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Fusion protein containing interferon alpha, recombinant strain for expressing fusion protein and preparation method of fusion protein

A fusion protein, interferon alpha technology, applied in the field of biomedicine, can solve problems such as reducing the activity of interferon alpha, and achieve the effects of blocking immune escape, improving the effect of virus clearance, and reducing morbidity and mortality.

Active Publication Date: 2022-05-27
山东仙普爱瑞科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods all reduce the activity of interferon α

Method used

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  • Fusion protein containing interferon alpha, recombinant strain for expressing fusion protein and preparation method of fusion protein
  • Fusion protein containing interferon alpha, recombinant strain for expressing fusion protein and preparation method of fusion protein
  • Fusion protein containing interferon alpha, recombinant strain for expressing fusion protein and preparation method of fusion protein

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1 A fusion protein containing interferon alpha, a recombinant strain expressing the fusion protein and its preparation method

[0039] 1. Insert the fusion gene fragment rpoALB-nsp5 site-IFNα into the secretion expression vector pPICZαA vector

[0040] Suggestions on the fusion protein module of rpoALB-nsp5 site-IFNα figure 1 (See appendix SEQ ID NO. 1 for the complete nucleic acid sequence and SEQ ID NO. 2 for the amino acid sequence).

[0041] Nucleic acid sequences of each module of the fusion protein of rpoALB-nsp5 site-IFNα

[0042] (1) rpoALB nucleic acid sequence (SEQ ID NO.3)

[0043] (3) nsp5 site nucleic acid sequence (SEQ ID NO.4)

[0044] (4) IFNα nucleic acid sequence (SEQ ID NO. 5).

[0045]According to the nucleic acid sequence of rpoALB, the nucleic acid sequence of nsp5 site, and the nucleic acid sequence of IFNα, Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to synthesize the entire expression cassette, and inserted into the ...

Embodiment 2

[0068] Example 2 Plasma stability detection of rpoALB-nsp5 site-IFNα

[0069] Blood was collected from healthy BALB / c mice, centrifuged to collect plasma, diluted with PBS to 50% plasma (V / V). 8) Add 50% plasma to a final concentration of 10µg / mL, incubate at 37°C for 0, 0.25, 0.5, 1.0, 3.0, 6.0, 12 and 24h, take 100µl each, centrifuge at 12000g for 30min, and collect the supernatant. According to the "2017 Version of Veterinary Drug Quality Standards", the MDBK-VSV detection system and the cytopathic inhibition method were used to detect the fusion protein activity. The results showed that the time for 50% decrease of IFNα activity was about 6 h, and the time for rpoALB-nsp5 site-IFNα activity to decrease by 50% was 24.5 h. For details, see Image 6 .

Embodiment 3

[0070] Example 3 Laboratory control test of rpoALB-nsp5 site-IFNα

[0071] 30 piglets that were born without colostrum were randomly divided into 3 groups, 10 pigs in each group. The first group was the rpoALB-nsp5site-IFNα treatment group; the second group was the IFNα treatment group (see patent CN201911058504.8 for specific preparation standards) ; Group 3 is no treatment group. Piglets in each group were injected with 3 mL of PED virus solution (the virus concentration was 1 × 10 6 TCID50 / mL), then immediately, the piglets of the experimental group were intramuscularly injected with the purified rpoALB-nsp5 site-IFNα obtained in Example 1 (the antiviral activity was 1 × 10 6 IU / mL), 2 ml each time, once a day for 3 consecutive days; the piglets in the second group were intramuscularly injected with IFNα ((the antiviral activity was 1 × 10 6 IU / mL), 2 ml each time, once a day for 3 consecutive days; piglets in the blank control group were intramuscularly injected with ...

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Abstract

The invention provides a fusion protein containing interferon alpha. The fusion protein is obtained by sequentially connecting the following modules in series: porcine serum albumin rpoALB, a coronavirus nsp5 cleavage site nsp5 site and interferon alpha fusion protein IFN alpha, wherein the porcine serum albumin rpoALB, the coronavirus nsp5 cleavage site nsp5 site and the interferon alpha fusion protein IFN alpha are sequentially connected in series; the invention also provides a recombinant strain for expressing the fusion protein and a preparation method of the fusion protein. The rpoALB-nsp5 site-IFN alpha fusion protein disclosed by the invention is high in antiviral activity and long in half-life period, and the morbidity and mortality of piglets infected with viruses can be reduced.

Description

technical field [0001] The invention relates to a fusion protein containing interferon alpha, a recombinant strain expressing the fusion protein and a preparation method thereof, belonging to the technical field of biomedicine. Background technique [0002] Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine transmissible gastroenteritis virus (TGEV) co-infection or single infection of three coronaviruses is the breeding industry. 's natural enemy. [0003] Interferon alpha (IFNα) has antiviral, antitumor, antiproliferative and immunomodulatory effects. At present, porcine α-interferon has been expressed in expression systems such as Escherichia coli, Pichia pastoris and animal cells. The activity analysis of recombinant porcine α-interferon protein showed that it has inhibitory effects on foot-and-mouth disease virus, porcine respiratory and reproductive syndrome virus, transmissible gastroenteritis virus, porcine delta coronavirus and por...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N1/19C12R1/84
CPCC07K14/765C07K14/005C07K14/56C12N1/16C12N2770/20022C07K2319/31Y02A50/30
Inventor 郭海岩刘刚王兴业韩国英苗玉和韩法杰王红军孙秀丽
Owner 山东仙普爱瑞科技股份有限公司
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