摘要
试验比较了玻璃珠层析法、ＤＥＡＥ纤维素层析法、Ｇ３漏斗过滤法３种提纯柔嫩艾美球虫（Ｅｉｍｅｒｉａｔｅｎｅｌｌａ）子孢子的方法。柱高８ｃｍ、直径２００μｍ的玻璃珠柱用ｐＨ８．０的Ｒｉｎｇｅｒ′ｓ缓冲液作洗脱液提纯子孢子，回收率为６４．１％，子孢子洗脱高峰期集中，且易于无菌操作；柱高５ｃｍ的ＤＥＡＥ纤维素柱用ｐＨ８．０的甘氨酸缓冲液作洗脱液，提纯子孢子回收率为６３．２％，但严格无菌操作困难；Ｇ３漏斗法易无菌操作，方法简便，但提纯效果不理想，回收率为２５％，有少量杂质。结果表明，玻璃珠柱层析法可作为细胞培养用提纯子孢子的首选方法。提纯的子孢子接种于体外培养的鸡原代肾细胞，在ＭＥＭ细胞形成培养液（含１０％的犊牛血清）和ＭＥＭ细胞维持培养液（含５％的犊牛血清和５％的水解乳蛋白）体外细胞培养系统中，能够发育到卵囊阶段；Ｅ．ｔｅｎｅｌａ在体外细胞培养中的发育与鸡体内发育的过程和时间基本相似，都经过子孢子→第一代裂殖体→第二代裂殖体→配子体→卵囊等阶段。试验也表明，水解乳蛋白作为营养补充物是Ｅ．ｔｅｎｅｌａ子孢子发育到卵囊的重要因素。
Three methods to purify Eimeria tenella sporozoites had been compared: column of 200 μm glass beads, column of DEAE celluose and G 3 funnel method. A 8 cm column of 200 μm glass beads with pH8.0 buffered Ringer′s solution as eluent was chosen as a optimum system for its higher purification efficiency and easier sterilized operation; Column of DEAE cellulose with pH 8.0 buffered glycine solution as an eluent also enable to purify E. tenella sporozoites, except for the difficulties in sterilization as compared with the former procedure. Sterilization was easy and the purification efficiency was low in the G 3 funnel method. Purified E. tenella sporoites were inoculated into partially confluent primary chicken kidney cells culture. The culture medium composed of 88% MEM medium and 10% fetal calf serum was used for the growth of cells. To maintain the infected cells after inoculation, an additional midium composed of 88% MEM medium, 5% fetal calf serum, 5% lactalbumin hydrolysate was used. The development from sporozoites to oocysts of E. tenella was observed daily by means of microscope. The results showed that the development of E. tenella in chicken kidney cells culture follows the pattern in vivo closely: First generation schizont, second generation schizont, gamont and oocyst.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1998年第2期151-155,共5页
Chinese Journal of Veterinary Science
关键词
柔嫩艾美球虫
提纯
鸡原代肾细胞
体外培养
Eimeria tenella
purification
primary chicken kidney cells
culture
