Endofree plasmid giga kit

The pVAX1 plasmids encoding T. gondii profilin (pVAX1-PF), rhoptry protein 16 (pVAX1-ROP16), rhoptry protein ROP18 (pVAX1-ROP18), microneme protein 6 (pVAX1-MIC6), and calcium-dependent protein kinase 3 (pVAX1-CDPK3) were constructed as previously reported (31 (link)–35 (link)), with the fidelity of all plasmids confirmed by sequencing (Sangon, China). The five eukaryotic plasmids were each transformed into E. coli DH5α for propagation and were isolated by anion exchange chromatography (EndoFree Plasmid Giga Kit, Qiagen Sciences, MD, USA) following the manufacturer’s instruction. Plasmid concentration and purity was determined by measuring the optical density ratio A₂₆₀/A₂₈₀. The purified plasmids were stored at −20°C until used in the mouse immunization protocols.

Vaccines were constructed by cloning the B. abortus BAB1_0267 and BAB1_0270 ORFs into the pVAX1 vector (Invitrogen). To achieve this, the ORFs were amplified by means of the polymerase chain reaction (PCR) using primers design in accordance with the appropriate sequences available at GenBank (CAJ10223.1 and CAJ10226). The final primer sequences, including the cutting sites for BamHI and XhoI, were: BAB1_0267 forward: 5′wordGCCGGATCCGCCACCATGTTGTCGAAGGCGAAAAACTGG3′ and reverse: 5′wordTTTCTCGAGCTAAACCACCCAGAGCGGT3′. The final sequence to BAB1_0270 was forward: 5′word-GCCGGATCCGCCACCATGAGCAGTCAGAATTACGTTGTC-3′, and reverse: 5′word-GCCCTCGAGTCAGATCCCTTTTTTATTGATCCA-3′. PCR product was cloned into the pVAX1 vector using T4 DNA ligase (Promega, Madison, WI). The resulting constructs were named pV267 and pV270, and used for DNA immunization. To obtain the quantity of DNA required for all immunizations, E. coli DH5α strain (Thermo Fisher Scientific Inc., MA) was transformed by electroporation with the constructs and recombinant plasmid DNA was isolated using the EndoFree Plasmid Giga kit (Qiagen, Valencia, CA).

HCV core expressing DNA vaccine was constructed and evaluated as previously described (20 (link)). Briefly, the core region of HCV genome was amplified from the sera with positive results for genotype 1 HCV and cloned into pcDNA3 (Invitrogen, CA) (summarized as pCore here). Murine MIP-3beta cDNA cloned into pcDNA3.1 + plasmid (Invitrogen, CA) was provided by Cytokine Bank, South Korea (summarized here as pMIP-3beta). The construct integrity was confirmed by restriction enzyme mapping, PCR and DNA sequencing (data not shown). Endofree plasmid Giga Kit (Qiagen, Germany) was used to prepare plasmids in large scale.

Human p62 (SQSTM, isoform 1) – encoding DNA vaccine (Elenagen) was previously described [31 (link)] and produced using EndoFree Plasmid Giga Kit (Qiagen).

The pVAX1-GRA24 plasmid constructed in the same manner as the above-described pET28a-GRA24 plasmid. The gra24 gene was inserted into the eukaryotic vector pVAX1, confirmed by sequencing, and named pVAX1-GRA24. pVAX1-GRA24 was then transformed into E. coli DH5a. An Endofree plasmid giga kit (Qiagen, Chatsworth, CA, USA) was used to purify the pVAX1-GRA24 plasmid. The DNA concentration was determined by a NanoDrop 2000 instrument. Finally, pVAX1-GRA24 was diluted to 1 mg/ml by sterile endotoxin-free phosphate-buffered saline (PBS) and stored at −20°C before use.
The pVAX1-GRA24 expression plasmid was transfected to HEK293 cells (human embryonic kidney cells) using the lipo2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 48 h, cell monolayers and supernatants were collected. pVAX1-GRA24 plasmid expression in the transfected cells was then detected by quantitative real-time PCR (qRT-PCR), western blotting and an indirect immunofluorescence assay (IFA).

Design of bicistronic pDNA carrying coding sequences of human HGF and VEGF165 is described in detail in our previous work [7 (link)]. HGF/VEGF pDNA was amplified in Escherichia coli (DH-5α strain) and purified to endotoxin-free grade using the EndoFree Plasmid Giga Kit (Qiagen, Germantown, MD, USA) as recommended by the manufacturer, followed by reconstitution in sterile saline (0.9% NaCl) for animal administration.

Competent E. coli Stbl3 cells were transformed with the pVAX-H5 plasmid. Recombinant E. coli Stbl3 cells were cultivated at 37 °C in LB medium with kanamycin (100 μg/mL). For analytical purposes, plasmid DNA was isolated from bacterial cells using the commercial “Hipure Plasmid Mini Kit” (Magen, Guangzhou, China) according to the manufacturer’s recommendations. For mice immunization, plasmid DNA was isolated using the EndoFree Plasmid Giga Kit (Qiagen, Hilden, Germany) and dissolved in saline. Quantitative and qualitative assessment of isolated plasmid DNA was carried out using a NanoDrop™ OneC spectrophotometer (Thermo Fisher Scientific, USA) at wavelengths of 260, 280, and 230 nm. Endotoxin detection was carried out using the LAL reagent Endosafe-PTS (Charles River Laboratories, Wilmington, MA, USA) according to the manufacturer’s instructions.