Cloning, Sequencing And Expression Of Phospholipase C Gene From Bacillus Cereus 754-1

Phospholipase C (PLC) is a kind of hydrolase for phosphoinositide (PI), which mainly exists in these membranes of blood platelet, and it can regulate the cellular-metabolization and transferring-communication, such as phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and so on. Many animal investigations showed that PLC can significantly inhibit platelet aggregation and adhesion, and can prevent coming into thrombus and cardiovascular and cerebrovascular disease. The PLC from bacterium is extracellular enzyme, which has much impurity- protein in fermentation broth. For simplifying the technics and reducing the difficulty of separation and purification, we design this experiment, and conceive the idea of constructing a functional strain farther upon. We change the Bacillus cereus 754-1 Wuhan strain from extra-cellular enzyme into intra-cellular enzyme, which is a good basic to the conceiving. The paper contains these main parts, as following:I . Designed four primers together to carry PCR early or late, and then the PCR products were purified by AGE. After purification and extraction, the products of primer I was cut by BamH I and Pst I , while the other primers were cut by BamH I and Xho I , and connected to pMD18-T vector and transformed to E.coli TG1 with white/Blue plaque color change and antibiotic ampicillin (Amp) to filtrate resistant colonies, then to carry PCR and cut by BamH I and Xho I . After sequencing we gained a gene segment which is 1623bp in length carried by primer I , the sequence comparability is very high with up to ninety-four percent, so this sequence is a part of Bacillus cereus 754-1 strain plc gene. And it is 1012bp by primer II, the sequence comparability is very low with down to forty-six percent, we find it has many mutations in the midst of PCR. And it is 929bp by primerIII, the sequence comparability is very high with up to ninety-three percent, but it has one mutation in the midst. And It is 1020bp by primerIV, the sequence comparability is very high with up to ninety-four percent, so this sequence is a part of Bacillus cereus 754-1 strain plc gene.II. The expression vector pMAL which was suit for the PCR products by primer 1 is so expensive, so we didn't continue. The PCR products by primer II and III all have mutation, so we didn't choose them. We chose the PCR products by primerIV to continue. And let it cut by BamH I and Xho I , then connected to pET28a vector and constructed a expression vector named pET-PLM, and then transformed to E. coli BL21. Using IPTG to induce it to expression, and Oxford cup agar scattering experiment proved it was expressed in cell.