[0060] Example 1
[0061] 1. Construction of Baculovirus Expression Vector System
[0062] Using the Gibson cloning method, replace the common replicons of pFastBac and pHelper with the temperature-sensitive pSC101ori replicon, and then insert the ccdA expression box on the basis of the temperature-sensitive helper plasmid to construct the optimized donor vector 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 electroporated into DB3.1 competence containing only the Bacmid vector (kanah-resistant Kan), and SOC medium was added to resuscitate and culture at 30°C for 1 hour .
[0064] The resuscitated culture product was spread on an LB plate containing Tc+Kan antibiotics, cultured at 30°C for two days, and clone-picking PCR identified positive clones containing both pRed/ET and Bacmid vectors.
[0065] The positive clones were picked and inoculated into LB liquid medium containing Tc+Kan antibiotics, and cultured overnight at 30°C.
[0066] The overnight culture was inoculated into fresh LB medium containing Tc+Kan antibiotics at a ratio of 1:100, cultured at 30°C to an OD600~0.3, added 0.3%~0.4% L-arabinose, and placed at 37°C to induce culture 45 minutes to 1 hour.
[0067] The induction product was prepared into electro-competent state, and the ccdB cassette-FRT-Ampcassette-FRT PCR product with homology arm was added for electrotransformation, and the SOC medium was added to resuscitate and culture at 37°C for 1 hour.
[0068] The resuscitated culture product was spread on an LB plate containing Kan+Amp antibiotics, incubated overnight at 37°C, and clone-picking PCR identified the recombinant positive clones.
[0069] The 707-FLPe (tetracycline-resistant Tc) was electroporated into the competent cells of the positive strain obtained in step 6, and the SOC medium was added to resuscitate and culture at 30°C for 1 hour.
[0070] The resuscitated culture product was spread on an LB plate containing Kan+Tc and cultured at 30°C for two days. Pick several clones separately and add them to the LB culture medium containing Kan antibiotics, and culture them at 30°C for 2 to 3 hours.
[0071] The resuscitated culture product was streaked on an LB plate containing Kan antibiotics and incubated overnight at 37°C.
[0072] Pick several single clones in duplicate, add them to two LB mediums containing Kan and Amp respectively, and cultivate overnight at 37°C.
[0073] The clones that are not long in LB containing Amp but grown in LB liquid containing Kan are used for PCR identification, and the PCR-positive clone is the modified Bacmid (ccdB) vector.
[0074] The purified Bacmid (ccdB) and pHelper (ts+ccdA) plasmids were co-electrotransformed into DH10B competent, and the positive clones containing the two plasmids were identified by PCR, and the optimized Bac-to-Bac clone strain VB UltraDH10Bac was obtained. The glycerol bacteria.
[0075] Two, application
[0076] First use Gibson cloning technology to clone the EGFP (enhanced green fluorescent protein) gene into the donor vector pFastBac(ts), construct pFastBac-EGFP(ts), and then transform it into the VB UltraDH10Bac strain for transposition recombination reaction, and extract the recombination After the DNA product was subjected to agarose gel electrophoresis, the DNA morphology was observed. Combine figure 2 As shown, the specific steps are as follows:
[0077] Gibson cloning technology was used to construct pFastBac-EGFP(ts).
[0078] VB UltraDH10Bac competence was prepared according to the conventional chemical competence preparation method.
[0079] Take 1μL of pFastBac-EGFP(ts) plasmid with a concentration of 10ng/μL into VB UltraDH10Bac competent and mix well.
[0080] Ice bath for 30 minutes, heat shock at 42°C for 45 seconds, and immediately ice bath for 2 minutes.
[0081] Add 900μL of SOC medium, shake culture at 30°C and 250rpm for 4 hours.
[0082] Take 100 μL of the resuscitation product and spread it on the LB plate containing Kan+Gen antibiotics, and incubate at 30°C for 20 hours.
[0083] Pick monoclonal PCR to identify positive recombinants.
[0084] The positive recombinant clones were picked and inoculated into liquid LB containing Kan+Gen antibiotics, and cultured overnight at 37°C.
[0085] The overnight culture product was subjected to plasmid extraction according to the conventional BAC extraction method.
[0086] Take 1μL of plasmid extract for agarose gel electrophoresis.
