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Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof

A technology of recombinant adenovirus and alpha interferon, applied in the field of interferon, can solve the problems of low titer of recombinant adenovirus and unsuitable for clinical application, and achieve high antiviral activity and high virus titer

Active Publication Date: 2014-08-13
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the titer of the recombinant adenovirus obtained by this method is low, and it has a GFP tag, so it is not suitable for clinical application
So far, there is no relevant report on the expression and biological function of goat interferon-alpha at home and abroad

Method used

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  • Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof
  • Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof
  • Recombinant adenovirus for expression of goat alpha interferon and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of recombinant adenovirus expressing goat interferon alpha

[0041] 1. Experimental method

[0042] 1.1 Codon optimization goat interferon alpha gene

[0043] Refer to the goat interferon alpha (GoIFN-α) gene sequence (FJ959074) published by GenBank, optimize the codon according to the codon preference of mammalian cells, and then manually optimize and synthesize the gene according to the GC content and secondary structure of the gene. , The nucleotide sequence of the optimized goat interferon alpha is shown in SEQ ID NO.1; the GC content of the optimized GoIFN-α gene is 57%, which is lower than the original sequence (60%).

[0044] 1.2 Construction of recombinant adenovirus shuttle vector

[0045] The optimized goat interferon alpha gene was amplified by PCR, the PCR product was glued and recovered, and then the recovered PCR product and the adenovirus shuttle vector pShuttle-CMV were digested with EcoR I and Not I. The gel was recovered and purified afte...

Embodiment 2

[0054] Example 2 Identification of recombinant adenovirus expressing goat interferon alpha

[0055] 1. Experimental method

[0056] 1.1 Recombinant adenovirus plasmid transfected cells

[0057] Three recombinant adenovirus plasmids pAd-GoIFN-α-1, pAd-GoIFN-α-2 and pAd-GoIFN-α-3 were transfected into HEK293 cells respectively, and the cytopathic condition was observed.

[0058] 1.2 Indirect immunofluorescence test

[0059] Spread HEK293 cells on a 96-well plate, and when they reach 90% monolayer, inoculate 10MOI (Multiplicities of infection) rAd-GoIFN-α-1, rAd-GoIFN-α-2, rAd-GoIFN-α-3 After 24h, discard the medium, wash the cells with PBS 3 times, fix the HEK293 cells infected with three recombinant adenoviruses with pre-chilled absolute ethanol for 15min, add mouse positive serum (500×) at 37℃ for 1h, use PBST After washing, add fluorescent-labeled goat anti-mouse IgG (Sigma) at 37°C for 1 h, wash with PBST and dry naturally, add buffered glycerin, and observe under a fluorescence mic...

experiment example 1

[0076] Experimental example 1 Stability and biological activity determination of recombinant adenovirus rAd-GoIFN-α-1

[0077] 1. Experimental method

[0078] 1.1 PCR detection of recombinant adenovirus rAd-GoIFN-α-1

[0079] The second, fourth, sixth, eighth and tenth generation viruses of rAd-GoIFN-α-1 were harvested, and the recombinant virus DNA was extracted with a DNA extraction kit for PCR detection.

[0080] 1.2 Western blot analysis

[0081] The HEK293 cells infected with rAd-GoIFN-α-1 were subjected to SDS-PAGE electrophoresis, and HEK293 cells infected with wild-type adenovirus were set up as a negative control, and then the separated protein was transferred to a nitrocellulose membrane and blocked with skim milk Then, add mouse anti-GoIFN-α serum (500×) and act at 37°C for 1h, wash with PBST, add horseradish peroxidase-labeled rabbit anti-mouse IgG (Sigma, diluted 1:5000), act at 37°C for 1h, After washing with PBST, DAB solution was added to develop color.

[0082] 1.3 Tit...

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PUM

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Abstract

The invention discloses recombinant adenovirus for expression of goat alpha interferon and a construction method and application thereof. The invention first provides an optimized goat alpha interferon gene with a nucleotide sequence shown as SEQ ID NO:1; the invention also provides the recombinant adenovirus for stable expression of the goat alpha interferon, and the microbial preservation number is: CGMCC (China General Microbiological Culture Collection Center) No.8757. The invention further provides a construction method of the recombinant adenovirus. The recombinant adenovirus constructed by the construction method is capable of stable expression of the goat alpha interferon, high in virus titer, and high in antiviral activity; the recombinant adenovirus and the goat alpha interferon expressed by the recombinant adenovirus both have antiviral activity in swine cells IBRS2, bovine cells MDBK and primary sheep skin cells, and can be used in the prevention or treatment of ruminant animal virus infectious diseases.

Description

Technical field [0001] The present invention relates to an optimized goat interferon alpha gene. The present invention also relates to a recombinant adenovirus stably expressing goat interferon alpha and a construction method thereof. The present invention further relates to the optimized goat interferon alpha gene and recombinant adenovirus in preparation The application of drugs for preventing or treating viral infectious diseases of ruminants such as cattle and sheep belongs to the construction and application field of recombinant adenovirus expressing goat interferon alpha. Background technique [0002] Animal infectious diseases caused by viruses have severely restricted the healthy development of aquaculture in various countries and regions, and also pose a serious threat to human health. The scale of China's sheep industry is huge and it is a pillar industry of animal husbandry. The biggest threat to the sheep industry is viral infectious diseases, such as sheep pox, blue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/21C12N15/85C12N7/01A61K48/00A61K35/76A61P31/14
Inventor 于力王芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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