人巨细胞病毒UL136基因在临床低传代分离株中多态性分析
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摘要
目的
人巨细胞病毒(Human cytomegalovirus，HCMV)是引起宫内感染进而导致婴儿先天性发育畸形的常见病原。HCMV感染可引起不同的临床表现，其发病机制不清，可能与宿主的体液和细胞免疫状态有关，亦可能与不同临床分离株基因及其编码产物的多态性有关。资料表明，HCMV UL／b’区在临床低传代分离株和实验室株中表现出极大的遗传多态性。本文主要研究该区基因之一UL136在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系，力图为揭示该基因编码产物的功能及先天性HCMV感染的致病机理奠定分子基础。
材料与方法
一、标本来源
48株低传代临床分离株均来自1988-1993年我院住院患儿，年龄(14个月，其中黄疸28株，小头畸形11株，先天性巨结肠9株。标本取自病毒分离实验，-70℃保存。低传代分离株传代少于10次。2000年应用荧光定量PCR方法检测HCMV-DNA，结果均为阳性。
二、实验方法
1、标本处理
取HCMV低传代分离株细胞培养上清液与等量裂解液混匀后，煮沸15分钟，提取DNA，作为扩增模板。
2、引物设计
按照Toledo株序列，应用引物设计软件primer premier5．0设计用于扩增HCMV UL136基因的全序列引物和鉴定引物。
全序列引物
上游引物 UL136ca：5’TCGGACATCGAGGAACTCI’tvi 3’
下游引物UL136Cb：5’GTGCCAGTGGTAAGCCAGATA3’
鉴定引物
上游引物 UL136UI：5’GAATGTCGGCTACGGGTG 3’
下游引物 UL136DI：5’TGTCTCGCCAAC’I’GTCTG 3’
下游引物 UL137DI：5’TCACGCCGATGAGGGTA 3’
3、UL136基因PCR扩增
PCR循环条件如下：预变性95℃，4分钟；96℃变性45秒，
55℃退火1分钟，72℃延伸至分钟30秒书个循环；72℃终延伸，
10分钟。＊5％经漠化已锭染色的琼脂糖凝胶电泳后，紫外检测
仪下观察扩增结果。
4、凝胶回收
应用PCR目的基因片段回收试剂盒对扩增阳性PCR产物进
行凝胶回收，纯化目的DNA片段。
5＊CR阳性扩增产物测序
6、序列分析
应用软件 DNAClub，BioEdit，Genedoc，DNASis，DNAStar完成
序列分析。
7、序列提交
用Setwn软件向 GenBank提交整理后的HCMV UL136 ORF
序歹。
实验结果
一JCR扩增结果
对 48株低传代临床分离株进行 HCMV UL136全序列 PCR扩
增，结果18株阳性，阳性率37．5％，其中黄疽12株，小头畸形4
株，先天性巨结肠2株。
·2·
二月136基因编码区及编码产物氨基酸序列的多态性
＼＊ 株临床低传代分离株UL136 ORF长度均与TOedo株相
同，为 723hp，预测编码 241个氨基酸的蛋白。
2山NA序列变异均为核耷酸替换，无插人和缺失突变。
3、不同临床分离株UL13。5基因与ToedO株进行同源性比较，
结果在核着酸水平为97．7％－99．3％，氨基酸水平为96．6％－
99．二％。UL136编码蛋白的氨基酸变异率为0．83％－3．3％。
4、氨基酸变异位点多集中于序列中部，两端变异较少。
5、绝大多数氨基酸变异为非保守替代，改变了氨基酸的极性
和电荷。
6、半既氨酸在三个位点存在变异。
三JL136编码蛋白翻译后修饰位点分析结果
翻译后修饰位点散在分布于整个氨基酸序列。大多数HCMV
UL136蛋白翻译后修饰位点在所有分离株中均高度保守，仅三个
位点在一些分离株中存在缺失或新增。
四刀L136编码蛋白性质预测结果
预测UL136编码蛋白分子量约为27．3kD，等电点为7．54－
8．sl，为弱碱性蛋白。7株临床分离株在175－185位之间氨基酸
序列形成一个新的转角，导致 UL136蛋白H级结构预测分为两种
构象。
五、ToledO株及18株临床分离株核着酸及氨基酸序列系统进
化树分析结果
未发现不同临床分离株UL136基因多态性与HCMV先天性
感染不同疾病表现的关系。
本 论
人巨细胞病毒基因组表现极大的遗传多态性，这种株依赖的
多态性可能与HCMV感染的组织细胞嗜性、毒力及免疫逃避机制
·3·
有关。人们曾推测，HCMV在体内广泛的组织分布可能与主要包
膜糖蛋白gB基因的多态性有关。根据gB蛋白基因断裂区PCR
产物的限制性片段长度多态将其分为4个基因型。但目前尚未证
实HCMV感染疾病表现与gB基因型有本质联系。
1999年，MacCormac等的实验结果表明：TOledo株及两株临床
分离株存在与实验室株不同的细胞嗜性，说明决定HCMV感染受
累细胞或器官不同的基因有可能定位于AD169等实验室株缺失
的19个ORF’中。正是这些新基因编码产物在HCMV感染中可能
存在的重要作用，促使我们去研究它们在不同分离株中多态性及
其与致病性的关系。
对48株临床分离株进行了UL136全序列PCR扩增，阳性18
株，阳性率 37．5％。阴性结果如果排除扩增条件的影响，可能为
模板与引物结合区DNA序列不完全互补，亦不能完全排除UL
Objective
Human cytomegalovirus ( HCMV) is the most common pathogen in congenital malformation due to intrauterine infection. HCMV infection can result in a wide variety of clinical manifestations, but the mechanisms remain unclear yet. Host factors such as celluar or humoral immune response might play an important role. On the other hand, genetic variability among different virus strains could influence clinical manifestations of HCMV infections. Recent data suggested that HCMV UL/b'region display considerable genetic polymorphisms in low passage clinical isolates and laboratory strains. The purpose of our study is to investigate UL136 gene polymorphism in low passage clinical isolates and try to find the relationship between the polymorphism and the outcome of congenital CMV infection. We hope our report could establish molecular basis for revealing the function of UL136 protein and pathogenesis of congenital CMV infection.
