目的:获得尘螨变应原第6组分Der f 6原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE结合率。方法:酶切质粒p ET28a(+)-Der f 6获得目的基因Der f 6,将其与p ET32a(+)载体连接成质粒p ET32a(+)-Der f 6,转化BL21细菌后,用异丙基...目的:获得尘螨变应原第6组分Der f 6原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE结合率。方法:酶切质粒p ET28a(+)-Der f 6获得目的基因Der f 6,将其与p ET32a(+)载体连接成质粒p ET32a(+)-Der f 6,转化BL21细菌后,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹实验(Western blot)和蛋白质串联质谱(MALDI-TOF/TOF)鉴定纯化产物。以纯化获得的产物为包被抗原建立间接ELISA法检测尘螨过敏性哮喘患儿血清抗体反应情况。结果:成功构建了原核表达质粒p ET32a(+)-Der f 6,将该质粒转化E.coli BL21诱导表达,亲和层析纯化后,SDS-PAGE显示获得目的蛋白,Western blot验证其能够与载体的组氨酸标签结合,质谱鉴定其Der f 6结构一致。以此产物为包被抗原建立间接ELISA检测尘螨过敏性哮喘患儿血清,阳性率为41.3%(19/46)。结论:成功构建了原核表达质粒p ET32a(+)-Der f 6,亲和纯化获得的目的蛋白具有良好的反应原性。展开更多
Objective:To examine the acaricidal effects of the essential oil of Cymbopogon citratits leaf extract(lemongrass)and ethanolic Azadirachta indica leaf extract(neem)against house dust mites Dermatophagoides farinae(D.f...Objective:To examine the acaricidal effects of the essential oil of Cymbopogon citratits leaf extract(lemongrass)and ethanolic Azadirachta indica leaf extract(neem)against house dust mites Dermatophagoides farinae(D.farinae)and Dermalophagoides pteronyssinus(D.pteronyssinus).Methods:Twenty-five adults mites were placed onto treated filter paper that is soaked with plant extract and been tested at different concentrations(50.00%,25.00%,12.50%,6.25%and 3.13%)and exposure times(24hrs,48hrs,72hrs and 96 hrs).All treatments were replicated 7 times,and the experiment repeated once.The topical and contact activities of the two herbs were investigated.Results:Mortalities from lemongrass extract were higher than neem for both topical and contact activities.At 50%concentration,both 24 hrs topical and contact exposures to lemongrass resulted in more than 91%mortalities for both species of inites.At the same concentration and exposure time,neem resulted in topical mortalities of 40.3%and 15.7%against D.pteronyssinus and D.farinae respectively;contact mortalities were 8.0%and 8.9%against the 2 mites,respectively.There was no difference in topical mortalities of D.pteronyssinus from exposure to concentrations oflemongrass and neem up to 12.50%;lemongrass was more effective than neem at the higher concentrations.Conclusions:Generally,topical mortalities of D.farinae due to lemongrass are higher than that due to neem.Contact mortalities of lemongrass are always higher that neem against both species of mites.展开更多
目的获得粉尘螨变应原第3组分Der f 3原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE反应性。方法酶切质粒pET28a(+)-Der f 3获得目的基因Der f 3,将其与pET32a载体连接成质粒pET32a(+)-Der f3,转化BL21细菌,用异丙基硫代半乳糖...目的获得粉尘螨变应原第3组分Der f 3原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE反应性。方法酶切质粒pET28a(+)-Der f 3获得目的基因Der f 3,将其与pET32a载体连接成质粒pET32a(+)-Der f3,转化BL21细菌,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot和质谱鉴定纯化产物。以纯化产物为包被抗原,采用间接ELISA检测尘螨过敏性哮喘患儿血清抗体反应情况。结果成功构建了原核表达质粒pET32a(+)-Der f 3。将该质粒转化E.coli BL21诱导表达,亲和层析纯化后经SDS-PAGE鉴定获得目的蛋白,Western blot验证该蛋白的能够与载体的组氨酸标签结合,质谱鉴定其结构与天然Der f 3一致。以此产物为包被抗原采用间接ELISA检测尘螨过敏性哮喘患儿血清,阳性率为29.73%(11/37)。结论成功构建了原核表达质粒pET32a(+)-Der f 3,亲和纯化获得的目的蛋白具有良好的反应原性。展开更多
Dermatophagoides farinae and D. pteronyssinus are the prevalent house dust mites (HDM) in tropical countries and are associated with allergic diseases. This investigation developed a time- resolved immunofluorometric ...Dermatophagoides farinae and D. pteronyssinus are the prevalent house dust mites (HDM) in tropical countries and are associated with allergic diseases. This investigation developed a time- resolved immunofluorometric assay (TR-IFMA) for the first time to detect specific IgE antibody in patients with skin prick test positive to HDM but no detectable IgE by other means. Levels of IgE to natural and recombinant HDM allergens were measured by TR-IFMA in 50 HDM-allergic patients and 19 healthy participants compared to sandwich enzyme-linked immunosorbent assay (ELISA). A recombinant allergen, rDerf2, showed a 14 kDa band corresponding to broad range proteins of natural HDM.TR-IFMA showed sensitivity lower than 0.35 kUA/l. TR-IFMA employing three HDM antigens showed good correlations with sandwich ELISA at R2 0.93-0.96. TR-IFMA detected HDM IgE in 62, 62, 25 percent of allergic patient serum sample compared to 28, 32, and 22 percent detected by ELISA result using three HDM allergen. TR-IFMA also detected 26.3, 31.6, and 5.3 percent positive samples from 19 healthy participants while ELISA showed 0, 5.3, and 0 percents IgE positive samples. The use of rDerf2 as an HDM allergen for the assay was verified with no statistically different from other HDM allergens. TR-IFMA showed lower detection limit than ELISA and yielded higher sensitivity for serum of people with allergic symptoms with no detectable HDM IgE. It is anticipated that TR-IFMA for HDM-specific IgE detection will play an important role in future diagnosis of HDM allergy in clinical laboratories and for different research purposes.展开更多
