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. 2019 May 14;10:923. doi: 10.3389/fimmu.2019.00923

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Copyright © 2019 Kilpeläinen, Saubi, Guitart, Moyo, Wee, Ravi, Hanke and Joseph.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Genetic characterization of the BCG.HIVconsv1&2^2auxo.intstrains. (A) Pasteur substrain identification of BCG.HIVconsv1&2^2auxo.intstrains by multiplex PCR assay. Lane 1 and 8: molecular weight marker; lane 2: BCG wild type Pasteur giving the bands of 472, 401, 276, and 196 bp; lane 3 and 4: BCG.HIVconsv1&2^2auxo.int Working Vaccine Stocks (WVS), respectively, giving the bands of 472, 401, 276, and 196 bp; lane 5: BCG Connaught giving the bands of 401, 252,199, and 196 bp; lane 6: MTBVAC.HIVA^2auxo.int (M.tuberculosis strain) WVS giving a distinctive band of 146 bp, and lane 7: negative control, distilled water. (B) Enzymatic restriction analysis of p2auxo.HIVconsv1^int (left) and p2auxo.HIVconsv2^int (right) plasmids DNA purified from E. coli M15ΔglyA cultures (pre-BCG transformation). Lane 1: Undigested plasmids; Lane 2: HindIII digestion; Lane 3: PstI digestion; Lane 4: StuI digestion; Lane 5: XbaI digestion; Lane 6: Molecular weight marker. (C) PCR analysis of E. coli glyA DNA and HIVconsv1 or HIVconsv2 DNA coding sequences. Left panel: glyA PCR. Lanes 1 and 6: Molecular weight marker; Lane 2: p2auxo.Ag85B, positive control; Lane 3: BCG.HIVconsv1^int (WVS); Lane 4: BCG.HIVconsv2^int (WVS); Lane 5: distilled water, negative control. Right panel: HIVconsv1 and HIVconsv2 PCR. Lanes 1 and 8 corresponds to the molecular weight marker; Lanes 2 and 3: p2auxo.HIVconsv1 and p2auxo.HIVconsv2 (episomal), positive controls; Lane 4: BCG.HIVconsv1^int (WVS); Lane 5: BCG.HIVconsv2^int (WVS); Lanes 6 and 7: distilled water, negative controls; (D) PCR analysis of attR and attL DNA region using as template the cultures of BCG wild type, negative control (lane2 and 3), BCG.HIVconsv1^2auxo.int WVS (lane 4 and 5), BCG.HIVconsv2^2auxo.int WVS (lanes 6, and 7) and distilled water as negative control (lane 8). Lane 1 corresponds to molecular weight marker.