Materials and methods
1 subject materials
48 low passage clinical strains were isolated from infants treated in our hospital between 1988 and 1993. They ranged in age from 0 to 14 months old. There are 28 strains isolated from patients with Jaundice , 11 strains from Microcephalus and 9 strains from Megacolon. The specimens were obtained through HCMV isolation experiment and stored at -70℃. Strains were passaged less than 10 times. In 2000, these isolates were proven containing detectable HCMV - DNA by u-
sing FQ - PCR. 2N Methods
1) Sample preparation
The supernatant of cultures were digested with identical volume lysis buffer at 100℃ for 15 min. DNA was isolated for use as template.
2) Primer design
According to Toledo sequence, the primers for complete coding region and for identification were designed by using software primer premier 5. 0.
Primers for complete coding region;
Upper primer UL136ca:5' TCGGACATCGAGGAACTCTTG 3' Lower primer UL136cb:5' GTGCCAGTGGTAAGCCAGATA 3' Primers for identification:
Upper primer UL136U1:5' GAATGTCGGCTACGGGTGT 3' Lower primer UL136D1:5' TGTCTCGCCAACTGTCCTG 3' Lower primer UL137D1:5' TCACGCCGATGATGGGTA 3' UL136ca/cb were designed to amplify a full length of UL136 ORF,while UL136U1/D1 were designed to be homologous to the internal region of ORF. These primers were also used for nucleotide sequencing of UL136 gene.
3) PCR amplification of UL136 gene
The amplification conditions were 951 for 4 min,followed by 35 cycles of 961 for 45 sec,551 for Imin, and 72℃ for Imin 30sec, and a final extension at 72℃ for 10 min. PCR products were detected on a 1.5% agerose gel, stained by ethidium bromide. The bands containing the amplified fragments were visualized under UV illumination.
4)Fragement recovery of PCR products
PCR products were separated on a 1. 5% agarose gel. The expected fragment was recovered by using PCR Fragment Recovery Kit according to the manufacturers instructions.
5 ) DNA sequencing
6) Sequence data analysis
Sequence analysis was performed by using programmes DNA-club, Bioedit, GeneDoc, DNAsis and DNAstar.
7) Sequence submission
UL136 open reading frames of 18 HCMV clinical isolates were submitted to GenBank by using program Sequin.
Results
1 PCR amplification result
18 among 48 isolates were amplified successfully and positive rate was 37.5%. Among them, there were 12 Jaundice strains, 4 Micro-cephalus strains and 2 Megacolon strains.
