甲型肝炎重组痘苗病毒(VMS11HAV25)的构建及其某些生物学性质

THE CONSTRUCTION AND BIOLOGICAL PROPERTIES OF A RECOMBINANT VACCINIA VIRUS(VMS11HAV25)EXPRESSING HEPATITIS A VIRUS ANTIGENS

将5′端经过剪切的甲型肝炎病毒全部开放读码框架cDNA连接于痘苗病毒晚期启动子P11下游,重组于痘苗病毒天坛株的HiodⅢM片段Spb Ⅰ位点获得了重组病毒VMS11HAV25。对其生物学性质的研究表明,该重组病毒诱生痘苗抗体的能力、对鸡红细胞的血凝性质、空斑大小及对温度的稳定性等均与原天坛株相同。重要的区别是,重组病毒在家兔皮内和小鼠脑内的毒力都比原天坛株低约1个对数。病毒在人胚肺二倍体细胞连续传15代的表达水平与传代早期者相同。连续传20代后提取病毒DNA做Southern blot杂交表明,甲型肝炎病毒基因仍稳定地存在于原插入位置。

A HAV recombinant vaccinia virus named VMS11HAV25 was constructed by inserting the entire open reading frame of HAV cDNA,driven by a vaccinia promotor P11, into Hind 1 M fragment of Tian Tan strain of vaccinia virus which had been used extensively for over 50 years as a vaccine to eradicate smallpox in China.The inserted HAV cDNA was modified by deleting the majority of 5' noncoding region to prevent the production of infectious hepatitis A viral particles.It is important to know if the insertion of HAV gene affects the biological properties of the vector virus. Results showed that there were not much difference between the recombinant virus and the parental virus. An obvious difference was that the virulence of the recombinant virus was about ten times lower than its parental virus when tested in rabbits intradermally and in mice intracerebrally.The expression of HAV antigen by the recombinant virus at the 15th passage in human lung diploid cells was as good as that at the 4th passage. Southern blot hybridization showed that the inserted HAV cDNA was still located at the correct site after the recombinant virus had undergone 20 passages in the diploid cell culture, indicating the stability of the HAV gene in the vector.