弓形虫表面抗原SAG4基因的克隆、表达和鉴定  

Cloning, Expression and Identification of Surface Antigen SAG4 of Toxoplasma gondii

作　　者：杨雯[1] 万孝玲[2] 田春林[1] 王卫群[1] 刘晓泉[1] 

机构地区：[1]广西医科大学寄生虫学教研室,南宁530021 [2]广西壮族自治区疾病预防控制中心,南宁530028

出　　处：《中国寄生虫学与寄生虫病杂志》2009年第4期340-343,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：广西科学研究与技术开发计划项目(No.0443004-36)~~

摘　　要：目的对弓形虫表面抗原SAG4基因进行克隆、表达和免疫反应性分析。方法根据弓形虫RH株SAG4基因序列(GenBank登录号为AF340224.1)设计引物,体外扩增目的基因,并将其克隆至pMD19-T载体,经PCR和双酶切鉴定并测序,亚克隆至pET28a(+)质粒,转化大肠埃希菌BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析SAG4重组蛋白的表达情况,蛋白质印迹(Westernblot-ting)分析该蛋白与弓形虫慢性感染小鼠血清的免疫反应性。结果扩增获得的目的基因片段为537bp。生物信息学分析表明重组基因编码产物为弓形虫SAG4抗原,是一种外膜蛋白。重组菌经IPTG诱导后,以包涵体的形式稳定表达SAG4。Westernblotting分析结果表明,诱导表达的蛋白能被弓形虫慢性感染小鼠血清识别,其相对分子质量(Mr)约为18740。结论重组蛋白SAG4有一定的免疫反应性。Objective To clone and express surface antigen SAG4 gene of Toxoplasma gondii, and analyze its immunoreactivity. Methods Specific primers were designed based on the reported SAG4 gene of T. gondii RH strain （GenBank Accession No： AF340224.1）. Using genomic DNA from T. gondii as templates, SAG4 gene was amplified by PCR. The PCR product was cloned into pMD19-T vector and identified by digestion with restriction enzyme and PCR. Then the target fragment was subcloned into pET28a（＋） vector, transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The protein was identified by Western blotting. Results The target gene was amplified with the length of 537 bp. Sequence analysis showed that the predicted amino acid sequence was identical with that of SAG4 as a membrane protein in T. gondii. After induced by IPTG, the recombinant SAG4 protein existed in an inclusion body form. The recombinant SAG4 （Mr 18 740） was recognized by serum of infected mice. Conclusion SAG4 has been expressed and shows certain immuno-response activity.

关 键 词：弓形虫 SAG4 克隆表达 免疫反应性 

分 类 号：R382.5[医药卫生—医学寄生虫学]