弓形虫P30基因-乳酸乳球菌重组质粒的构建及其表达

目的构建表达弓形虫昆山分离株P30基因的乳酸乳球菌表达载体L2-Ps-P30-T,并在乳酸乳球菌中表达P30蛋白。方法用BamHⅠ/XhoⅠ将P30从质粒pSK-P30中切出并克隆至质粒pSK-PsT,构建pSK-Ps-P30-T质粒;用PvuⅡ将表达元件-Ps-P30-T-从pSK-Ps-P30-T质粒中切出,以相同的酶切位点导入大肠埃希菌与乳酸乳球菌穿梭质粒pTRKL2,构建L2-Ps-P30-T质粒;通过电转化将L2-Ps-P30-T导入乳酸乳球菌中,以Western blot鉴定P30蛋白的表达。结果酶切及PCR鉴定质粒L2-Ps-P30-T构建正确,并在乳酸乳球菌中表达能被弓形虫病患者血清识别的分子质量单位为30ku的蛋白。结论重组载体L2-Ps-P30-T构建正确,电转化入乳酸乳球菌后能表达具有反应原性的P30蛋白。

Expression of P30 of the Toxoplasma gondii Kunshan strain in Lactococcus lactis

Objective To construct the Lactococcus lactis expression plasmid L2-Ps-P30-T that contains the P30 gene of the Toxoplasma gondii Kunshan strain and express the P30 gene in Lactococcus lactis LM 2345.Methods The P30 gene was obtained from the pSK-P30 plasmid using BamHI and XhoI restriction enzymes cleavage.The P30 gene was then cloned into psk-PsT,resulting in construction of psk-Ps-P30-T.The expression element-Ps-P30-T was obtained from the psk-Ps-P30-T vector using PvuⅡrestriction enzyme cleavage and subcloned into the same clone site of the shuttle plasmid L2-PTRK,resulting in construction of L2-Ps-P30-T.The plasmid L2-Ps-P30-T was then transformed into L.lactis LM 2345 by electroporation.The recombinant L.Lactis was cultured in GM17 medium overnight and expression of P30 in L.Lactis was then detected by SDS-PAGE and Western blot according to standard protocols.Result Restriction enzyme cleavage,PCR,and DNA sequencing confirmed that the L2-Ps-P30-T vector was correctly structured.A 30 ku protein was expressed in L.Lactis and reacted with the antisera from a patient with acute toxoplasmosis.Conclusion L2-Ps-P30-T was correctly constructed.P30 was expressed in L.Lactis and reacted with antisera from a patient with acute toxoplasmosis.