Collins P L, Mink M A, Hill M G, Camargo E, Grosfeld H, Stec D S
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Virology. 1993 Jul;195(1):252-6. doi: 10.1006/viro.1993.1368.
A cDNA was constructed to encode an internally-truncated version of negative-sense genomic RNA (vRNA) of respiratory syncytial virus (RSV) containing (in 3' to 5' order) the 3' vRNA leader region, the complement of the open reading frame for bacterial chloramphenicol acetyl transferase (CAT) under the control of RSV gene-start and gene-end signals, an intergenic nucleotide, the complete L gene including the gene-start and gene-end signals, and the 5' vRNA trailer region. The encoded vRNA analog (RSV-CAT-L) would be 7502 nucleotides (nt) in length, 49.3% of the complete 15,222-nt parental vRNA. RSV-CAT-L vRNA was synthesized in vitro from HgaI-digested cDNA and transfected into RSV-infected cells. The vRNA was "rescued" such that it was expressed to yield CAT and was packaged into particles that could be passaged to express CAT in fresh cells. The efficiency of rescue was greatly improved by a single nucleotide substitution in the leader region that had been found to increase the efficiency of rescue and passage of the previously described 935-nt RSV-CAT vRNA. Compared to the 8-fold smaller RSV-CAT vRNA, and adjusted to molar equivalence of transfected RNA, RSV-CAT-L vRNA was 119- to 347-fold less efficient in expressing CAT upon transfection, whereas RSV-CAT-L vRNA containing the above-mentioned nucleotide substitution was 15.8-fold less efficient.
构建了一个cDNA，用于编码呼吸道合胞病毒（RSV）负链基因组RNA（vRNA）的内部截短版本，该版本包含（从3'到5'顺序）3' vRNA前导区、在RSV基因起始和基因终止信号控制下的细菌氯霉素乙酰转移酶（CAT）开放阅读框的互补序列、一个基因间核苷酸、包括基因起始和基因终止信号的完整L基因以及5' vRNA尾区。编码的vRNA类似物（RSV-CAT-L）长度为7502个核苷酸（nt），占完整的15222 nt亲本vRNA的49.3%。RSV-CAT-L vRNA由经HgaI消化的cDNA体外合成，并转染到RSV感染的细胞中。vRNA被“拯救”，从而表达产生CAT，并被包装成颗粒，这些颗粒可以传代以在新鲜细胞中表达CAT。通过在前导区的一个单核苷酸取代，拯救效率大大提高，该取代已被发现可提高先前描述的935 nt RSV-CAT vRNA的拯救和传代效率。与小8倍的RSV-CAT vRNA相比，并调整到转染RNA的摩尔当量，RSV-CAT-L vRNA转染后表达CAT的效率低119至347倍，而含有上述核苷酸取代的RSV-CAT-L vRNA效率低15.8倍。
