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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。
Objective To construct Lactococcus lactis expression vector L2-Ps-P30-T expressing P30 gene of Kuwania isolates and express P30 protein in Lactococcus lactis. Methods P30 was cut out from plasmid pSK-P30 by BamHⅠ / XhoⅠ and cloned into plasmid pSK-PsT to construct pSK-Ps-P30-T plasmid. The pSK-Ps-P30- P30-T plasmids were introduced into Escherichia coli and Lactococcus lactis shuttle plasmid pTRKL2 to construct L2-Ps-P30-T plasmids; the L2-Ps-P30-T was introduced by electrotransformation Lactococcus lactis, P30 protein was identified by Western blot. Results The restriction endonuclease digestion and PCR identification of the plasmid L2-Ps-P30-T were correctly constructed and expressed in Lactococcus lactis as a 30ku molecular weight unit recognized by the serum of toxoplasmosis patients. Conclusion The recombinant vector L2-Ps-P30-T is constructed correctly and electropositively transformed into Lactococcus lactis to express the reactive P30 protein.