Studies On Cloining And Expression Of PL-PLC Gene And The Effect Of Anti-coccidiosis In Chicken

Bacterial PI-PLC is a small,water-soluble enzyme that can catalyze the hydrolysis of phosphatidylinositol phosphodiester bonding to producea water-soluble inositol phosphates and fat-soluble diacylglycerol?DAG?.Studies have shown that bacterial enzyme PI-PLC can hydrolyze glycosylation?GPI?proteins anchored on cell surface in most pathogenic parasite,and deprive of the parasite invading and proliferation in the host cells.Thus,bacterial enzyme PI-PLC showed significant anti-infection properties.Since most of bacteria that can produce PI-PLC are belonged to pathogens,and the yield of PI-PLC is low,it is desirable to produce PI-PLC in probiotics by genetic manipulation method.In this paper,the PI-PLC gene from Bacillus cereus was expressed in E.coli and Lactococcus lactis to explore the effect of anticoccidiosis in chicken.The main research include as followed:Based on the nucleotide sequence of Bacillus cereus PI-PLC published on Genbank,the sequence of PI-PLC gene was optimized and synthesised to construct the recombinant expression vector pET28 a,giving rise to pET28a-PIPLC.The completed construct was transformed into E.coli BL21 cells.After induction by IPTG?isopropyl ?-D-1-thiogalactopyranoside?,an approximate 35 kDa protein was detected by SDS-PAGE and cell lysates showed significant activity of PI-PLC.Under the optimal induction condition: 1 mmol/L IPTG added when cells reached OD600 s of 0.4 and induced for 6 hours at 37?,maximum PI-PLC enzyme producing(0.688 mg·L^-1)was detected.Lactic acid-producing bacteria?LAB?are generally regarded as safe organisms and are therefore widely used in the production and preservation of fermented products by the food industry.Lactococcus lactis has been considered a good candidate to serve as acost-effective live vaccine delivery vehicle for mucosal immunization.Therefore the PLPLC gene was cloned into E.coli-Lactococcus lactis shuttle vector p AMJ399,and then transformed to L.lactis cells by electroporation.The result of SDS-PAGE showed that the recombinant protein was secreted extracellar in the form of soluble proteins and the molecular weight of PI-PLC protein was about 35 kDa.Meanwhile,the recombinant protein showed significant enzyme activity on PI-Listeria chromogenic plate.The results indicated that PI-PLC was successfully expressed in L.lactis.The optimum growth condition for PI-PLC enzyme producing 1.092 ?g/m L was at 32? for 24 h without shaking in GM17 Medium with 1 ug/m L erythromycin.The efficiency of PI-PLC expressed in L.lactis was evaluated in chicken with anticoccidial assay,After challenging the chicken with 5×10⁴ oocysts/bird,the body weights of chickens fed with PI-PLC containing L.lactis was increased,but the result was not statistics significant.In term of of cecal lesion and oocyst scores,a significantly reduction was observed,suggesting that the enzyme PI-PLC played a significant role in resisting to coccidia infection;In summary,PI-PLC have anticoccidial effect,and anticoccidial effect was close to the antibotics such as diclazuril.