摘要
目的采用PCR方法从刚地弓形虫RH株基因组DNA中扩增弓形虫ROP18基因,构建原核表达载体pET-ROP18,并检测融合蛋白ROP18的免疫反应原性。方法采用PCR技术扩增弓形虫ROP18基因,将其插入原核表达载体pET-28a中,构建原核表达质粒pET-ROP18,PCR和双酶切鉴定正确的重组质粒pET-ROP18转入大肠埃希菌BL21中,在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下表达融合蛋白,诱导产物裂解后进行SDS-PAGE电泳,观察重组蛋白的诱导表达情况,Western blot分析重组蛋白的反应原性。结果以弓形虫基因组DNA为模板,PCR扩增出约1.6kb的ROP18基因片段。将其定向插入原核表达载体pET-28a,构建原核表达质粒pET-ROP18,经PCR和双酶切鉴定后测序,显示重组质粒包含ROP18蛋白基因读码框内的完整序列,能完整表达ROP18抗原蛋白。重组质粒转化菌经IPTG诱导后表达分子质量单位约63ku的融合蛋白,以37℃1mmol/L IPTG诱导4h表达量最大。Western blot分析重组蛋白能被鼠抗弓形虫血清识别。结论成功构建了原核表达载pET-ROP18,重组蛋白ROP18具有免疫反应原性。
Objective To use PCR to amplify ROP18 from the genomic DNA of the RH strain of Toxoplasma gondii and construct the prokaryotic expression vector pET-ROP18 in order to test recombinant protein immunoreactivity using Western blotting. Methods A ROP18 gene fragment was amplified with PCR and subcloned into a pET-28a vector. It was then digested with BamH I/HindI]] and subjected to PCR to create the recombinant plasmid pET-ROP18. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG to express the target protein. The relative molecular mass, solubility, and antigenicity of the expressed products were analyzed using SDS-PAGE and Western blot- ting. Results The PCR product of ROP18 was about 1. 6 kb in length, and the prokaryotic expression vector pET- ROP18 was successfully constructed. Sequence analysis indicated that the full coding length of ROP18 was correct. SDS- PAGE results revealed expression of a 63-ku fusion protein, and a high level of expression was noted at lmmol/L IPTG after incubation at 37℃for 4 h. Western blot analysis indicated that the recombinant protein reacted with serum with antibodies against T. gondii. Conclusion The prokaryotic expression vector pET-ROP18 was successfully constructed and the recombinant protein had good immunoreactivity.
出处
《中国病原生物学杂志》
CSCD
北大核心
2013年第6期527-529,534,共4页
Journal of Pathogen Biology
基金
海南省自然科学基金项目(No.809018)
关键词
弓形虫
ROP18
原核表达
Toxoplasma gondii
rhoptry proteinl8 （ROP18）
prokaryotic expression
