弓形虫P30基因MBP融合蛋白的表达  

EXPRESSION OF P30 GENE OF TOXOPLASMA GONDII AS MBP FUSION PROTEIN

作　　者：丛华[1] 古钦民[1] 黄勇[1] 李瑛[1] 禹卉[1] 

机构地区：[1]山东医科大学寄生虫教研室,济南250012

出　　处：《中国人兽共患病杂志》2000年第1期29-31,共3页Chinese Journal of Zoonoses

基　　金：山东省卫生厅科研资助

摘　　要：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 中表达。方法　根据已发表的弓形虫Ｐ３０基因序列，自行设计合成一对引物并在引物５’端分别引入限制性内切酶ＥｃｏＲＩ、ＳａｌＩ酶切位点，用ＰＣＲ技术从弓形虫ＲＨ 株基因组ＤＮＡ中扩增Ｐ３０ 基因片段，插入ｐＭＡＬＰ２ 质粒转化大肠杆菌ＤＨ５ａα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。将阳性重组子经ＩＰＴＧ诱导表达，ＳＤＳ－ ＰＡＧＥ及免疫印迹分析。结果　从弓形虫ＲＨ 株ＤＮＡ中扩增出９７６ｂｐ 的Ｐ３０ 基因，构建成功ｐＭＡＬＰ２ － Ｐ３０ 重组质粒；ＳＤＳ－ ＰＡＧＥ电泳及Ｗｅｓｔｅｒｎ － ｂｌｏｔ 显示ＭＢＰ／Ｐ３０ 融合蛋白条带的分子量约为７７－５ｋＤ，减去ＭＢＰ的分子量４３ｋＤ，得出Ｐ３０ 蛋白分子量为３４－５ｋＤ，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ｐＭＡＬＰ２ － Ｐ３０ 重组质粒，诱导表达了Ｐ３０ 的融合蛋白。为进一步Ｐ３０ 蛋白的分离纯化及其对动物的免疫原性研究作好准备。Aim Construction of Prokaryotic expression vector contained a gene encoding P30 gene of T gondii and expression in E coli Methods According to the published P30 sequence,a pair of primer was designed and at the 5’ end E coRI and SalI enzyme sites were added respectively Using PCR technique,a fragment of P30 gene was obstained by amplification of genomic DNA of tachyzoites:By direct cloning,P30 gene was inserted into pMALP 2 plasmid,then transformed into E coli DH5 a The recombinant clones were induced by IPTG concentration A high level expression of MBP fusion protein was analyzed by means of SDS-PAGE and Western-blot Results The gene encoding P30 was successfully amplified from the genomic DNA of RH strain of Toxoplasma gondii by PCR,and the recombinant plasmid pMALP 2-P30was constructed successfully The results of SDS-PAGE and Western-blot revealed that the molecular weight of recombinant protein MBP/P30 was approximately 77 5KD,and P30 is 34 5KD out of MBP 43KD and can be specifically recognized by rat antiserum of Toxoplasma Conclusion P30 gene was obtained from genomic DNA of Toxoplasma and pMALP 2-P30 recombinant was successfully constructed The expression of P30 fusion protein will be used to purify P30 protein and study it's immunity in to animal

关 键 词：弓形虫 P30基因 融合蛋白 基因表达 MBP 

分 类 号：R531.802[医药卫生—内科学]