弓形虫棒状体蛋白18的原核表达及免疫分析

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1 665 bp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60 kD的位置出现目的蛋白条带,pET32a-ROP18在相对分子质量约83 kD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。

Prokaryotic expression and antigenicity analysis on rhoptryprotein 18 of Toxoplasma gondii

To clone and express the rhoptry protein 18(ROP18) gene of RH strain of Toxoplasma 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 expression vector pET-28a(+) or pET-32a(+) with restriction enzymes BamHⅠ and HindⅢ. The recombinant vector pET28a-ROP18 or pET32a-ROP18 was then transformed into E. coli BL21(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 antibody 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.