摘要
为提高传染性法氏囊病病毒(IBDV)多表位基因5epis的免疫效力,本研究应用重叠延伸PCR技术,在乙型肝炎病毒核心抗原(HBcAg)基因的231位～232位核苷酸(77位～78位氨基酸残基)之间插入BamHⅠ和EcoRⅠ酶切位点,构建基于HBcAg修饰基因(mHBc)的抗原表位嵌合体基因分子载体pET-mHBc。将编码IBDV多表位基因5epis定向插入到pET-mHBc的mHBc基因中,获得嵌合体基因mHBc-5epis表达重组质粒pET-mHBc-5epis,并在大肠杆菌中表达。经SDS-PAGE分析,重组嵌合体蛋白的分子量约29 ku,表达量约占菌体总蛋白的47.6%;Western blot结果表明,重组嵌合体蛋白可与IBDV抗体反应形成1条特异性蛋白带;重组嵌合体蛋白呈病毒样颗粒(VLP),直径约100 nm～120 nm;用IBDV抗体夹心ELISA检测,抗原效价达到1∶200。本实验成功构建了5epis与HBcAg嵌合体基因,并在大肠杆菌中高效表达,具有IBDV抗原反应性的重组病毒样颗粒嵌合体蛋白,为研究其表位型基因工程疫苗提供了新途径。
To enhance the immune efficacy of multi-mimotopes gene 5epis of infectious bursal disease virus（IBDV）,the multi-mimotopes gene 5epis was inserted into mHBc of pET28-mHBc expression vector by overlapping PCR.The recombinant plasmid pET-mHBc-5epis was transformed into E.coli BL21（DE3） and a chimeric mHBc-5epis protein was expressed by inducing with IPTG.SDS-PAGE showed that the expressed protein was approximately 29 ku and up to 47.6% of the total bacterial proteins.Western blot confirmed that the recombinant protein could be recognized by anti-IBDV polyclonal antibody.In addition,self-assembled virus-like particles（VLP） with a diameter of 60 nm to 80 nm was observed under electron microscopy in the bacterial lysate.The recombinant mimotope-based chimeric construction reported here could be used as an effective strategy for
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第11期879-883,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金资助项目(30571371)
江苏省自然科学基金资助项目(BK2008065
BK2008352
BK2009041)
