弓形虫P30基因非融合蛋白表达的研究

EXPRESSION OF P30 GENE OF TOXOPLASMA GONDII AS NON-FUSION PROTEIN

目的 :构建弓形虫主要表面抗原P30基因的原核表达载体并在E .coli中进行表达。方法 :采用定向克隆的方法 ,将PCR扩增得到的P30片段 ,插入原核表达质粒 pBV2 2 0上 ,转化大肠杆菌DH5α感受态细胞 ,于氨苄LB培养平板上筛选阳性克隆 ,经过酶切及PCR扩增鉴定重组子。将含阳性重组子 pBV2 2 0 P30的工程菌经温度诱导表达 ,SDS PAGE电泳及免疫印迹分析。结果 :成功构建了 pBV2 2 0 P30重组质粒 ;SDS PAGE电泳及Western blot显示表达P30非融合蛋白的分子量为 2 9kD ,且能被弓形虫高免鼠血清识别。结论 :从弓形虫基因组DNA中获取P30基因 ,并成功构建了 pBV2 2 0 P30重组质粒 ,诱导表达了P30非融合蛋白。对弓形虫病基因疫苗研制奠定了基础。

Objective:To construct Prokaryotic expression vector containing a gene encoding P30 gene of T.gondii and express its protein in E.coli. Methods:By direct cloning,P30 gene which was amplified by PCR was inserted into prokaryotic expression vector pBV220 and then was transformed into E.coli DH5α.The positive clones were primarily confirmed by anti ampicillin selecting,restriction enzymes digestion and PCR.The recombinant clones were induced by temperature.The expression of non fusion protein was analyzed by means of SDS PAGE and Western blot.Results:The recombinant plasmid pBV220 P30 was constructed successfully.The results of SDS PAGE and Western blot revealed that the molecular weight of non fusion P30 protein was 29kD and can be specifically recognized by rat antiserum of Toxoplasma. Conclusion:pBV220 P30 is constructed and P30 non fusion protein expressed successfully.