Research for developinga multivalent vaccine based onvaccinia virus.

为了构建基于牛痘病毒的多价载体，我们研究了牛痘病毒血凝素（HA）基因和a型包涵体（ATI）基因。我们用两种方法插入成人T细胞白血病病毒（HTLV-I）的env基因，并检测其表达模式。得到的结果如下：1)含有HA基因中env基因的痘苗病毒在体内和体外都能繁殖。2)用鸡红细胞染色可分离到重组痘苗病毒；野生型病毒的空斑被染成红色，而重组病毒的空斑则保持颜色。3) HA启动子的强度与目前广泛应用的p7.5启动子相当。这些结果表明，HA基因位点适合外源基因的植入。研究了这些重组痘苗病毒的接种效果。1)它们在注射兔体内引起了高滴度的抗env抗体。2) HTLV-I攻毒后（27周）未检出HTLV-I抗原阳性淋巴细胞。由此可见，接种表达env基因的重组病毒可保护家兔免受htlv - 1感染。为了克隆ATI基因，构建了用Sal I酶切产生的牛痘DNA片段文库。然后，通过ATI基因转染和瞬时表达的方法对文库进行筛选，获得含有ATI基因的质粒。进一步分析发现ATI基因具有以下特征：1) ATI编码框上游区域的核苷酸序列与其他痘苗病毒晚期启动子相似。2)开放阅读框编码150000道尔顿的蛋白质。3)牛痘病毒也有mw94000的ati相关蛋白。

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.