Although the rapid progress of genetic techniques has allowed investigators to genetically manipulate probiotics to improve, introduce, or knockout some of their phenotypes, the stability of modulated strains is still the foundation for the advanced development of recombinant oral vaccines (Ou et al. 2016). Genetic modulation usually involves in introducing exogenous genes encoding target antigen protein or just as DNA antigen (Gahan et al. 2007), into suitable recipients, and their subsequent replication and expression. In order to achieve this goal, plasmid-based overexpression, a very convenient method while with some disadvantages, has been widely performed. Taking the EcN strain as an instance, studies on the EcN have found the existence of two small cryptic plasmids termed pMUT1 and pMUT2 which are genetically stable and unable to transfer to other strains of E. coli (Sonnenborn and Schulze 2009). Based on that, Remer et al. (2009) explored the method of expressing the K88 fimbrial adhesin from the ETEC on the EcN. They attempted to insert the corresponding gene into pMUT2 and then orally administrated the recombinant strain into mice. Meanwhile, Buddenborg et al. (2008) have also developed a tripartite vector system which used the adhesin involved in diffuse adherence (AIDA) system to express heterologous antigens on the surface of EcN. The stability of this system has been evaluated through subculture on agar plates and the result showed that neither pMUT1 nor pMUT2 could contribute to the stability of the segregational plasmid, which indicated that although pMUT1 and pMUT2 are stable plasmids, their ability to facilitate stable heterologous gene expression remains invalid. In addition to the EcN, studies on pSIP expression vectors in Lactobacillus plantarum demonstrated that the pSIP expression system is applicable to intracellular expression, secretion, and surface anchoring of heterologous proteins (Anbazhagan et al. 2013; Bohmer et al. 2012; Fredriksen et al. 2012). However, when it was applied to other Lactobacillus strains to produce NucA (model protein), unstable transformants were observed and one possible explanation for that is due to the expression of NucA that may lead strains highly burdensome (Karlskas et al. 2014), a widely shared potential downside of the plasmid-based overexpression strategy.
COTD – Cryptic Countries
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