摘要
应用RT-PCR获得新城疫病毒(NDV)La Sota株的血凝素-神经氨酸酶(HN)基因片段,将HN基因片段定向插入毕赤酵母表达载体pPICZαA,构建了分泌型表达载体pPICZαA-HN,将其用SacⅠ线性化后电转化入P. pastorisX-33。经高抗性及阳性转化子筛选得到重组菌株X-33/pPICZαA-HN,甲醇诱导后,表达产物的上清液经SDS-PAGE、Western-blot和血凝活性检测,结果约在97 ku处有目的条带,比预期分子质量大,占总蛋白的53%,与NDV阳性血清发生特异性免疫反应,并具有较高的血凝活性。表明,HN基因在毕赤酵母中成功地进行了表达且表达产物具有良好的免疫原性。
A cDNA fragment encoding hemagglutinin-neuraminidase（HN）was amplified from Newcas tle disease virus（NDV） La Sota strain by RT-PCR. The fragment was inserted into pPICZaA by a direc tional link to construct secreting expression vector pPICZaA HN. The constructed pPICZαA HN plasrnid was linearized by Sac i and then transformed into Pichia pastoris X-33 by electroporation. Through selec tion of X-33 resistant transformation and expression clones,a highly expression transformant was obtained. The expressed product from the recombinant strain X-33/pPICZαA-HN induced with methanol was ana lyzed by SDS PAGE, Western-blot and hemagglutination tests. The expressed protein was approximately 97 ku in molecular mass in the supernatant,which was a little larger than that of the expected one. The tar geted protein made up approximately 53% of the total yeast body proteins. The expressed HN had strong antigen immune reactions with the positive antiserum against NDV which had high hemagglutinative titer. These results demonstrated that the HN gene was expressed successfully in P. pastoris and the expressed product had immunogenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第10期860-865,共6页
Chinese Veterinary Science
基金
河南省自然科学基金项目(0511032300)
