摘要
应用RT-PCR技术扩增柔嫩艾美耳球虫哈尔滨株子孢子表面抗原3-1E基因,再克隆至质粒载体pMD18-T中,获得重组质粒pMD18-T-3-1E,采用EcoRⅠ+HindⅢ双酶切法及PCR扩增鉴定正确后,将3-1E基因cDNA目的片段亚克隆到原核表达载体pET-32a(+)中,获得重组质粒pET-32a-3-1E,用EcoRⅠ+HindⅢ双酶切法及DNA测序证明cDNA序列完全正确。表达蛋白的SDS-PAGE分析表明,表达产物与预期大小(36.47 ku)一致,为可溶性表达。对诱导时间以及IPTG浓度的优化结果表明,当诱导6 h且IPTG浓度在0.8 mmol/L时表达量最大。Western-blot分析表明,表达的蛋白可被感染柔嫩艾美耳球虫鸡的阳性血清特异性识别。将重组3-1E蛋白纯化后分为肌注蛋白组和肌注蛋白加弗氏完全佐剂组免疫AA鸡,通过计算抗球虫指数检测其保护力。结果显示,肌注重组蛋白组与肌注重组蛋白加弗氏完全佐剂组均能刺激鸡体产生一定的免疫反应,对柔嫩艾美耳球虫感染产生有力的保护,其中后者优于前者,表明该重组3-1E蛋白具有研制成亚单位疫苗的潜力。
3-1E antigen gene was amplified by PCR from Eimeria tenella Harbin strain and cloned into a vector pMD18-T.The positive plasmid containing 31E antigen cDNA was determined by restriction enzyme analysis and PCR.The plasmid pMD18-T-3-1E and the vector pET-32a（＋） were both digested with EcoRⅠand Hind Ⅲ.The cDNA encoding 3-1E antigen was subcloned into pET-32a（＋）.In result,it was confirmed by restriction enzyme analysis and sequence determination that the positive recombinant plasmid pET-32a-3-1E contained 3-1E gene.In the positive transformants,the 3-1E antigen was expressed in a soluble fusion protein,which was verified by SDS-PAGE.That the optimized inducing time and IPTG concentration was selected showed that 6 h and 0.8mmol/L were the optimal expression conditions.The immune reactivity of the recombinant protein was confirmed by Western-blot.The AA chickens were injected with the 3-1E recombinant protein using the 3-1E recombinant protein＋Freund＇s complete adjuvant（FCA） as control.Then the anticoccidia index was detected.The result showed that both the 3-1E recombinant protein and the protein＋FCA could induce immune response and immunoprotection.The protein＋FCA was better than the others.In conclusion,the 3-1E recombinant protein is the potential subunit vaccine against E.tenella.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第4期400-405,共6页
Chinese Veterinary Science
基金
国家"十一五"科技支撑计划重大项目(2006BAD06A08)
哈尔滨市科技创新人才研究专项(2006RFQXN016)
