To analyse the –2.3- to –2.1-kb promoter region with mutations, three constructs were designed with base substitutions at Box I, Box II, or Box III, designated constructs M1, M2, and M3, respectively (Fig. 5, constructs M1 M3).
RTA3'UTR was then inserted into the pGL3 vector downstream from the luciferase gene, and we designated this construct pGL3 RTA3'UTR.
A random peptide phage library designated T7S constructed by G. Froman (Department of Microbiology, Uppsala University, Sweden), was used previously for the determination and characterization of a mimotope that bound to MAb to mouse polyomavirus large T-antigen (Houshmand et al. 1999) and a mimotope of MAb specific to Leptospira spp. (Tungtrakanpong et al., in press).
HeLa cells exogenously transfected with GFP-Hippi construct (designated as 'HeLa-Hippi') was taken as test sample while HeLa cells transfected with empty GFP vector (designated as 'HeLa-Gfp') served as control and relative level of transcripts were compared between these cells by microarray.
The construct designated V5PB pDNA (6.4 kbp) was made as follows: the 3′UTR fragment of the Xenopus laevis β-globin gene (designated U3, 317 bp) was removed from pBSSK/SB10 (kindly supplied by Dr.Z. Ivics, Max Delbrück Center, Germany), and inserted into pCS2+ already containing the 5′UTR (designated U5) (Invitrogen/Life Technologies, Paisley, UK).
The entire CgPAD gene was amplified by PCR and cloned into the Nde I/Hin dIII site of pET-22b (Novagen, Darmstadt, Germany), yielding the construct designated pPAD22b.
The GP64 of BmNPV (BmGP64) was replaced with the GP64 of AcMNPV (AcGP64); this construct, designated BmNPVΔbgp/AcGP64, displays AcGP64 on the surface of BmNPV.
The resultant BCG construct, designated AERAS-401 (BCG1331 ΔureC::ΩpfoAG137Q) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice.
We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins.
As a result, the expression of the gene fusion was under the control of CaMV 35S promoter (the construct designated as pBI121-PparoA1optimized, Fig. 2).
