of primers to amplify VP2 33-221aa Gene fragment.

[0053] Using the previously constructed pMD18-T-VP2 as a template, PCR amplifies IBDV VP2 33-221aa fragment. After the amplified product was identified by 1% agarose gel electrophoresis, the target band was excised, recovered and purified using a common agarose gel DNA recovery kit, and the purified product was VP2 33-221aa Gene fragments were sent to Dalian Bao Biological Engineering Co., Ltd. for sequencing;

[0054] ② Acquisition of NDV HN gene

[0055] According to the nucleotide sequence of NDV HN gene in GenBank (No: JF343538), a pair of primers were de...

Embodiment 1

[0066] 1. HN-VP2 33-221aa Acquisition of fusion protein coding gene sequence

[0067] (1) Acquisition of Newcastle disease virus HN gene

[0068] According to the nucleotide sequence of the HN gene of Newcastle disease virus in GenBank (No: JF343538), a pair of specific primers were designed using DNAStar7.0 and Primer premier6.0 software to amplify the HN gene fragment of Newcastle disease virus.

[0069] P1 and P2 amplify the HN gene of Newcastle disease virus, and the 5' end of primer P1 is introduced Bam HI endonuclease site, P2 5' end introduces a flexible linker. The primer sequences are as follows:

[0070] Table 1 Primer sequences used in PCR amplification

[0071]

[0072] The frozen NDV strain was thawed in a 37°C water bath, and then inoculated into 9-10 day-old non-immune chicken embryos (0.2 mL per embryo) through the allantoic cavity, and incubated at 37°C. Aseptically collect the allantoic fluid of dead chicken embryos within 24 to 48 hours (discard the...

Embodiment 2

[0092] rHN-VP2 33-221aa Applications of fusion proteins:

[0093] 1) Animal immunity

[0094] With above recombinant HN-VP2 33-221aa Chickens were immunized with the fusion protein, and its immune characteristics were evaluated. 240 14-day-old healthy chicks were randomly divided into 4 groups, 60 per group. Group 1 was a negative control, immunized with 200 μL PBS; group 2 was immunized with 200 μg of HN-VP2 recombinant fusion protein (diluted with 200 μL PBS), and immunized by chest muscle injection. Group 3 and Group 4 were immunized against Newcastle disease Vaccine and bursa vaccine (B87 strain) were used as the traditional vaccine control group, and 200 μL was immunized by nasal drops and eye drops; all experimental groups were immunized once every 2 weeks, and immunized 3 times in total. And on the 7th, 21d, and 35th day after the first immunization, 8 chicks were randomly selected from each group for heart blood collection and serum separation. At the same time,...

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Abstract

The invention discloses HN-VP233-221aa fusion protein as well as a preparation method and application thereof. The HN-VP233-221aa fusion protein comprises an amino acid sequence of infectious bursal disease virus VP233-221aa, an amino acid sequence of Newcastle disease virus hemagglutinin-neuraminidase (HN protein) and a flexible Linker peptide amino acid sequence located between the amino acid sequence of the VP233-221aa and the amino acid sequence of the HN protein of Newcastle disease virus; the HN-VP233-221aa fusion protein is formed by connecting a main protective gene segment of VP2 and an HN gene of the Newcastle disease virus in series through a flexible Linker peptide (-G-S-). The recombinant HN-VP233-221aa fusion protein disclosed by the invention can be used for effectively inducing an organism to generate body fluid and cell immune response and can be used for inducing immunized chickens to generate high-level HN specific antibodies and VP2 antibodies and accelerating a proliferation and activation capability of chicken T lymphocytes; the HN-VP233-221aa fusion protein has a very good protection effect on attacks of infectious bursal disease virulent strains and Newcastle disease virulent strains so that the effect of preventing two diseases through one injection is realized.

Description

technical field [0001] The invention relates to the field of genetic engineering production of vaccines and immune adjuvants in the biopharmaceutical industry, in particular to a HN-VP2 33-221aa Fusion protein and its preparation method and application. Background technique [0002] Newcastle disease (ND) is an OIE class A disease, and infectious bursal disease (IBD) is a class B infectious disease, causing huge economic losses to the global poultry industry. In my country, Newcastle disease and infectious bursal disease have been important infectious diseases that seriously endanger the development of poultry industry. Chicken Newcastle disease is highly contagious and has a high mortality rate, and the range of infected hosts is becoming wider and wider. In 2009, several Newcastle disease outbreaks occurred in Sweden, the Netherlands, Peru and Belgium, causing huge economic losses. In recent years, there are still reports of strong NDV infection in chickens or quails, p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61K39/295A61K39/17A61K39/12A61P31/14
CPCA61K39/12A61K2039/552A61K2039/572A61K2039/575A61K2039/70C07K14/005C07K2319/00C12N9/2402C12N2720/10022C12N2720/10034C12N2760/18134C12Y302/01018
Inventor 贾艳艳张春杰刘莎莎廖成水何雷高冰李静郁川余祖华张晓洁程相朝丁轲
Owner HENAN UNIV OF SCI & TECH
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