DEVELOPMENT OF POLYVALENT RECOMBINANT VACCINES FOR CATS.

本研究的目的是开发对猫安全、经济、有效的上级多价重组（rec）疫苗，并将其投入实际应用。主要研究结果如下：1.成功地进行了胸苷激酶（TK）缺陷突变株的减毒猫疱疹病毒1型（FHV-1）C7301 dlTK基因的克隆及重组FHV-1的构建（C7301 dlTK-帽）将猫杯状病毒（FCV）的前体衣壳基因插入C7301 dlTK的TK缺失基因座中。Cap，然后用强毒FHV-1和FCV攻击，在很大程度上保护了由这两种病毒引起的疾病的表现。2.为了进一步完善重组FHV-1，我们构建了一个改良的重组FHV-1（dlTK（gCp）-Cap），它在C7301 dlTK-Cap的FCV前体衣壳基因上游携带一个推定的FHV-1 gC启动子序列。细胞培养物中dlTK（gCp）-Cap的生长动力学与C7301 dlTK和C7301 dlTK-Cap相似。通过间接免疫荧光和酶联免疫吸附测定证实dlTK（gCp）-Cap强烈表达FCV免疫原性抗原。3.将编码猫免疫缺陷病毒（FIV）Gag蛋白的cDNA插入C7301 ddlTK的TK缺失位点，构建了表达FIV Gag蛋白的重组FHV-1，命名为C7301 ddlTK-gag。重组病毒在Crandell猫肾细胞中的生长动力学与亲本C7301株相似。通过免疫印迹分析，证实C7301 ddlTK-gag在细胞中表达FIV Gag前体蛋白。

The aim of the present studies is to develop polyvalent recombinant (rec) vaccines which are superior to safeness, inexpensiveness and effctiveness to cats, and to put the vaccines to practical use. The following results are obtained.1.We succeeded in the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinat FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calicivirus (FCV) into the TK deletion locus of the C7301dlTK.Cats vaccinated with C7301dlTK-Cap and then challenged with virulent FHV-1 and FCV were protected to a significant degree against the manifestations of the illness caused by both viruses. However, immune presponse to FCV was weak.2.Therfore to improve further on the recombinat FHV-1, we constructed an improved recombinant FHV-1 (dlTK (gCp) -Cap) carring a putative FHV-1 gC promoter sequence on the upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK (gCp) -Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK (gCp) -Cap was confirmed by indirect immunofluorescence and enzime-linked immunosorbent assays. In addition, one vaccination with dlTK (gCp) -Cap protected cats more effectively against subsequent virulent FCV challenge than that with C7301dlTK-Cap.3.We constructed a recombinat FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of C7301ddlTK,and was designated as C7301ddlTK-gag. Growth kinetics of the reconbinat viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK-gag was confirmed to express the FIV Gag precursor protein in the cells.