人巨细胞病毒cDNA表达文库的构建及pp65阳性克隆的筛选
摘要
目的：构建人巨细胞病毒（HCMV）AD169株及地方株基因组cDNA表达文库，
为进一步研究其基因组的结构特点及功能创立基本条件。方法：从MOI=10人巨
细胞病毒（HCMV）感染HF细胞96小时，提取mRNA OD260/OD280比值为2.1，
逆转录合成cDNA片段，重组入λgtll EcoR酶切位点之间，包装蛋白包装，转
染宿主菌LE392和Y1090；经IPTG诱导和x-gal颜色选择测定。采用鼠多克隆抗
血清进行免疫学筛选和地高辛标记特异性寡核苷酸探针进行原位噬斑杂交筛选阳
性克隆，经PCR扩增鉴定。结果：构建的cDNA表达文库的库容量分别为3.6×
10~6pfu/ml和3.3×10~6pfu/ml，经检测HCMV AD169 cDNA文库的重组效率为
76%；免疫学筛选出阳性克隆为168；pp65核酸探针进行噬斑杂交筛选出34个阳
性克隆；HCMV pp65全长PCR扩增出3个阳性克隆。结论：通过抗血清和核酸
杂交筛选证实成功的构建了HCMV AD169株感染96小时cDNA表达文库，并
从AD169文库中筛选出3个pp65阳性克隆。本研究结果将为阐明HCMV在机
体内的免疫逃逸机制；病毒基因及产物与细胞相互作用；探索新型疫苗奠定基础。
Objective: Construct HCMV AD 169 strain and isolated strain genome cDNA
expressing library, which provide a sound basis for further studying of the structural
character and function of their genome Methods: HF cell which were infected by
1-ICMV AD169 sftain(MON1O) and HCMV isolated strain(MOI=1O)cells were
collected at 96h p.i, mRNA contained poly(A) was isolated by chronimatography on
oligo(dT) cellulose, whose ratio of 0D260/0D280 was 2.1, then reverse transcripted
into eDNA, second- strand DNA synthesis was done by using Rnase H and DNA
polymerase 1, and E. co/i ligase to replace the RNA strand with deoxynucleotides . After
methylation and EcoR I linker addition the cDNA was cloned into EcoR I-digested
lambda gtl 1 .The ligated DNA was packaged , then transinfected host cells ,ie LE392
and Yl 090.The libray was screened by IPTG inducing and X-gal colouring , immune
blot with anti-HCMV mouse convalescent sera , nucleic acid hybridization with DIG-
labled HCMV pp65 gene probe and confirmed by PCR. Results: HCMV eDNA
expressing libraries were constructed , their volume were 3.6 X lO6pfulml and 3.3 X
1 O6pfuIml respectively,recombinatant efficiency of AD 169 was up to 76%, 168 positive
clones of ADI 69 were tested by immune blot, 34 positive clones were obtained by dot
nucleic acid hybridization with pp65 probe, 3 positive clones were amplified by HCMV
pp65 all length primer. Conclusion:HCMV AD169 strain and isolated strain cDNA
expressing library have been constructed and 3 pp65 positive clones frome AD 169
cDNA expressing library had been screened.These results should be valuable for
studying HCMV immunology mechanism, effect of viral genes on cells and new
vaccines.
为进一步研究其基因组的结构特点及功能创立基本条件。方法：从MOI=10人巨
细胞病毒（HCMV）感染HF细胞96小时，提取mRNA OD260/OD280比值为2.1，
逆转录合成cDNA片段，重组入λgtll EcoR酶切位点之间，包装蛋白包装，转
染宿主菌LE392和Y1090；经IPTG诱导和x-gal颜色选择测定。采用鼠多克隆抗
血清进行免疫学筛选和地高辛标记特异性寡核苷酸探针进行原位噬斑杂交筛选阳
性克隆，经PCR扩增鉴定。结果：构建的cDNA表达文库的库容量分别为3.6×
10~6pfu/ml和3.3×10~6pfu/ml，经检测HCMV AD169 cDNA文库的重组效率为
76%；免疫学筛选出阳性克隆为168；pp65核酸探针进行噬斑杂交筛选出34个阳
性克隆；HCMV pp65全长PCR扩增出3个阳性克隆。结论：通过抗血清和核酸
杂交筛选证实成功的构建了HCMV AD169株感染96小时cDNA表达文库，并
从AD169文库中筛选出3个pp65阳性克隆。本研究结果将为阐明HCMV在机
体内的免疫逃逸机制；病毒基因及产物与细胞相互作用；探索新型疫苗奠定基础。
Objective: Construct HCMV AD 169 strain and isolated strain genome cDNA
expressing library, which provide a sound basis for further studying of the structural
character and function of their genome Methods: HF cell which were infected by
1-ICMV AD169 sftain(MON1O) and HCMV isolated strain(MOI=1O)cells were
collected at 96h p.i, mRNA contained poly(A) was isolated by chronimatography on
oligo(dT) cellulose, whose ratio of 0D260/0D280 was 2.1, then reverse transcripted
into eDNA, second- strand DNA synthesis was done by using Rnase H and DNA
polymerase 1, and E. co/i ligase to replace the RNA strand with deoxynucleotides . After
methylation and EcoR I linker addition the cDNA was cloned into EcoR I-digested
lambda gtl 1 .The ligated DNA was packaged , then transinfected host cells ,ie LE392
and Yl 090.The libray was screened by IPTG inducing and X-gal colouring , immune
blot with anti-HCMV mouse convalescent sera , nucleic acid hybridization with DIG-
labled HCMV pp65 gene probe and confirmed by PCR. Results: HCMV eDNA
expressing libraries were constructed , their volume were 3.6 X lO6pfulml and 3.3 X
1 O6pfuIml respectively,recombinatant efficiency of AD 169 was up to 76%, 168 positive
clones of ADI 69 were tested by immune blot, 34 positive clones were obtained by dot
nucleic acid hybridization with pp65 probe, 3 positive clones were amplified by HCMV
pp65 all length primer. Conclusion:HCMV AD169 strain and isolated strain cDNA
expressing library have been constructed and 3 pp65 positive clones frome AD 169
cDNA expressing library had been screened.These results should be valuable for
studying HCMV immunology mechanism, effect of viral genes on cells and new
vaccines.
