蛋白质沉淀剂对棉铃虫谷胱甘肽S_转移酶的部分纯化  

Partial purification of glutathione S-transferases by protein precipitators in the cotton bollworm, Helicoverpa armigera (Hübner)

作　　者：汤方[1] 张常忠[1] 梁沛[1] 史雪岩[1] 高希武[1] 

机构地区：[1]中国农业大学农学及生物技术学院昆虫学系,北京100094

出　　处：《昆虫学报》2005年第2期172-178,共7页Acta Entomologica Sinica

基　　金：国家重点基础研究发展规划"973"项目 (G2000016207);国家自然科学基金项目 (30471153; 30170621)

摘　　要：通过用聚乙烯亚胺(PEI)、硫酸铵、聚乙二醇(PEG)沉淀技术和GSH-Sepharose 4B亲和柱对棉铃虫Helicoverpa armigera(Hübner)幼虫中谷胱甘肽S-转移酶进行了部分纯化研究.结果表明PEG10000和PEG20000的纯化效果优于硫酸铵的沉淀效果.通过PEI沉淀去核酸后,再用硫酸铵沉淀,中肠和脂肪体GST活性分布在70%～75%和60%～65%沉淀段,比活力分别为1081.49和596.41 nmol/(min·mg),纯化倍数分别为2.53和2.2.在6种PEG中,PEG10000和PEG20000的纯化效果较好.在中肠和脂肪体中PEG10000沉淀的GST活性峰分别在40%～45%和30%～40%,GST比活力分别为795.11和1 080.18 nmol/(min·mg),纯化倍数分别是2.4和3.97.PEG20000沉淀中肠和脂肪体GST的活性峰分别在25%～40%和25%～45%,比活力分别是767.57和945.96 nmol/(min·mg),纯化倍数分别是2.81和3.05.用GSH-Sepharose 4B纯化中肠GST,GST比活力达到5 888.44nmol/(min·mg),纯化倍数达到107.38.The partial purification of glutathione S-transferases (GST) in larvae of the cotton bollworm, Helicoverpa armigera (Hübner) was studied using ammonium sulfate fractionation after polyethylene imine (PEI) fractionation, polyethyleneglycol (PEG) fractionation and GSH-Sepharose 4B. The results showed that efficacy of purification by PEG10000 and PEG20000 was better than that of ammonium sulfate fractionation after PEI fractionation. After wiping off nucleic acid using PEI fractionation, the peaks of GST activities were in 70%-75% and 60%-65% of ammonium sulfate fractionation for midgut and fat body, respectively; the specific activities were 1081.49 and 596.41 nmol/(min·mg), and purification factors were 2.53-fold and 2.2-fold, respectively. The efficacy of purification by PEG10000 and PEG20000 was the best in six kinds of PEG tested. The activity peaks of GST were in 40%-45% and 30%-40% of PEG10000 fractionation for midgut and fat body, respectively; the specific activities were 795.11 and 1 080.18 nmol/(min·mg), and purification factors were 2.4-fold and 3.97-fold, respectively; the activities of GST were the highest in 25%-40% and 25%-45% of PEG20000 fractionation for midgut and fat body, respectively. The specific activities of GST were 767.57 and 945.96 nmol/(min·mg) respectively, and the purification factors were 2.81-fold and 3.05-fold. The specific activity of GST from midgut reached \{5 888.44\} (nmol/(min·mg)) by GSH-Sepharose 4B column chromatography and the purification factor increased to 107.38-fold.

关 键 词：棉铃虫 谷胱甘肽S-转移酶 硫酸铵沉淀 聚乙烯亚胺沉淀 聚乙二醇沉淀 GSH—Sephamse 4B亲和柱 

分 类 号：Q965.9[生物学—昆虫学]