Structure and expression of antigen genes from Toxoplasma gondii

克隆了弓形虫抗原蛋白的编码基因，并将其作为诊断抗原。从弓形虫速殖子中提取总RNA，用兔网织红细胞裂解液进行体外翻译，以鉴定抗原蛋白。翻译产物与弓形虫感染的人血清进行免疫沉淀。SDS-PAGE放射自显影检测到分子量分别为27,000，31,000和35,000的3种主要抗原蛋白。其中主要的抗原性多肽分子量为27,000。构建了gtll表达载体中的cDNA文库。用感染的人血清免疫筛选编码27 kDa抗原多肽的cDNA克隆。从4×10~(-4)中分离到26个阳性克隆。从多克隆抗血清中筛选出针对各克隆的单特异性抗体，并与体外翻译产物反应，鉴定编码27 kDa抗原多肽的cDNA克隆。用十二烷基硫酸钠-PAGE放射自显影测定抗原蛋白的相对分子质量。结果表明，Tg18为编码2M kDa抗原性多肽的cDNA克隆。Southern印迹分析表明，编码27 kDa抗原的基因位于基因组DNA的约13kb的EcoRI片段上。Northern印迹分析表明，27 kDa抗原的mRNA大小为1400个核苷酸。免疫印迹检测到分子量分别为32,000和27,000的天然抗原。此外，在大肠杆菌中合成的GT11-半乳糖苷酶融合蛋白与其他寄生虫感染的血清没有交叉反应。Tg18基因全长739bp，含有174个氨基酸的开放阅读框。GenBank核酸数据库和NBRF数据库显示没有序列同源性。

The cDNA encoding antigenic protein of Toxoplasma gondii was cloned to utilize as a diagnostic antigen. Total RNA extracted from T. gondii tachyzoite was in vitro translated using rabbit reticulocyte lysate for the identification of antigenic protein. Translation products were immunoprecipitated with T. gondii-infected human serum. Three major antigenic proteins with molecular weights of 27,000, 31,000 and 35,000 were detected by the SDS-PAGE autoradiography. The predominant antigenic polyprptide among them has a molecular weight of 27,000. cDNA library was constructed in gtll expression vector. cDNA clones encoding 27 kDa antigenic polypeptide were immunoscreened using infected human serum. Twenty six positive clones were isolated from 4x10^4. Monospecific antibody to respective clone was epitope-selected from polyclonal antiserum and reacted with in vitro translation products to identify cDNA clone encoding 27 kDa-antigenic polypeptide. The molecular weight of antigenic protein was determined by the SDS-PAGE autoradiography. As a result, Tg18 was identified as a cDNA clone encoding 2m kDa-antigenic polypeptide. Southern blot assay demonstrated that the gene encoding 27 kDa antigen was located in approximately 13 kb EcoRI fragment of genomic DNA. Northern blot assay revealed that the mRNA for 27 kDa antigen was 1400 nucleotides in size. Native antigens with molecular weights of 32,000 and 27,000 were detected by the Western blot assay. Furthermore, gtll -galactosidase fusion protein synthesized in E. coli had no cross-reactivity with the other parasite-infected serum. Tg18 cDNA is 739 bp in length containing open reading frame encoding 174 amino acids. Genbank nucleic acid and NBRF data banks reveal no sequence homology.