2 The polymorphism of UL136 gene DNA and amino acid sequence
1) The length of UL136 ORF in all 18 clinical isolates was similar to that of Toledo, 723 bp in size. They hade the potential to encode 241 amino acid protein.
2) DNA sequence variations were all nucleotide substitutions and neither insertions nor deletions were detected.
3 ) Alignment comparison revealed that UL136 sequences of various clinical isolates contained 97. 7% -99. 3% of nucleoptide (nt) and 96.6% -99. 1% of amino acid (aa) sequence homologies compared with those of Toledo, respectively. Amino acid variability rate of UL136 protein was 0.83% - 3.3%.
4) Most of amino aci
人巨细胞病毒(Human cytomegalovirus，HCMV)是引起宫内感染进而导致婴儿先天性发育畸形的常见病原。HCMV感染可引起不同的临床表现，其发病机制不清，可能与宿主的体液和细胞免疫状态有关，亦可能与不同临床分离株基因及其编码产物的多态性有关。资料表明，HCMV UL／b’区在临床低传代分离株和实验室株中表现出极大的遗传多态性。本文主要研究该区基因之一UL136在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系，力图为揭示该基因编码产物的功能及先天性HCMV感染的致病机理奠定分子基础。
材料与方法
一、标本来源
48株低传代临床分离株均来自1988-1993年我院住院患儿，年龄(14个月，其中黄疸28株，小头畸形11株，先天性巨结肠9株。标本取自病毒分离实验，-70℃保存。低传代分离株传代少于10次。2000年应用荧光定量PCR方法检测HCMV-DNA，结果均为阳性。
二、实验方法
1、标本处理
取HCMV低传代分离株细胞培养上清液与等量裂解液混匀后，煮沸15分钟，提取DNA，作为扩增模板。
2、引物设计
按照Toledo株序列，应用引物设计软件primer premier5．0设计用于扩增HCMV UL136基因的全序列引物和鉴定引物。
全序列引物
上游引物 UL136ca：5’TCGGACATCGAGGAACTCI’tvi 3’
下游引物UL136Cb：5’GTGCCAGTGGTAAGCCAGATA3’
鉴定引物
上游引物 UL136UI：5’GAATGTCGGCTACGGGTG 3’
下游引物 UL136DI：5’TGTCTCGCCAAC’I’GTCTG 3’
下游引物 UL137DI：5’TCACGCCGATGAGGGTA 3’
3、UL136基因PCR扩增
PCR循环条件如下：预变性95℃，4分钟；96℃变性45秒，
55℃退火1分钟，72℃延伸至分钟30秒书个循环；72℃终延伸，
10分钟。＊5％经漠化已锭染色的琼脂糖凝胶电泳后，紫外检测
仪下观察扩增结果。
4、凝胶回收
应用PCR目的基因片段回收试剂盒对扩增阳性PCR产物进
行凝胶回收，纯化目的DNA片段。
5＊CR阳性扩增产物测序
6、序列分析
应用软件 DNAClub，BioEdit，Genedoc，DNASis，DNAStar完成
序列分析。
7、序列提交
用Setwn软件向 GenBank提交整理后的HCMV UL136 ORF
序歹。
实验结果
一JCR扩增结果
对 48株低传代临床分离株进行 HCMV UL136全序列 PCR扩
增，结果18株阳性，阳性率37．5％，其中黄疽12株，小头畸形4
株，先天性巨结肠2株。
·2·
二月136基因编码区及编码产物氨基酸序列的多态性
＼＊ 株临床低传代分离株UL136 ORF长度均与TOedo株相
同，为 723hp，预测编码 241个氨基酸的蛋白。
2山NA序列变异均为核耷酸替换，无插人和缺失突变。
3、不同临床分离株UL13。5基因与ToedO株进行同源性比较，
结果在核着酸水平为97．7％－99．3％，氨基酸水平为96．6％－
99．二％。UL136编码蛋白的氨基酸变异率为0．83％－3．3％。
4、氨基酸变异位点多集中于序列中部，两端变异较少。
5、绝大多数氨基酸变异为非保守替代，改变了氨基酸的极性
和电荷。
6、半既氨酸在三个位点存在变异。
三JL136编码蛋白翻译后修饰位点分析结果
翻译后修饰位点散在分布于整个氨基酸序列。大多数HCMV
UL136蛋白翻译后修饰位点在所有分离株中均高度保守，仅三个
位点在一些分离株中存在缺失或新增。
四刀L136编码蛋白性质预测结果
预测UL136编码蛋白分子量约为27．3kD，等电点为7．54－
8．sl，为弱碱性蛋白。7株临床分离株在175－185位之间氨基酸
序列形成一个新的转角，导致 UL136蛋白H级结构预测分为两种
构象。
五、ToledO株及18株临床分离株核着酸及氨基酸序列系统进
化树分析结果
未发现不同临床分离株UL136基因多态性与HCMV先天性
感染不同疾病表现的关系。
本 论
人巨细胞病毒基因组表现极大的遗传多态性，这种株依赖的
多态性可能与HCMV感染的组织细胞嗜性、毒力及免疫逃避机制
·3·
有关。人们曾推测，HCMV在体内广泛的组织分布可能与主要包
膜糖蛋白gB基因的多态性有关。根据gB蛋白基因断裂区PCR
产物的限制性片段长度多态将其分为4个基因型。但目前尚未证
实HCMV感染疾病表现与gB基因型有本质联系。
1999年，MacCormac等的实验结果表明：TOledo株及两株临床
分离株存在与实验室株不同的细胞嗜性，说明决定HCMV感染受
累细胞或器官不同的基因有可能定位于AD169等实验室株缺失
的19个ORF’中。正是这些新基因编码产物在HCMV感染中可能
存在的重要作用，促使我们去研究它们在不同分离株中多态性及
其与致病性的关系。
对48株临床分离株进行了UL136全序列PCR扩增，阳性18
株，阳性率 37．5％。阴性结果如果排除扩增条件的影响，可能为
模板与引物结合区DNA序列不完全互补，亦不能完全排除UL
Objective
