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用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。
Abstract：cDNAs were prepared from virion RNAs extracted from the purified viruses as described previously,using poly A tailing and Oligo-dT as a pri-mer. After treatment and repair, double stranded cDNA were ligated to dephosphorylated pUC 18 plasmid DNA, and then transfe.cted into E, coli MC1061. The transformants were selected by resistence to ampicilin. The resulting colonies were collected and the specificity of cDNAs was identi-fied as follows.1. Virus specificity and virai RNA segment specificity. Colonies were screened for the presence of R22 cDNA inserts by in situ hybridization assay using α32p labeled virai RNA as probe. 18 hybridization-positive colonies were characterized by comparing their Hinf I restriction fragments to those of pUC18.Five clones were further analysed by nick translation of the plasmid DNA and hybridization to purified virai RNAs on nitrocellulose paper ( northern blot analysis ) . Three clones ( designated R3, R9 and R11 ) hy-bridized with the M ( medium ) segment, while R7 and Rl3 hybridized with L ( large ) and S ( small ) segments respectively.2. DNA sequence analysis: The 3'-terminal nucleotide sequence of R3 clone of R22 virus was found to be similar to that of Hantaan virus, but a slight difference of sequences between Hantaan and R22 viruses was found to be present in a few nucleotides upstream of ATG.
[1] 杭长寿等:病毒学报,3:120,1987
[2] David D A et al: Virology, 134: 208, 1984
[3] Verma L M et al: Nature(New Biol), 235:163, 1972
[4] Efstratiadis A et al: Cell, 7: 279, 1976
[5] Wickens M P et al: J Biol Chem, 253:2483, 1978
[6] Buell C et al: J Biol Chem, 253:2471, 1978
[7] Gubler U et al: Gene, 25:263, 1983
[8] Okayama H et al: Mol Cell Biol, 2:161, 1983
[9] David D A et al:Virology, 154:155, 1986
[10] Suteliffe G et al: Cold Spring Harbor Symp Quant Biol, 43:73, 1979
[11] Grunstein M et al: Proc Natl Acad Sci (USA), 72: 3967, 1975
[12] Bishop D H L et al: Nucl Acids Res, 10: 3703, 1982
[13] Bishop D H L et al: Nucl Acids Res, 11: 6409, 1983
[14] Schmaljohn C S et al: Virology, 157: 31, 1987
[15] Maniatis T et al: Molecular cloning, A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor New York, 1982
[16] Maxam A et al: Methods in Enzymology(L Grossmen and K Moldave, eds), 105: 499, Academic Press Orlando Fla, 1980
[17] Messing J et al: Methods in Enzymology (L Grossmen and K Mo ldave, eds), 101:20, Academic Press Orando Fla, 1983
[18] Sanger F et al: Proc Natl Acad Sci(USA), 71:5463, 1977
[19] Schmaljohn C S et al: Science, 227: 1041, 1085
[20] Schmaljohn C S et al, Virology, 155:633, 1986
[21] Land H et al: Nucl Acids Res, 9:2251, 1981
[22] Heidecker G et al: Nucl Acids Res, 11: 4891, 1983
[23] Leis P et al: Proc Natl Acid Sci(USA), 70:466, 1973
[24] Song G et al: J Infect Dis, 150:889, 1984
[25] 廖化新等:中华传染病杂志,3:27,1987
[26] 邢峥等:病毒学报,3:27,1987
[27] Lee P W et al:J Clini Microbio, 22: 940, 1985
[28] Dantas J R et al: Intervirol, 27:161, 1987
基本信息:
引用信息:
[1]杭长寿,Sanchez,A,陈思毅,Kiley,M. P,David,D.A.,McCormick,J.B.,宋干.流行性出血热病毒R22株cDNA克隆及其特异性鉴定[J].病毒学报,1988(03):208-212.
基金信息:
国家自然科学基金
1988-09-30
1988-09-30