Lalek Hüsniye, Gürbüz Esra, Karaca Serkan, Yazar Süleyman, Kuk Salih
Erciyes Üniversitesi Tıp Fakültesi, Parazitoloji Anabilim Dalı, Kayseri, Türkiye.
Turkiye Parazitol Derg. 2015 Dec;39(4):255-9. doi: 10.5152/tpd.2015.3760.
Toxoplasma gondii, which is observed in our country and worldwide, can cause mortality and is an important public health problem because of engaging babies from pregnant women. An effective vaccine against toxoplasmosis has not yet been developed. SAG1 protein is released from the bradyzoites and tachyzoites stages of T. gondii and is important at the pathogenesis of the disease. This study aimed to clone a promising DNA vaccine candidate, SAG1 gene, of T. gondii.
T. gondii genomic DNA was isolated from tachyzoites of T. gondii. SAG1 gene was amplified with specific primers and then cloned into the pJET1.2 vector. Recombinant plasmids were transformed into Escherichia coli cells. The presence of recombinant plasmids was determined by polymerase chain reaction (PCR) screening. Following the purification of the recombinant plasmid from positive colonies, cloning was confirmed by PCR, restriction enzyme assays, and DNA sequence analysis.
After PCR with SAG1 gene-specific primers, 1010-bp PCR products were obtained. Recombinant plasmid, which was transformed into competent E. coli cells, was verified by PCR screening. Moreover, PCR verified that the recombinant plasmids contained the SAG1 gene. The DNA sequence was analyzed, and the DNA sequence was obtained.
One of the promising DNA vaccine candidates against toxoplasmosis, SAG1 gene, has been cloned.
弓形虫在我国及全球均有发现，可导致死亡，且因感染孕妇腹中胎儿而成为重要的公共卫生问题。目前尚未研发出有效的抗弓形虫病疫苗。SAG1蛋白由弓形虫的缓殖子和速殖子阶段释放，在该疾病的发病机制中起重要作用。本研究旨在克隆一种有前景的弓形虫DNA疫苗候选基因SAG1基因。
从弓形虫速殖子中分离出弓形虫基因组DNA。用特异性引物扩增SAG1基因，然后将其克隆到pJET1.2载体中。将重组质粒转化到大肠杆菌细胞中。通过聚合酶链反应（PCR）筛选确定重组质粒的存在。从阳性菌落中纯化重组质粒后，通过PCR、限制性内切酶分析和DNA序列分析确认克隆。
用SAG1基因特异性引物进行PCR后，获得了1010 bp的PCR产物。通过PCR筛选验证了转化到感受态大肠杆菌细胞中的重组质粒。此外，PCR证实重组质粒含有SAG1基因。对DNA序列进行了分析并获得了DNA序列。
已克隆出一种有前景的抗弓形虫病DNA疫苗候选基因SAG1基因。
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