Human cytomegalovirus ( HCMV) is the most common pathogen in congenital malformation due to intrauterine infection. HCMV infection can result in a wide variety of clinical manifestations, but the mechanisms remain unclear yet. Host factors such as celluar or humoral immune response might play an important role. On the other hand, genetic variability among different virus strains could influence clinical manifestations of HCMV infections. Recent data suggested that HCMV UL/b'region display considerable genetic polymorphisms in low passage clinical isolates and laboratory strains. The purpose of our study is to investigate UL136 gene polymorphism in low passage clinical isolates and try to find the relationship between the polymorphism and the outcome of congenital CMV infection. We hope our report could establish molecular basis for revealing the function of UL136 protein and pathogenesis of congenital CMV infection.
Materials and methods
1 subject materials
48 low passage clinical strains were isolated from infants treated in our hospital between 1988 and 1993. They ranged in age from 0 to 14 months old. There are 28 strains isolated from patients with Jaundice , 11 strains from Microcephalus and 9 strains from Megacolon. The specimens were obtained through HCMV isolation experiment and stored at -70℃. Strains were passaged less than 10 times. In 2000, these isolates were proven containing detectable HCMV - DNA by u-
sing FQ - PCR. 2N Methods
1) Sample preparation
The supernatant of cultures were digested with identical volume lysis buffer at 100℃ for 15 min. DNA was isolated for use as template.
2) Primer design
According to Toledo sequence, the primers for complete coding region and for identification were designed by using software primer premier 5. 0.
Primers for complete coding region;
Upper primer UL136ca:5' TCGGACATCGAGGAACTCTTG 3' Lower primer UL136cb:5' GTGCCAGTGGTAAGCCAGATA 3' Primers for identification:
Upper primer UL136U1:5' GAATGTCGGCTACGGGTGT 3' Lower primer UL136D1:5' TGTCTCGCCAACTGTCCTG 3' Lower primer UL137D1:5' TCACGCCGATGATGGGTA 3' UL136ca/cb were designed to amplify a full length of UL136 ORF,while UL136U1/D1 were designed to be homologous to the internal region of ORF. These primers were also used for nucleotide sequencing of UL136 gene.
3) PCR amplification of UL136 gene
The amplification conditions were 951 for 4 min,followed by 35 cycles of 961 for 45 sec,551 for Imin, and 72℃ for Imin 30sec, and a final extension at 72℃ for 10 min. PCR products were detected on a 1.5% agerose gel, stained by ethidium bromide. The bands containing the amplified fragments were visualized under UV illumination.
4)Fragement recovery of PCR products
PCR products were separated on a 1. 5% agarose gel. The expected fragment was recovered by using PCR Fragment Recovery Kit according to the manufacturers instructions.
