弓形虫棒状体蛋白18的原核表达及免疫分析

Prokaryotic expression and antigenicity analysis on rhoptry protein 18 of Toxoplasma gondii

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.