引文
1． Gole E and Nankervis GA. Cytomegaloviruse In: Viral infetions of humans: Epidemiology and Control(Evans AS ed). New York: Plenum Publishing Corp. 1982; 167-186．
2． 傅建林．中国医学科学院学报，1981：5(3) ：182)
3． Sissons JGP, Borysiewicz LK, Rodgers B, et al. Cytomegalovirus its celluar immunology and biology. Immuno Today, 1986(2) ; 7:57-61．
4． Pomeroy C, Englund J A, Cytomegalovirus: epidemiology and infection control. Am J Infect Control. 1987; 15: 107．
5． 现代微生物学．上海：闻玉梅；上海医科大学出版社..1999；929-931
6． Brook GF, Butel JS, Morss SA. Herpesviruses (Cytomegalovirus). In: Jawetz, Melnick, and Adelberg' s Medical Microbiology (21st Edition). Prentice Hall Internatiional Inc. (printed in USA). 1998; 401-405 (6Lee H et al. Intervirology,1996;39(4) 223-229) .
7． Spaete RR,A recombinant subunit vaccine approach to HCMV vaccine developwent. Transpant Proc 1991;23(suppl 3) :90-6．
8． Pachl C, Probert WS, Hermsen KM, Masiarz FR et al. The human Cytomegaloviruse strain Towne glycoprotein H gene encodes glycoprotein p86 Virology. 1989; 162: 478-82．
9． Navarro D, Paz P, Tugizov S. Topp K, et al. Glycoprotein B of human Cytomegalovirus promotes penetration into cells, transmission of infection from cell tocell, and fusion of infected cells. Virology 1993; (in press)
10． Lee H et al. Intervirology . 1996; 39(4) : 223-229
11． Jault FM et al. J Virol. 1995; 69(11) : 6697-6704
12． Susan K et al. J Gen Virol. 1996; 77(10) : 2597-2604
13． Dal Monte P et al．J Gen Virol．1996；77(10) ：2591-2595
14． St．Jeor S et al．Disease．Amsterrdam．Eisevier Science
15． 王明丽，唐久来，胡闻等．模拟人类巨细胞病毒先天性中枢神经系统感染小鼠 模型的建立．动物学报．1999；15(2) ：136-142
16． Gibson W．Structural and nonstructural proteins of strain Colbnrn cytomegalovirus． Virology,1981；111：516-537
17． Plola dal monte et al．Stably expressed antisense RNA to cytomegaloviruse UL83 inhibits viral replication．J virology．1996；70(4) ：2086-2094
2． 傅建林．中国医学科学院学报，1981：5(3) ：182)
3． Sissons JGP, Borysiewicz LK, Rodgers B, et al. Cytomegalovirus its celluar immunology and biology. Immuno Today, 1986(2) ; 7:57-61．
4． Pomeroy C, Englund J A, Cytomegalovirus: epidemiology and infection control. Am J Infect Control. 1987; 15: 107．
5． 现代微生物学．上海：闻玉梅；上海医科大学出版社..1999；929-931
6． Brook GF, Butel JS, Morss SA. Herpesviruses (Cytomegalovirus). In: Jawetz, Melnick, and Adelberg' s Medical Microbiology (21st Edition). Prentice Hall Internatiional Inc. (printed in USA). 1998; 401-405 (6Lee H et al. Intervirology,1996;39(4) 223-229) .
7． Spaete RR,A recombinant subunit vaccine approach to HCMV vaccine developwent. Transpant Proc 1991;23(suppl 3) :90-6．
8． Pachl C, Probert WS, Hermsen KM, Masiarz FR et al. The human Cytomegaloviruse strain Towne glycoprotein H gene encodes glycoprotein p86 Virology. 1989; 162: 478-82．
9． Navarro D, Paz P, Tugizov S. Topp K, et al. Glycoprotein B of human Cytomegalovirus promotes penetration into cells, transmission of infection from cell tocell, and fusion of infected cells. Virology 1993; (in press)
10． Lee H et al. Intervirology . 1996; 39(4) : 223-229
11． Jault FM et al. J Virol. 1995; 69(11) : 6697-6704
12． Susan K et al. J Gen Virol. 1996; 77(10) : 2597-2604
13． Dal Monte P et al．J Gen Virol．1996；77(10) ：2591-2595
14． St．Jeor S et al．Disease．Amsterrdam．Eisevier Science
15． 王明丽，唐久来，胡闻等．模拟人类巨细胞病毒先天性中枢神经系统感染小鼠 模型的建立．动物学报．1999；15(2) ：136-142
16． Gibson W．Structural and nonstructural proteins of strain Colbnrn cytomegalovirus． Virology,1981；111：516-537
17． Plola dal monte et al．Stably expressed antisense RNA to cytomegaloviruse UL83 inhibits viral replication．J virology．1996；70(4) ：2086-2094