Recently, in the therapies of a malignant tumor, methods of using a transformed anaerobic bacterium as a carrier for gene transfer have been highlighted. For instance, methods of such as using a transformed Clostridium for transferring to the tumor site a gene that expresses nitroreductase, an enzyme that transforms a prodrug of an antitumor substance to the antitumor substance, has been proposed (see Patent Literatures 1 to 3).
Furthermore, methods of using invasive anaerobic bacteria such as Salmonella, enteroinvasive Escherichia coli, Listeria and Shigella for transferring a gene encoding a nucleic acid that abolishes or interferes the expression of a gene involved in an anaerobic disease by RNA interfering to tumor cells, such as small interfering RNAs (siRNAs), short interfering RNAs and short hairpin RNAs, have been investigated (see Patent Literatures 4 to 6).
Nevertheless, since all these microorganisms are pathogenic bacteria which have been mutated to be avirulent, the possibility cannot be denied that back mutation might be happened to return to the original pathogenic bacteria and exert harmfulness. Furthermore, for their motility and invasiveness, these bacteria might express their effect not only in the disease tissue but also in a normal tissue, causing a systemic side effect. Thus, their safety is still a matter of concern.
The inventors focused on Bifidobacterium which is a non-pathogenic enteric bacterium being present in human intestine to form a flora and which is known to be an extremely safe obligate anaerobe, and developed a method for treating a malignant tumor using a transformed bacterium of this Bifidobacterium. 
The inventors then developed a Bifidobacterium longum 105A which have been transformed to express cytosine deaminase (hereinbelow referred to as CD), which is an enzyme that converts 5-fluorocytosine (hereinbelow referred to as 5-FC) (a prodrug of an antitumor substance 5-fluorouracil (hereinbelow referred to as 5-FU)) to 5-FU (see Patent Literatures 7 and 8).
This transformed Bifidobacterium is characterized in that when being administered into a model animal of solid tumor, which is an anaerobic disease, it specifically colonizes and proliferates in the anaerobic disease tissue which is in hypoxic condition, whereas it quickly disappears in a normal tissue which is not in a hypoxic environment (see non-Patent Literatures 1 and 2).
Furthermore, this transformed Bifidobacterium is also characterized in that it does not exhibit antigenicity even when being administered intravenously. It may therefore be expected as an excellent therapeutic for malignant tumor.
Since these transformed bifidobacteria have been transformed using an Escherichia coli (E. coli)-Bifidobacterium shuttle plasmid such as pBLES100-S-eCD and pAV001-HU-eCD-M968, if they are horizontally transferred to an E. coli, they might be replicated in that E. coli. Therefore, the inventors improved the plasmid to solve this problem and developed a non-shuttle plasmid pBifiCD which does not have a replication origin that functions in E. coli (see Patent Literature 9).
On the hand, since these non-shuttle plasmids did not possess a secretory signal, the transformed bifidobacteria could not secrete expressed CD extracellularly.
Therefore, it has been desired to develop a secretory signal peptide that is capable of functioning in Bifidobacterium and secreting expressed proteins from the bacteria cell.
As examples of secretory proteins of Bifidobacterium, amylase of Bifidobacterium adolescentis, and Sec1, Sec2 and Sec 3 of Bifidobacterium breve have been reported, and plasmids introduced their secretory signals have also been reported.
For example, Bifidobacterium longum MG1 has been reported, which has been transformed with an E. coli-Bifidobacterium shuttle plasmid pYBamy59 in which a secretory signal peptide gene of Bifidobacterium adolescentis amylase have been transferred (see Patent Literature 3).
Also, Bifidobacterium breve UCC2003 has been reported, which has been transformed with an E. coli-Bifidobacterium shuttle plasmid such as pESH86 or pESH87 in which a fusion gene of a secretory signal peptide of Sec2 of B. breve and human fibroblast growth factor 2 (FGF-2) have been transferred (see Patent Literature 4).
Furthermore, there have been reports of an expression cassette containing a promoter and a signal sequence derived from Bifidobacterium, in particular an expression cassette containing a signal of BL1181 gene product or a signal sequence of amyB gene product; indeed, a significant secretion of the expressed protein was confirmed in B. breve and B. longum (see, Patent Literature 10).
Nevertheless, said plasmids are all E. coli-Bifidobacterium shuttle plasmid. A non-shuttle plasmid that does not possess a replication origin that functions in E. coli and, that has a secretory signal that functions in Bifidobacterium, such as a plasmid of the present invention, was not known. Moreover, it has not been ascertained whether any of these secretory signals function in a bacterial strain other than those already confirmed. Furthermore, the secretion of target protein by the transformed bacterium is expected to be small. Therefore, it was also desired to develop a secretory signal peptide for practical use that is capable of exerting a good secretory function.
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