弓形虫RH株微线体蛋白MIC3的原核表达及其免疫反应性

目的 克隆表达弓形虫RH株MIC3基因,并对重组蛋白进行免疫反应性分析.方法 应用PCR技术扩增弓形虫RH株MIC3基因,将目的基因片段分别克隆至pET28a(+)和pET32a(+)两种表达载体中构建重组质粒,重组质粒转化至大肠杆菌体BL21(DE3)中,经IPTG诱导表达后,表达产物进行SDS-PAGE电泳分析和Western-blot鉴定.结果 成功从弓形虫RH株基因组DNA中克隆出大小约1080bp MIC3基因片段；重组质粒pET28a-MIC3和pET32a-MIC3经过酶切和PCR鉴定,及测序分析,表明重组质粒构建正确.两种重组质粒分别诱导表达后进行SDS-PAGE电泳分析,诱导表达产物分别在50 KD及70KD左右出现目的条带,Westem-blot分析显示重组蛋白对弓形虫慢性感染小鼠血清具有特异免疫反应性.结论 原核表达了弓形虫MIC3重组蛋白,所表达的重组蛋白具有免疫反应性,为下一步利用重组蛋白进行弓形虫病的诊断和疫苗研究奠定基础.

Prokarytic expression and antigenicity analysis of MIC3 gene of Toxoplasma gondii RH strain

Objective To clone and express the MIC3 gene of Toxop]asma gondii RH strain and to analyze the antigenecity of the recombinant protein. Methods The genomic DNA, extracted from tachyzoites of T. gondff RH strain was amplified by PCR with a pair of primers designed according to the encoding sequence of MIC3 gene. The PCR product about 1080 bp was cloned into prokaryotic expression vector pET-28a（＋） or pET-32a（＋）. The recombinant vector pET28a-MIC3 or pET32a-MIC3 was transformed into E. coli BL21（DE3） and induced with IPTG for expression. The expression product then was analyzed by means of SDS-PAGE and Western-blot. Results Recombinant pET28a-MIC3 and pET2a-MIC3 were constructed successfully. SDS-PAGE and Western blot showed that the expression product was a non-fusion protein about 50 KD with pET28a-MIC3 and a fusion protein about 70 KD with pET32a-MIC3. The antigenicity of MIC3 was detected by Western-blot with primary antibody of prepared mice antiserum against T. gondii. Conclusiorl pET28a-MIC3 and pET32a-MIC3 strongly reacted with the mice antiserum against T. gondii, the present study might provide foundation for further study of diagnosis of toxoplasmosis and preparation of vaccine with MIC3.