重组刚地弓形虫GRA10蛋白的表达纯化与鉴定

Cloning and Expression of the GRA10 Truncated Gene of Toxoplasma gondii in Eschericha coli

为构建表达刚地弓形虫(Toxoplasma gondii)致密颗粒蛋白10(dense granule protein 10,GRA10)N端(AA 4-478)的重组质粒并在大肠杆菌中表达纯化该重组蛋白,以用于后续的诊断检测试剂盒设计。根据GRA10的基因序列设计并合成特异性引物,通过PCR方法从弓形虫ME49虫株cDNA中扩增GRA10(AA 4-487)的编码片段,利用同源重组的方法将其连接到pE-SUMO载体上。经测序鉴定后,将重组质粒转化大肠埃希氏菌BL21(DE3)后并用IPTG诱导SUMO-GRA10的表达,产物通过SDS-PAGE分析并用亲和层析方法纯化,然后采用Western blot分析其免疫反应性。结果表明,已成功构建了表达弓形虫GRA10蛋白的重组质粒并利用原核表达系统表达纯化了具有良好免疫反应性的蛋白,为弓形虫病诊断奠定了基础。

In order to construct a recombinant plasmid expressing the N terminal part of Toxoplasma granule protein 10(GRA10)and purify it from Escherichia coli,the target fragment of GRA10(AA 4-487)was amplified from the complementary DNA of the ME49 strain of Toxoplasma gondii and cloned into the pE-SUMO vector by homologous recombination.The resulting pE-SUMOGRA10(AA 4-487)plasmid was then sequenced and transformed into E.coli BL21(DE3)competent cells.The recombinant protein expression was induced with IPTG and subsequently the product was purified by affinity chromatography and then analyzed by using SDS-PAGE.In order to evaluate the immuno-reactivity of the recombinant protein,the product was analyzed using Western blotting.The results showed that the recombinant plasmid pE-SUMO-GRA10(AA 4-487)was successfully constructed.SDS-PAGE results indicated that the SUMO-GRA10(AA 4-487)fusion protein was efficiently expressed with a molecular weight of about 64 ku.The Western blotting indicated that GRA10(AA4-487)fusion protein was recognized by sera from mice infected with Toxoplasma gondii.It indicated that the GRA10(AA 4-487)gene of T.gondii was successfully cloned and expressed in BL21(DE3)cells.The GRA10(AA 4-487)recombinant protein had decent immuno-reactivity,which might be useful for the design of diagnostic kits to detect T.gondii infections.