5 ) DNA sequencing
6) Sequence data analysis
Sequence analysis was performed by using programmes DNA-club, Bioedit, GeneDoc, DNAsis and DNAstar.
7) Sequence submission
UL136 open reading frames of 18 HCMV clinical isolates were submitted to GenBank by using program Sequin.
Results
1 PCR amplification result
18 among 48 isolates were amplified successfully and positive rate was 37.5%. Among them, there were 12 Jaundice strains, 4 Micro-cephalus strains and 2 Megacolon strains.
2 The polymorphism of UL136 gene DNA and amino acid sequence
1) The length of UL136 ORF in all 18 clinical isolates was similar to that of Toledo, 723 bp in size. They hade the potential to encode 241 amino acid protein.
2) DNA sequence variations were all nucleotide substitutions and neither insertions nor deletions were detected.
3 ) Alignment comparison revealed that UL136 sequences of various clinical isolates contained 97. 7% -99. 3% of nucleoptide (nt) and 96.6% -99. 1% of amino acid (aa) sequence homologies compared with those of Toledo, respectively. Amino acid variability rate of UL136 protein was 0.83% - 3.3%.
4) Most of amino aci
引文
1．阮强，吕绳敏，刘兰青，等．婴儿先天性巨结肠与巨细胞病毒感染相关性的研究．中华微生物和免疫学杂志，1992，12(2)：103-105．
2. Cha TA,Tom E, Kemble GW, et al. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J. virol, 1996,70: 78-83.
3. Brown JM, Kaneshime H, Mocarski ES, et al. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J. infect. Dis, 1995,171:1599-1603.
4. Lurain NS, Kapell KS, Huang DD,et al. Human cytomegalovirus UL144 open reading frame: sequence hytervariability in low-passage clinical isolates. J. Virol, 1999,73: 10040-10050.
5. Bale JF, Jr, Petharam SJ, et al. Human cytomegalovims a sequence and UL144 variability in strains from infected children. J. Med. Virol,2001, 65: 90-96.
6. Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus (CMV)-Encoded Tumor Necrosis Factor-α and β-Chemokine Receptors in Congenital CMV Disease. J. infect. Dis,2002,186:1057-64
7．何蓉，阮强，吉耀华，等．人类巨细胞病毒UL144基因在临床低传代分离株中的多态性研究．中华微生物学和免疫学杂志，2003，23：42-48
8．卢颖，阮强，何蓉，等．人类巨细胞病毒UL145基因在临床低传代分离株中的多态性研究．中华微生物学和免疫学杂志，2003，23：97
9. Penfold ME, Dairghi DJ, Duke GM, et al. Cytomegalovvirus en-
codes a potent alpha chemokine. Proc. Natl. Acad. Sci. USA, 1999,96 : 9839-9844．
10． 何蓉，刘兰清，吕绳敏，等．荧光定量PCR方法检测婴儿尿液 中人类巨细胞病毒基因的含量．中华儿科杂志，2001，39 (12) ：739-742
11． Navarro D, Paz P, Tugizov S et al. Glycoprotein B of human cyto-megalovirus promotes virion penetration into cells, transmission of infection from cell to cell, and fusion of infected cells. Virology. 1993,197: 143
12． Chou SW,Dennison KW. Analysis of interstrain variation in cyto-megalovirus glycoprotein B sequencs encoding neutralization-related epitopes. J. infect. Dis. 1991,163 : 1229-1234．
13． MacCormac LP,Grundy JE. Two clinical isolates and Toledo strain of cytomegalovirus contain endothelial cell tropic variants that are not present in the AD169, Towne or Davis strains. J. Med. Virol, 1999, 57 : 298-307
2. Cha TA,Tom E, Kemble GW, et al. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J. virol, 1996,70: 78-83.
3. Brown JM, Kaneshime H, Mocarski ES, et al. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J. infect. Dis, 1995,171:1599-1603.
4. Lurain NS, Kapell KS, Huang DD,et al. Human cytomegalovirus UL144 open reading frame: sequence hytervariability in low-passage clinical isolates. J. Virol, 1999,73: 10040-10050.
5. Bale JF, Jr, Petharam SJ, et al. Human cytomegalovims a sequence and UL144 variability in strains from infected children. J. Med. Virol,2001, 65: 90-96.
6. Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus (CMV)-Encoded Tumor Necrosis Factor-α and β-Chemokine Receptors in Congenital CMV Disease. J. infect. Dis,2002,186:1057-64
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