Expression Of A Novel Fibrinolytic Enzyme Gene In Lactobacillus Casei

Lactic acid bacteria (LAB) are vital members of food microbes. Because they are healthy for human beings and animals, they have important application future in food and medicine fields. The genetic elements, existed outside the chromosome of LAB are excellent material for developing their vector systems. Recent years, with the endogenous plasmids of LAB removed, electrotransformation established and the expression signal elements cloned, a serial of vector-host systems with different usages have been successfully constructed. These vectors include basic cloning vector,various integrative and express vectors. Food-graded vectors, which use food-graded selective markers to substitute antibiotics resistant gene markers, are highlight point in LAB researches. It can avoid the release of antibiotic-resistant genes into environment, including human beings and animals. Thus, exploring new LAB vectors has considerable meaning.This study was aimed at expression of a novel fibrinolytic gene from Douchi in Lactobacillus casei using LacF selective marker. We firstly constructed an integrative vector pRVGF by cloning the fragments of LacF and LacG into pRV300 and eliminated the Sph I site of LacF by T4DNA polymerase to get plasmid pRVGΔF. The plasmid pRVGΔF was electrotransformated into L.casei, the LacF~- deficient strain MBL25 was successfully.obtained. This strain was further confirmed by the.PCR analysis and Lac phenotype examination. In addition, an autoreplicative expression vector pIAbeta-plac was constructed by using the promoter of lactose operon of L.casei. Three fragments encoding different peptides of DouChi fibrinolytic enzyme were cloned into pRVGF and pIAbeta-plac, respectively, and six recombinant plasmids were constructed.Finally, the plasmid pRVGF-BADFE containing the mature peptide-codying fragment of fibrinolytic enzyme were electrotransformated L.casei into MBL25. PCR analysis demonstrated that the BADFE gene fragment had integrated into the chromosome of the MBL25. The phenotype examination indicated that the Lac~+ had been restorated. After induction overnight by 0.5% lactose, SDS-PAGE analysis showed that 28KD protein was successfully expressed.