【作者】 李海涛；

【导师】 扈荣良；

【作者基本信息】 吉林大学 ， 预防兽医学， 2005， 硕士

【摘要】 本研究在前期获得狂犬病病毒糖蛋白重组犬2 型腺病毒的基础上,以狂犬病病毒(Rabies Virus,RV)SRV9株的核蛋白基因为目的基因,构建了狂犬病病毒核蛋白重组犬2 型腺病毒,并与狂犬病病毒糖蛋白重组犬2 型腺病毒共同进行了犬科动物的免疫试验。首先对犬2 型腺病毒全基因组的E3 区进行了部分缺失,插入了由CMV 启动子、狂犬病病毒核蛋白基因、牛生长激素的ployA 组成的表达盒,构建了含有狂犬病病毒核蛋白基因的重组犬2 型腺病毒基因组,将构建的重组全基因组脂质体介导转染MDCK 细胞,获得了携带狂犬病病毒核蛋白基因的重组病毒。对获得的重组病毒和狂犬病病毒糖蛋白重组犬2 型腺病毒进行病毒形态学观察,反应原性、遗传稳定性分析,以及病毒其他生物学特性的研究。结果显示该两株重组病毒具有典型的腺病毒粒子形态、良好的安全性和反应原性,能够与抗狂犬病病毒的多抗血清反应,进一步的免疫实验研究表明,该重组病毒在犬、狐狸和貂等野生动物体内均能产生针对狂犬病病毒糖蛋白和核蛋白的抗体,小鼠中和试验和荧光中和试验结果显示,狂犬病病毒糖蛋白重组犬2 型腺病毒能够刺激机体产生针对狂犬病病毒的中和抗体,而狂犬病病毒核蛋白重组2 型腺病毒仅能刺激机体产生微弱的中和抗体,但是病毒增殖抑制试验表明狂犬病病毒核蛋白重组犬2 型腺病毒免疫动物的血清可在感染后24-72h 对病毒的增殖产生抑制,攻毒试验显示,抗狂犬病病毒糖蛋白抗体能够保护动物抵抗狂犬病病毒攻击,狂犬病病毒核蛋白抗体能够抵抗低剂量的狂犬病病毒的攻击。更多还原

【Abstract】 Rabies continues to be a serious problem in China due to the reservoirof rabies virus(RV) in dogs. The control and eradication of rabiesdepends on the development of safe, effective and economical vaccine thatmight be used in preexposure vaccination programs for dogs. Fiveproteins are encoded by the rabies virus genome. The glycoprotein isgenerally regarded as the only protein that can stimulate neutralizingantibody. Several studies have also shown that the nucleoprotein protectsmice and dongs against lethal rabies virus infections. To construct arecombinant canine adenovirus against Rabies, the N gene of RV virus andcanine adenovirus type 2(CAV-2) were chosen as foreign gene and thevector, respectively. The expression cassette of nucleoprotein consists of thehuman cytomegalovirus(CMV) immediate promoter, rabies virus strainSRV9 nucleoprotein cDNA and polyadenylation signal of the bovine growthhormone. Using two Ssp Ιsites in the E3 region of canine adenovirustype 2 strain YCA18, a 859 bp DNA fragment was replaced by the 2 392 bpexpression cassette of the nucleoprotein. The recombinant genome wasreleased by AscΙand PvuΙdouble digestion and was transfected intoMDCK cells by Lipofectamine ? . Typical cytopathogenic effect wasobserved 5 passages after transfection. Using the restriction endonucleasedigestion and Southern blot, we confirmed that the recombinant canineadenovirus type 2 expressing rabies nucleoprotein (CAV-2-RN) had been更多还原