Identification of an epitope encoded in the env gene of Friend murine leukemia virus recognized by anti-Friend virus cytotoxic T lymphocytes.
Identification of an epitope encoded in the env gene of Friend murine leukemia virus recognized by anti-Friend virus cytotoxic T lymphocytes.
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DOI：
10.1016/0042-6822(91)90473-o
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发表时间：
1991-03
期刊：
影响因子：
3.7
通讯作者：
K. Ruan;F. Lilly
中科院分区：
文献类型：
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作者：
K. Ruan;F. Lilly
我们先前已经证明，H-2b小鼠中抗Friend病毒（FV）细胞毒性T淋巴细胞（CTL）识别的强表位编码在辅助Friend小鼠白血病病毒基因组的env和gag/polregions中。已经使用两种方法来鉴定这些表位。在核酸水平上，我们构建了两种读框内缺失的env基因：pKR 2，在gp 70区缺失681-bp的env基因，插入到pSV 2-gpt-1表达载体中; pKR 1，在p15 E区缺失81-bp的env基因，插入到pSV 2-gpt-1表达载体中。用pDb（H-2D限制元件）、PNEO（用于G418筛选）和pKR 1或pKR 2分离Fisher大鼠胚胎细胞，建立细胞克隆。用多克隆抗体免疫沉淀或用北方印迹检测病毒RNA表达。用多克隆抗FV/DbCTL在~（51）Cr释放试验中检测表达细胞克隆对裂解的敏感性。表达pKR 1的细胞与表达完整env基因的细胞裂解程度相同，而表达pKR 2的细胞对裂解具有抗性，这表明所有可检测的env表位都在681-bp缺失内编码。合成代表该区段中编码的两个最可能的候选表位的多肽，并测试它们使表达Dbalone的FRE细胞对CTL裂解敏感的能力。一个17-mer多肽AGTGNRCCNCYEGAYEA在该测定中作为强CTL表位起作用，但另一个18-mer多肽无活性。该表位在对候选病毒疫苗的免疫应答中的作用的研究正在进行中。
We have previously shown that strong epitopes recognized by anti-Friend virus (FV) cytotoxic T lymphocytes (CTL) inH-2bmice are encoded in both the env andgag/polregions of the helper friend murine leukemia virus genome. Two approaches have been used to identify these epitopes. At the nucleic acid level, we have constructedenvgenes with either of two in-frame deletions: pKR2, anenvgene with a 681-bp deletion in the gp70 region and inserted into the pSV2-gpt-1 expression vector; and pKR1, anenvgene with an 81-bp deletion in the pl5E region and inserted into pSV2-gpt-1. Cell clones were established by transfecting Fisher rat embryo cells with pDb(the H-2Dbrestriction element), PNEO (for G418 selection) and either pKR1 or pKR2.Dbandenvgene expression was monitored by immunoprecipitation with polyclonal antibodies or by detection of viral RNA on Northern blots. Expressor cell clones were tested for susceptibility to lysis by polyclonal anti-FV/DbCTL in51Cr-release assays. Whereas cells expressing pKR1 were lysed to the same extent as cells expressing the intactenvgene, cells expressing pKR2 were resistant to lysis, suggesting that all detectable env epitopes are encoded within the 681-bp deletion. Polypeptides representing the two most likely candidate epitopes encoded in this segment were synthesized and tested for their abilities to sensitize FRE cells expressing Dbalone for lysis by the CTL. One 17-mer polypeptide, AGTGNRCCNCYEGAYEA, functioned as a strong CTL epitope in this assay, but the other 18-mer polypeptide was inactive. Studies of the role of this epitope in the immune response to candidate viral vaccines are in progress.