期刊论文

Cloning and prokaryotic expression of Cryptosporidium mouse genotype oocyst wall protein CP41 gene

中文

中国预防兽医学报

1008-0589

2010

32

8

622-626

10.3969/j.issn.1008-0589.2010.08.11

国家高技术研究发展计划（2006AA10A207） 国家“十一五”科技支撑计划（2007BAD40B05） 上海市科技兴农重点攻关项目[沪农科攻字（2005）第3-4号]

本校为其他机构

为克隆和表达编码隐孢子虫鼠基因型卵囊壁蛋白CP41基因,以隐孢子虫鼠基因型卵囊总RNA作为模板,RT-PCR扩增CP41基因,克隆到pMD18-T载体并进行序列测定,构建重组质粒pGEX-5x-3-CP41,转化BL21（DE3）感受态细胞进行诱导,表达产物SDS-PAGE检测目的蛋白,westernblot分析该重组蛋白的免疫活性。结果显示,克隆的目的基因核苷酸序列与GenBank登录中的序列比较,同源性为90.5%,氨基酸序列同源性为87.57%。重组质粒转化菌在IPTG诱导下以包涵体形式高效表达,表达的融合蛋白大小约为46ku,westernblot分析显示,纯化复性后的重组蛋白可被辣根过氧化物酶标记的抗谷胱甘肽巯基转移酶（GST）单克隆抗体、隐孢子虫兔...

The oocyst wall protein CP41 gene of Cryptosporidium mouse genotype was amplified from total RNA by RT-PCR, sequenced and compared with that from C. parvum in GenBank. The sequences shared 90.5 % nucleotide and 87.57 % amino acid identities with the published sequences. The gene fragment was subcloned into pGEX-5x-3 vector and transformed into E.coli BL21 （DE3）. Recombinant protein was expressed after IPTG induction and identified by SDS-PAGE and western blot. The expressed proteins were used to immunize rabbit, specific antibodies were detec...