TENDLER, Miriam,VILAR, Mônica Magno,ARMÔA, Geraldo Rodrigues Garcia,MCINTOSH, Douglas,SIMPSON, Andrew
申请号：
ES10750256
公开号：
ES2595372T3
申请日:
2010.03.10
申请国别(地区):
ES
年份:
2016
代理人:
摘要：
An auxotrophic and recombinant BCG Pasteur vaccine strain supplemented by the amino acid leucine expressing a Schistossoma mansoni Sm14 protein, ie rBCG Pasteur [Delta] LeuD / p [Delta] K410-hsp60 * - Sm14, obtaining said vaccine strain according to with the following steps: (a) PCR amplification of a fragment containing a Sm14 expression cassette containing a modified hsp60 * promoter, using - a pAU5-Sm14 vector as a template, said vector having a modified hsp60 sequence containing a deletion of four base pairs immediately upstream of the promoter region in combination with the loss of the first base pair of the hsp60 promoter sequence and accordingly is called hsp60 *, and - the PSm14F primers, that is, TAT ATA TAT ATA TAG GCG CCA CCA CAA CGA CGC GC - SEQ ID NO: 3 and PSm14R, that is, CGC GGG CGC CTT AGG ATG ATC GTT TAT A - SEQ ID NO: 4； (b) digestion with a Narl enzyme of an 816 bp amplicon, ie hsp60 * -Sm14, obtained by PCR amplification of step (a), providing an hsp60 * -Sm14 amplicon； (c) cloning of the hsp60 * -Sm14 amplicon obtained in step (b) in a pUP410 vector digested with the Narl enzyme, resulting in a pUP410-hsp60 * -Sm14 construct； (d) evaluation of the capacity of the pUP410-hsp60 * -Sm14 construct to express the Sm14 protein in Mycobacterium vaccae； (e) elimination of the kanamycin resistance gene from the pUP410-hsp60 * -Sm14 construct by digestion with a HindIII restriction endonuclease； and (f) re-circularization of the pUP410-hsp60 * -Sm14 digested construct without the kanamycin resistance gene using T4 ligase to produce the digested pUP410-hsp60 * -Sm14 construct, which is used to transform a BCG Pasteur strain auxotroph, that is, BCG [Delta] LeuD, to produce a recombinant auxotrophic BCG Pasteur strain supplemented with the amino acid leucine expressing a Smistos protein of Schistossoma mansoni, ie BCG Pasteur [Delta] LeuD / p [Delta] K410- hsp60 * -Sm14Una cepa vacunal Pasteur de BCG auxótrofa y recombinante complementada por el aminoácido
