Expression And Purification The Protein Of Plnc And Plnd Which Regulate The Synthesis Of Bacteriocin Produced By Lactobacillus Plantarum ZJ316

Lactobacillus plantarum is a kind of Lactobacillus which isolate from pickles, yogurt and the gastrointestinal tract of animals and the L. plantarum can widely ferment plant carbon source, tolerated cholate and low pH. Some of the L. plantarum can produce bacteriocin which belong to the class Ⅱ, and they can kill Escherichia coli, Salmonella, Shigella and other intestinal pathogenic bacteria. The class Ⅱ bacteriocin produced by L. plantarum was a thermal-stability-hydrophobic peptide, consists by natural amino acid, without modification after transcription. This bacteriocin synthesis is regulated by quorum-sensing system.L. plantarum ZJ316was isolated from infant feces, and it could produce the bacteriocin with molecular weight4.0KD. In this paper, we using the method of gene clone the quorum-sensing system (QS) which regulate the bacteriocin synthesis. What’s more, we study the regulation factors of plnC and plnD (RR) which regulate the QS, including:(1) Design the primers according to QS genes from Genbank, serve the L. plantarum ZJ316chromosome DNA as template and amplify by PCR. It shows, the plnABCD operon which encode the quorum sensing system exists in ZJ316. Furthermore, the proteins of PlnC and PlnD were predicted, including primary structure, physical and chemical properties, secondary structure and space structure. The results show that the structures of PlnC and PlnD are similar, and their function realized by combination to the DNA on the specific sites respectively.(2) plnC and plnD genes were constructed into the prokaryotic expression vector pET-32a-SUMO respectively, building the pET-plnC and pET-plnD recombinant plasmids, transforming the recombinant plasmids into E. coli BL21(DE3), which express induced by IPTG. The results show that, the recombinant genes express better in the condition of100μg/mL Kana,37℃,3h,0.2mM IPTG. In this expressing condition, both the recombinant proteins are existing in the form of inclusion.(3) The bacterial suspension which manage with ultrasonic is dealed with Ni-NTA affinity chromatography, and the balance buffer is composed of20mM PBS,500mM NaCl,20mM imidazole, pH8.0and elution buffer is composed of20mM PBS,500mM NaCl,150-300mM imidazole, pH8.0; after renaturation of the inclusions, purify those target proteins by Ni-NTA affinity chromatography and sephadexG200chromatography; then fellowed digestion and desalting purification by DEAE ion exchange column; at last, purify the target proteins by sephadexG75chromatography, get the proteins of PlnC and PlnD which molecular weight are both about25KD.The proteins of PlnC and PlnD purified are groundwork for studying the regulate factors in QS and the regulation of biosynthesis the bacteriocin.