Cloning Of Bile Salt Hydrolase Gene Of Lactobacillus Plantarum And The Expression In L. Lactis ML23

Cholesterol is a kind of steroids originally got from gall-stone. It exists widely in animals. The content of cholesterol is high in kidney, spleen, skin, heparin and bile, especially in brain and nerve tissues. Cholesterol plays an indispensable part in animal tissues and cells; it joins the formation of cell membrane, and also is the starting material for the synthesis of the bile acid, vitamin D and steroidal hormones. Otherwise,some negative effects are accompanied,such as atherosclerosis, coronary heart disease,high blood pressure and other cardiovascular or cerebrovascular diseases. Reducing serum cholesterol affects directly human health, and also is one of the hot issues in the current study. Many clinical trials confirmed that orally taking probiotics and their related products could not only reduce the serum cholesterol concentration but also reduce the risk of suffering from cardiovascular disease.Yet cholesterol-reducing activity of probiotics is closely related to its bile salt hydrolase, so the characteristic researches on bile salt hydrolase are essential to reveal the action mechanism of the cholesterol-reducing activity of probiotics.BSH is the metabolism outcome of probiotics during their growth and reproduction phase. It is a kind of intracelluar enzyme and appears to be most active during pH 5 to 6.The bound state of taurine bile salts and glycine bile salt can be hydrolyzed by BSH and finally turn into a mino acids and free cholic acid, reducing the serum cholesterol concentrations.At present BSHs activity has been detected in many bacteria like Lactobacillus, Bifidobacteria, Enterococcus, Clostridium, Bacteroides and Streptococcus. Lactobacillus and Bifidobacteria are often viewed as probiotics; Enterococcus, Clostridium and Bacteroides usually exist in the gastrointestinal tract of hosts in the form of commensalism. So far, BSHs activity has not been detected in the bacteria that are separated from environment without bile salt. And till now, all the BSH positive bacteria found except for two Bacteroides strains, are Gram-positive bacteria.Based on Lactobacillus plantarum genome as template and bsh sequence published in Genbank(EU477374.1), we designed primers and amplified bsh gene (972bp), then used Sac I and Hind III to cut bsh gene and connectted it to the multiple cloning sites in pMG36e with T4 ligase, finally established recombinant vector pMG36e-bsh.The recombinant vector pMG36e-bsh was electrotransformed into L. lactis ML23, and the analyses of SDS-PAGE verified that bsh gene can be expressed, and its molecular weight is 35 kDa.In this experiment, bsh gene in Lactobacillus plantarum was successfully cloned, and BSHs was successfully expressed in Lactococcus lactis ML23. We lay a foundation for expression of bsh gene and construction of probiotic strains with a efficiently cholesterol-reducing fuction; as well as provide a theoretical guide for developing cholesterol-lowering probiotic preparations.