痘苗病毒天坛株非必需区与病毒生物学性状的关系

RELATIONSHIP BETWEEN THE NON-ESSENTIAL REGIONS OF VACCINIA VIRUS ( TIANTAN STRAIN ) AND ITS BIOLOGICAL PROPERTIES

将位于痘苗病毒启动子p25下游的lαc基因插入到5个非必需区中,用X-gal筛选获得表达β-半乳糖苷酶的相应重组病毒。Southern印迹杂交证实,lαc基因准确地插入到相应的非必需区中。5株重组病毒与天坛野毒株的生物学性状比较表明,M片段SphⅠ位点插入lαc基因对重组病毒在兔子皮内毒力和小鼠脑内毒力没有明显影响,重组病毒的宿主范围和温度稳定性与亲本株相似,lαc基因能够稳定遗传和表达;K片段左起第一个BglⅡ位点插入lαc基因后,病毒在兔子皮内和小鼠脑内毒力均下降1～2个对数,但宿主范围和温度稳定性没有改变,lαc基因能稳定遗传并表达;K片段左起第三、四个BglⅡ及K/F交界处的HindⅡ位点插入lαc基因对病毒毒力亦无明显影响。这些结果说明M片段sphⅠ位点、K片段左起第三、四个BglⅡ位点及K/F交界处HindⅢ位点适合于外源基因表达和基因工程疫苗研究。

Five non-essential sites on the left end of the genome of vaccinia virus ( Tian Tan strain ) have been studied regarding their effects on viral biological properties. The lac gene controlled by vaccinia virus promoter p25 was inserted into each of the five sites by marker rescue. Recombinants expressing β-galactosidase were obtained and purified.Correct insertion of the lac gene into recombinant viruses was confirmed by Southern blot.The insertion of lac into Sph I of M fragment has no significant effect on both intracerebral ( i.c. ) virulence in mice and intradermal(i. d. ) virulence in rabbits. There is no difference between the parental strain and the recombinant in host range and thermostability. Expression of lac is genetically stable in the recombinant.The insertion of lac into the left most Bgl I site in K fragment results in the attenuation of virulence both in mice ( i.c.) and in rabbits (i. p. ) , but does not alter the viral host range, thermostability and genetic stability in the recombinant.Insertions at the third, fourth Bgl I sites from the left in K fragment and at the Hindi site between K and F have no appreciable influence on viral virulece in mice ( i.c. ) and rabbits (i.p.).In conclusion,the Sph I site of the M fragment is a suitable insertion site for foreign genes without appreciable effect on the biological properties of the resulting recombinants. The third, fourth Bgl Ⅱ sites from the left in K. fragment and at the Hind Ⅲ site between K and F may also be good insertion sites,but need more detailed further study.The possile mechanism of foreign gene insertion at different nones sential sites on the biological properties ( including virulence ) of the resulting recombinant vaccinia virus and the advantages of chosing the M-Sph I insertion site are discussed. The need of further study concerning the possible effects of insertion of foreign viral genes instead of lac and the possible additive effect of insertion at multiple sites in the construction of a polyvalent live vaccine are mentioned.