To overcome these problems much research is still needed, specifically focused on cloning and testing of new c-DNA sequences in the following: genome projects: different ways of delivery: design of vectors containing appropriate immunostimulatory sequences and very detailed studies on safety.
Leaf explants were transformed using the pBin vector containing the appropriate cDNA in sense orientation.
Cell were transfected 24 h later, with either a pEP-MIR vector expressing a pre-miRNA or with the pEP-mir Null control and with the pMIR-REPORT luciferase reporter vector containing the appropriate 3'UTR linked to luciferase.
For expression of recombinant proteins, each pET-5b vector containing the appropriate odorant-binding protein (OBP) sequence was used to transform BL21 DE3 pLysS and BL21 DE3 Rosetta-gami E. coli cells, for OBL21 DE3 Rosetta-gamitivE.y.
Cells were co-transfected with 2 μg of pβRFP vector containing the appropriate 4q25 fragment and 1 μg of pCAGGS-GFP (a kind gift from Joaquín Rodríguez-León, University of Extremadura, Badajoz, Spain) as an internal transfection efficiency control; co-transfections were performed with 6 μL of Lipofectamine 2000 Invitrogen (Waltham, Massachusetts, USA).
The following primers containing appropriate restriction sites for cloning to the pMCG161 vector were used: for Pina (forward) 5′-TTCGGACCGACTAGTATGAAGGCCCTCTTCCTCATA-3′, (reverse) 5′-TTCCTAGGCCCGGGTCACCAGTAATAGCCAATAGTG-3′; for Pinb (forward) 5′-TTCGGACCGACTAGTATGAAGACCTTATTCCTCCTA-3′, (reverse) 5′-TTCCTAGGCCCGGGAGTAATAGCCACTAGGGAACTT-3′.
Spread on plates containing appropriate antibiotic selection.
In conclusion, high level production of soluble recombinant dengue-2 NS1 can be produced from E. coli by choosing appropriate vector containing suitable fusion partners, promoter (tightly control and regulatable), and other production strategies like optimization of temperature.
Briefly, variable regions were synthesized and subcloned into expression vectors containing the appropriate IgG1 H- and L-chain constant region sequences.
All other plasmids were generated via the Gateway technology (life technologies) using the pENTR/D-TOPO entry vector (life technologies) and pcDNA3.1-based destination vectors containing the appropriate N-terminal tags.
