Baculovirus expression system and construction method and application thereof
A baculovirus and expression system technology, applied in the field of protein expression, can solve problems such as time-consuming, labor-intensive, reduced work efficiency, and cumbersome operations
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[0061] 1. Construction of baculovirus expression vector system
[0062] The common replicons of pFastBac and pHelper were replaced by the temperature-sensitive pSC101ori replicons by the Gibson cloning method, and then the ccdA expression cassette was inserted on the basis of the temperature-sensitive helper plasmid to construct optimized donor vectors pFastBac(ts) and Helper vector pHelper(ts+ccdA).
[0063] Such as figure 1 As shown, the homologous recombinase expression plasmid pRed / ET (tetracycline-resistant Tc) was electrotransferred into the competent DB3.1 containing only the Bacmid vector (Kana-resistant Kan), added to SOC culture and incubated at 30°C for 1 hour .
[0064] The resuscitated culture products were spread on LB plates containing Tc+Kan antibiotics, cultured at 30°C for two days, and positive clones containing both pRed / ET and Bacmid vectors were identified by PCR.
[0065] Pick positive clones and inoculate them into LB liquid medium containing Tc+Kan ...
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