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
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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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