重组弓形虫致密颗粒蛋白7的表达及免疫活性分析

Recombinant Expression and Immunological Activites of Dense Ggranule Antigen 7 from Toxoplasma Gondii

目的:重组表达弓形虫致密颗粒蛋白7(GRA7),分析其免疫活性。方法:采用聚合酶链反应(PCR)从刚地弓形虫基因组DNA中扩增GRA7基因,经克隆和测序分析后,亚克隆至表达载体pET-23a(+),转化大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达GRA7,用His-bindTM亲和层析柱纯化,免疫印迹和小鼠免疫分析其抗原性。结果:PCR扩增出大小约660bp的GRA7基因片段,并被亚克隆到原核表达载体pET-23a(+),构建了原核重组表达质粒pET-GRA7;表达纯化获得分子量约29,000的重组GRA7;重组GRA7能被弓形虫感染兔血清所识别,并诱导小鼠产生抗体滴度达1:12800。结论:获得了具有良好免疫学活性的重组抗原GRA7。

Objective To express recombinant Toxoplasma gondii(T.gondii) Dense granule antigen 7(GRA7)in E.coli and analyze its immunological activites.Methods DNA fragment encoding the GRA7 antigen of T.gondii was obtained from the total DNA of T.gondii RH strains by polymerase chain reaction(PCR).The gene was cloned and the DNA sequence was determined subsequently.The gene with correct sequence was subcloned into a expression vector pET-23a(+)and was transformed into Escherichia coli BL21 for expression.The expressed fusion protein induced by IPTG was purified by His-bindTM affinity chromatography,and the purified fusion protein was analyzed by Western-blot and immunization in mice for its antigenicity.Results A 660bp gene fragment encoding GRA7 was amplified by PCR from T.gondii genomic DNA;The insert of GRA7 gene in positive clone was subcloned into pET-23a(+)properly to construct a recombinant expression plasmid pET-GRA7.The fusion protein with molecular weight about 29 000 was purified by His-bindTM affinity chromatography from lysate of the induced E.coli BL21 containing recombinant expression plasmid pET-GRA7.Recombinant protein GRA7 was recognized by rabbit sera infected with T.gondii tachyzoites and induced antibodies with a titre of 1:12 800.Conclusion Recombinant protein GRA7 with good immunological activites was obtained through cloning and expression.