中文摘要：

目的筛选Vero细胞无血清培养制备弓形虫排泄-分泌抗原（ESA）的适宜条件。方法采用拉丁方析因设计。第一部分：在血清浓度为0.5%、1.0%、2.0%、4.0%的培养基中,共培养Vero细胞与弓形虫速殖子,分别于培养3、6、12、24h时,改为无血清培养基,继续培养至第7天,收集培养上清液,制备ESA,并检测蛋白含量。第二部分：在含1.0%血清培养基中共培养Vero细胞与弓形虫速殖子,分别于培养12、24、48、72h时,改为无血清培养基,继续培养至第7、9、11、13天,收集培养上清液,制备ESA,并检测蛋白含量。结果Vero细胞与弓形虫速殖子在1.0%血清浓度培养基中培养12或24h时,无血清培养基继续培养7d,制备的ESA蛋白含量显著高于其他血清浓度（0.5%、2.0%、4.0%）及含血清培养时间（3、6h）。Vero细胞与弓形虫速殖子在含1.0%血清培养基中共培养12、24h,无血清培养基继续培养至第13天,制备的ESA蛋白含量显著高于其他培养时间（48、72h）和无血清培养基继续培养时间（7、9、11d）。结论培养基的血清浓度以及含血清和无血清培养时间是影响体外制备ESA的重要因素。Vero细胞与弓形虫速殖子在1.0%血清浓度培养基中培养12h,无血清培养基继续培养13d,为制备ESA的适宜条件。

英文摘要：

Objective To optimize the condition for preparation of Toxoplasma gondii excreted-secreted antigen （ESA） by serum-free culture in Vero cells. Methods Latin square factorial design was adopted in this study. In part 1, T. gondii tachyzoites were co-cultured with Vero cells in the media containing 0. 5%, 1. 0%, 2. 0% and 4. 0% fetal bovine serum （FBS）, and the media containing each concentration of FBS were changed into serum-free ones 3, 6, 12 and 24 h later respectively. The culture supernatant were collected on day 7 after co-culture to prepare ESA and determined for protein content. However, in part 2, T. gondii tachyzoites were co-cultured with Vero cells in the media containing 1. 0% FBS, and the media were changed into serum-free ones 12, 24, 48 and 72 h later respectively. The culture supernatant was collected on days 7, 9, 11 and 13 after co-culture to prepare ESA and determined for protein content. Results The protein contents in ESA prepared with T. gondii tachyzoite co-cultured with Vero cells in the media containing 1. 0% FBS for 12 and 24 h and further co-cultured in serum-free medium until day 7 were significantly higher than those in the media containing FBS at other concentrations（0. 5%, 2. 0% and 4. 0%）for 3 and 6 h. However, the protein contents in ESA prepared with T. gondii tachyzoites co-cultured with Vero cells in the medium containing 1. 0% FBS for 12 and 24 h and further co-cultured in serum-free media until day 13 were significantly higher than those co-cultured for 48 and 72 h in the media containing 1. 0% FBS and further co-cultured until days 7, 9 and 11 in serum-free media. Conclusion The serum concentration in medium and the culture time in serum-containing and serum-free media were the important influencing factors on in vitro preparation of ESA. The condition for preparation was optimized as co-culture of T. gondii tachyzoite with Vero cells in the medium containing 1. 0% FBS for 12 h and further co-culture in serum-free medium until day 13.