反相高效液相色谱法分离纯化野蚕黑卵蜂寄主识别利它素  

Separation and Purification of Host Recognition Kairomone of Telenomus Theophilae by Reversed Phase High Performance Liquid Chromatography

作　　者：孙守慧[1] 楼兵干[2] 杨彤彤[1] 高其康[3] 

机构地区：[1]沈阳农业大学,沈阳110160 [2]浙江大学农业与生物技术学院生物技术研究所,杭州310027 [3]浙江大学分析测试中心,杭州310027

出　　处：《分析化学》2007年第7期1004-1007,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金资助项目(No30270896)

摘　　要：采用反相高效液相色谱和酶联免疫分析的方法进行了野蚕黑卵蜂寄主识别利它素分离和免疫活性测定。RP-HPLC的分离条件是：C8反相色谱柱，在柱温30℃、流量1mL／min的条件下，用0～2min，100％A（5％乙腈＋0．05％三氟乙酸）；2～6min，100％A→100％B（95％乙腈＋0．05％三氟乙酸）线性洗脱；6—8min，100％B进行洗脱，DAD检测波长为280nm。通过比较家蚕和野桑蚕色谱图发现，无论是粗提物还是上清液两者的峰形极其相似，有5个主要的馏分。通过粗提物和低温物理快速分离上清液的色谱图比较，确定了2号峰为低温物理快速法沉淀部分的馏分。收集家蚕的5种主要馏分，分别用利它素抗血清进行ELISA检测，证实2号峰是野蚕黑卵蜂寄主识别利它素，其免疫活性分别是上清液中其它馏分的100倍至1000倍。The host recognition kairomone of Telenomus theophilae was separated by RP-HPLC and the immune-activity was detected by ELISA. The isolated conductions with RP-HPLC were following： Cs protein reversed phase column （ 150 mm × 4. 6 mm i. d. , 5μm ） ; column temperature, 30℃ and flow rate 1 mL/min. Elution conditions ： 0 - 2 min, 100% A, 2 - 6 min 100% A to 100% B gradient elution, 6 - 8 min 100% B, and detection with DAD detector at 280 nm. The result showed that the chromatogram curves of extract and supernatant from B. mor/and T. mandarina were similar, there were five primary fractions to be separated, the second fraction was confirmed to be the same precipitation by using low-temperature rapid method. The five primary fractions, separated by RP-HPLC from B. mori, were collected and detected with kairomone antiserum, results indicated that the immune activity of the precipitation fraction was 100 - 1000 times than that of other fractions in supernatant.

关 键 词：野蚕黑卵蜂 寄主识别利它素 反相高效液相色谱 酶联免疫分析 

分 类 号：O657.72[理学—分析化学]