摘要
参照GenBank中登录的鸡白介素5(IL-5)基因的核苷酸序列,设计1对特异性引物。用聚合酶链反应(PCR)以克隆载体pMD18-T-IL-5为模板扩增IL-5基因,然后将IL-5基因克隆到酵母表达载体pPICZαA中并电转化入酵母X-33,在Zeocin作为筛选标记的YPD培养基上筛选阳性菌落。经甲醇诱导表达鸡IL-5蛋白,对表达产物进行SDS-PAGE和Western-bolt分析。以纯化的鸡白介素5蛋白作为包被抗原,建立了检测鸡白介素5蛋白抗体的间接ELISA。结果显示,鸡白介素5基因获得成功表达,诱导表达培养物上清中表达出具有反应活性的26ku左右的重组鸡白介素5蛋白。从而实现了鸡白介素5蛋白高效的酵母表达。确定了间接ELISA的最佳反应条件:抗原包被浓度为10.00μg/mL,包被时间为37℃2h,血清稀释度为1∶1 600,HRP标记的山羊抗小鼠IgG抗体的稀释度为1∶1 000,血清与抗原在37℃反应1h,血清和二抗于37℃反应1h。应用该方法对试验小鼠血清进行检测,结果显示建立的ELISA具有较好的重复性,适用于检测抗鸡白介素5蛋白的抗体。
According to the sequence of chicken interleukin-5（ChIL-5） gene in GenBank,a pair of primers was designed and used for amplifying ChIL-5 gene from the plasmid pMD18-T-IL-5. The amplified product was cloned into Pichia pastoris expression plasmid pPICZaA,and transformed into P. pastoris cell X-33. The P. pastoris protein was isolated, and analyzed by SDS-PAGE and Western-bolt. Using the purified recombinant ChIL-5 protein as coating antigen,an indirect ELISA was developed for the detection of antibody against ChIL-5 protein. The result showed that ChIL-5 gene was expressed successfully and the expressed ChIL-5 protein was 26 ku in molecular mass. The expressed ChIL-5 protein existed in supernatant and had a good reactionogenicity. In the indirect ELISA, the optimal concentration for coating antigen was 10.00μg/mL and the optimal condition was at 37 ℃ for 2 h,the optimal dilution of serum sam- ple was 1 ： 1 600, the optimal dilution of the goat anti-mice IgG labeled with HRP was 1 ： 1 000. The serum sample should be incubated with ChIL-5 protein at 37 ℃ for 1 h, and the HRP-labeled goat anti-mice IgG should be incubated with serum sample at 37 ℃ for 1 h. The murine serum samples were examined by the indirect ELISA to monitor antibody titer. The results revealed that the indirect ELISA had good reproducibility, and was suitable to detect the specific antibody against ChIL-5 protein.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第8期854-860,共7页
Chinese Veterinary Science
基金
河南省重点科技攻关项目(132102110117)
河南科技大学培育基金项目(2013ZCX010)
