Cloning Of β-GALACTOSIDASE Gene From Lactobacillus Acidophilus And Study Of Its Activity In Lactococcus Lactis As A Food-grade Selection Marker

Objective:Cloning and expressingβ-galactosidase gene (lacZ) from Lactobacillus acidophilus in Lactococcus lactis, and observing the selective activity and stability of lacZ gene as a food-grade selection marker for the construction of a food-grade vector.Methods:1. According to the lacZ sequence of Lactobacillus acidophilus ATCC4356 in GenBank, a pair of primers was designed. And then the lacZ gene was amplified from L. acidophilus ATCC4356-DNA by PCR techniques. After purification, the lacZ gene fragment was digested with restriction endonucleases PstI and XbaI, and then linked with the lactic acid bacterial expression vector pMG36e digested with the same enzymes to construct the recombinant plasmid pMG36e-lacZ. The positive recombinant from Escherichia coli DH5αwas selected with blue colonies on X-gal-containing LB medium.2. Optimize the electroportion procedures for L. lactis MG1363, And the recombinant plasmid pMG36e-lacZ was transformed into MG1363 by electroportion. Selected the positive transformant MG1363-lacZ by selecting blue colonies on X-gal- and lactose-containing M17 medium.3. MG1363-lacZ was grown at 30℃on M17 medium containing lactose and induced for expressing ofβ-galactosidase. The proteins expression andβ-galactosidase activity was analysed with SDS-PAGE and native-PAGE respectively. The activity and specific activity ofβ-galactosidase were detected after a period of 60 generations with X-gal as the coloration for the stability observation.Results :1. Restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-lacZ was successful. The positive recombinant from Escherichia coli DH5αwas selected successfully with blue colonies on X-gal-containing LB medium.2. The electroportion procedures of L. lactis MG1363 was optimized, and the recombinant plasmid pMG36e-lacZ was transformed into MG1363 at 1.9 kv of voltage and 2.8 ms of duration. The positive transformant MG1363-lacZ was selected successfully by selecting blue colonies on X-gal- and lactose-containing M17 medium. And the PCR identification and restriction enzyme digestion also confirmed that the recombinant plasmid pMG36e-lacZ was electroporated into L. lactis MG1363 successfully. The recombinant L. lactis strain MG1363-lacZ was obtained.3. SDS-PAGE showed the genetic engineering L. lactis strain MG1363-lacZ could express a protein, about 70kDa, which was confirmed to be ofβ-galactosidase activity by native-PAGE analysis. Theβ-galactosidase specific activity of recombinant L. lactis MG1363-lacZ after a period of 60 generations selected with X-gal was similar to the 2nd generation (P=0.592>0.05), and also similar to the 61st generation selected with Erythromycin (P=0.882>0.05). These indicated that theβ-galactosidase lacZ gene had good activity and stability as a selection marker.Conclusion:The positive transformant DH5α-lacZ and MG1363-lacZ was selected successfully by expressing L. acidophilus ATCC4356β-galactosidase and selecting blue colonies on X-gal-containing medium. Theβ-galactosidase lacZ gene had good activity and stability as a selection marker, and it could be used as a selection marker for constructing of a food-grade vector.