文摘目的:获得尘螨变应原第6组分Der f 6原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE结合率。方法:酶切质粒p ET28a(+)-Der f 6获得目的基因Der f 6,将其与p ET32a(+)载体连接成质粒p ET32a(+)-Der f 6,转化BL21细菌后,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹实验(Western blot)和蛋白质串联质谱(MALDI-TOF/TOF)鉴定纯化产物。以纯化获得的产物为包被抗原建立间接ELISA法检测尘螨过敏性哮喘患儿血清抗体反应情况。结果:成功构建了原核表达质粒p ET32a(+)-Der f 6,将该质粒转化E.coli BL21诱导表达,亲和层析纯化后,SDS-PAGE显示获得目的蛋白,Western blot验证其能够与载体的组氨酸标签结合,质谱鉴定其Der f 6结构一致。以此产物为包被抗原建立间接ELISA检测尘螨过敏性哮喘患儿血清,阳性率为41.3%(19/46)。结论:成功构建了原核表达质粒p ET32a(+)-Der f 6,亲和纯化获得的目的蛋白具有良好的反应原性。
文摘Objective:To examine the acaricidal effects of the essential oil of Cymbopogon citratits leaf extract(lemongrass)and ethanolic Azadirachta indica leaf extract(neem)against house dust mites Dermatophagoides farinae(D.farinae)and Dermalophagoides pteronyssinus(D.pteronyssinus).Methods:Twenty-five adults mites were placed onto treated filter paper that is soaked with plant extract and been tested at different concentrations(50.00%,25.00%,12.50%,6.25%and 3.13%)and exposure times(24hrs,48hrs,72hrs and 96 hrs).All treatments were replicated 7 times,and the experiment repeated once.The topical and contact activities of the two herbs were investigated.Results:Mortalities from lemongrass extract were higher than neem for both topical and contact activities.At 50%concentration,both 24 hrs topical and contact exposures to lemongrass resulted in more than 91%mortalities for both species of inites.At the same concentration and exposure time,neem resulted in topical mortalities of 40.3%and 15.7%against D.pteronyssinus and D.farinae respectively;contact mortalities were 8.0%and 8.9%against the 2 mites,respectively.There was no difference in topical mortalities of D.pteronyssinus from exposure to concentrations oflemongrass and neem up to 12.50%;lemongrass was more effective than neem at the higher concentrations.Conclusions:Generally,topical mortalities of D.farinae due to lemongrass are higher than that due to neem.Contact mortalities of lemongrass are always higher that neem against both species of mites.
文摘目的获得粉尘螨变应原第3组分Der f 3原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE反应性。方法酶切质粒pET28a(+)-Der f 3获得目的基因Der f 3,将其与pET32a载体连接成质粒pET32a(+)-Der f3,转化BL21细菌,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot和质谱鉴定纯化产物。以纯化产物为包被抗原,采用间接ELISA检测尘螨过敏性哮喘患儿血清抗体反应情况。结果成功构建了原核表达质粒pET32a(+)-Der f 3。将该质粒转化E.coli BL21诱导表达,亲和层析纯化后经SDS-PAGE鉴定获得目的蛋白,Western blot验证该蛋白的能够与载体的组氨酸标签结合,质谱鉴定其结构与天然Der f 3一致。以此产物为包被抗原采用间接ELISA检测尘螨过敏性哮喘患儿血清,阳性率为29.73%(11/37)。结论成功构建了原核表达质粒pET32a(+)-Der f 3,亲和纯化获得的目的蛋白具有良好的反应原性。
文摘Dermatophagoides farinae and D. pteronyssinus are the prevalent house dust mites (HDM) in tropical countries and are associated with allergic diseases. This investigation developed a time- resolved immunofluorometric assay (TR-IFMA) for the first time to detect specific IgE antibody in patients with skin prick test positive to HDM but no detectable IgE by other means. Levels of IgE to natural and recombinant HDM allergens were measured by TR-IFMA in 50 HDM-allergic patients and 19 healthy participants compared to sandwich enzyme-linked immunosorbent assay (ELISA). A recombinant allergen, rDerf2, showed a 14 kDa band corresponding to broad range proteins of natural HDM.TR-IFMA showed sensitivity lower than 0.35 kUA/l. TR-IFMA employing three HDM antigens showed good correlations with sandwich ELISA at R2 0.93-0.96. TR-IFMA detected HDM IgE in 62, 62, 25 percent of allergic patient serum sample compared to 28, 32, and 22 percent detected by ELISA result using three HDM allergen. TR-IFMA also detected 26.3, 31.6, and 5.3 percent positive samples from 19 healthy participants while ELISA showed 0, 5.3, and 0 percents IgE positive samples. The use of rDerf2 as an HDM allergen for the assay was verified with no statistically different from other HDM allergens. TR-IFMA showed lower detection limit than ELISA and yielded higher sensitivity for serum of people with allergic symptoms with no detectable HDM IgE. It is anticipated that TR-IFMA for HDM-specific IgE detection will play an important role in future diagnosis of HDM allergy in clinical laboratories and for different research purposes.
