摘要
目的克隆刚地弓形虫ROP18基因,进行原核表达及重组蛋白的免疫分析。方法提取弓形虫RH株基因组DNA,经PCR获得ROP18基因片段,经双酶切分别连入原核表达载体pET-28a(+)和pET-32a(+),构建重组表达载体pET28a-ROP18和pET32a-ROP18,并转化大肠杆菌BL21(DE3)。IPTG诱导表达后,收集菌体进行SDS-PAGE电泳及Western-blot检测。结果 PCR得到约1 665bp目的基因片段,单、双酶切及PCR鉴定结果显示成功构建重组表达载体pET28a-ROP18和pET32a-ROP18;经IPTG诱导后,ROP18基因在大肠杆菌中高效表达;SDS-PAGE电泳及Western-blot分析显示,pET28a-ROP18在相对分子质量约60kD的位置出现目的蛋白条带,pET32a-ROP18在相对分子质量约83kD的位置出现目的蛋白条带,均与理论值相符;Western-blot结果显示重组蛋白与弓形虫慢性感染小鼠血清具有特异免疫反应性。结论成功克隆和表达了ROP18基因,所表达的重组蛋白具有免疫效应,为其功能研究奠定基础。
To clone and express the rhoptry protein 18（ROP18） gene of R H strain of Toxoplasrna gondii, the genomic DNA, extracted from tachyzoites of RH strain of T. gondii, was amplified by PCR with a pair of specific primers, which was designed according to the encoding sequence of ROP18 gene. The PCR product about 1 665 bp was cloned into prokaryotic ex pression vector pET 28a （-） or pET-32a （＋） with restriction enzymes BamH i and Hind [11. The recombinant vector pET28a ROP18 or pET32a-ROP18 was then transformed into E. coli BI.21（DE3） and induced with IPTG for expression. SDS- PAGE and western blotting showed that the expression product was a non-fusion protein about 60 kD with pET28a-ROP18 and a fusion protein about 83 kD with pET32a-ROP18. The antigenicity of ROP18 was detected by western blot with primary anti- body of prepared mice antiserum against T. gondii. With the immunological effect of this expressed recombinant protein, the present study might provide the foundation for the further study of ROP18.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第3期211-215,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金(No.30972578
31030066)
教育部博士点项目(No.20094433120013
20094433120016)
广东省自然科学基金(No.9151051501000075)
广东省"大学生创新实验计划"项目(No.1212110025
1212110027)
南方医科大学大学生课外科研项目(Nos.2010kw082
GWXS20110101
GWXS20110205)~~
关键词
刚地弓形虫
棒状体蛋白18
基因克隆
原核表达
免疫分析
coplasma gondii
rhoptry protein 18
gene clone
prokaryotic expression
antigenicity analysis
