Research for developinga multivalent vaccine based onvaccinia virus.

为了构建基于牛痘病毒的多价表达载体，我们对牛痘病毒血凝素（HA）基因和牛痘病毒A型包涵体（ATI）基因进行了研究.用这两种方法将成人T细胞白血病病毒（HTLV-Ⅰ）的env基因插入细胞中，观察其表达模式。获得的结果如下。1)在HA基因中含有env基因的痘苗病毒可以在体外和体内繁殖。2)重组痘苗病毒可通过鸡红细胞染色空斑而容易地分离;野生型病毒的空斑被染成红色，而重组病毒的空斑则保持着颜色。3)HA启动子的活性与目前广泛使用的p7.5启动子相当。这些结果表明，HA基因位点适合于插入外源基因.检测了这些重组牛痘病毒的接种效果。1)它们在注射的兔子中引发了高滴度的抗env抗体。2)HTLV-I激发后，迄今为止（激发后27周）未检测到HTLV-I抗原阳性淋巴细胞。因此，表达env基因的重组病毒免疫家兔可保护家兔免受HTLV-I感染.为了克隆ATI基因，构建了用Sal I消化产生的牛痘DNA片段文库。然后，通过涉及ATI基因的转染和瞬时表达的方法筛选文库，以获得含有ATI基因的质粒。进一步的分析揭示了ATI基因的以下特征。1)ATI编码框的上游区域具有与牛痘病毒的其它晚期启动子相似的核苷酸序列。2)开放阅读框编码分子量为150，000道尔顿的蛋白。94,000.

To develop a multivalent vector based on vaccinia virus, we studied the vaccinia virus hemagglutinin (HA) gene and the A-type inclusion body (ATI) gene of cowpox virus.1. We inserted the env gene of adult T cell leukemia virus (HTLV-I) by the two mwthods and examined the modes of their expressions. The obtained results follow. 1) Vaccinia virus containing the env gene in the HA gene can propagates in vtro and in vivo. 2) The recombinant vaccinia virus could be easily isolated by staining the plaques by chicken erythrocytes; the plaques of the wild-type virus were stained red whereas those of the recombinant viruses remained coloress. 3) The HA promoter was as strong as p7.5 promoter which had been used extensively. These results suggest that the HA gene site is suitable for inserting the foreigen genes.2. The effect of vaccination by these recombinant vaccinia viruses was examined. 1) They elicited a high titer of anti-env antibodies in injected rabbits. 2) After challenge of HTLV-I no HTLV-I antigen-positive lymphocytes could be detected so far (27 weeks after challenge). Thus, the vaccination of the recombinant virus expressing the env gene protected the rabbits from HTLV-I infection.3. To clone the ATI gene, the library of cowpox DNA fragments generated by digestion with Sal I was constructed. Then, the library was screened by the methods involving the transfection and transient expression of the ATI gene so as to obtain the plasmid harboring the ATI gene. Further analyses revealed the following characters of the ATI gene. 1) The region upstream of the ATI coding frame has the nucleotide sequence similar to the other late promoters of the vaccinia virus. 2) The open reading frame codes for the protein of 150,000dalton.3) Vaccinia virus also has ATI-related protein of M.W. 94,000.