基因芯片用于鼠疫耶尔森菌检测的研究
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摘要
鼠疫是由鼠疫耶尔森菌所致的烈性传染病，曾在人类历史上三次大流行，并造成约2亿人死亡，全世界为之战栗。过去几十年鼠疫在全球范围内的流行都已经得到有效的控制，但近年陆续又有一些自然疫源地复燃和新自然疫源地出现，以及可能被生物恐怖分子用作武器，使其仍然是公众健康安全的隐患。因而我们需用分子生物学手段对该疾病重新认识，并在快速诊断和疫苗研制上取得了新的突破，才能从根本上遏制住它的再次大流行，现在利用基因芯片技术对鼠疫菌做进一步的研究仍具有现实意义。
基因芯片技术是上世纪90年代兴起，源于计算机集成化芯片理念的一门新技术。实质就是利用Southern blot原理，以可寻址的方式在载体表面，有序地点阵排列大量DNA双链或Oligo探针。这些被固定在基质的探针上就形成了高密度DNA微阵列。样品DNA或RNA经过荧光标记，与阵列上的探针进行杂交反应，按照碱基互补配对原则，探针特异性地结合相应被荧光标记的分子。用激光激发荧光标记物，就可获得样品分子的信息，从而对基因序列及功能进行大规模、高通量、平行地研究。
鼠疫耶尔森菌含有3个公认与致病性有关的质粒，分别是9.5kb、70kb和110kb，已知的序列提示它们的开放阅读框编码密度很高在70％以上，并且集中了多数已知的鼠疫致病性基因，我们直接利用其限制性片段，采用我们建立的巢式PCR方法进行扩增，制备出供芯片打印的探针，并结合马文丽、郑文岭等建立的限制性显示(Restriction Display，简称为RD)技术标记样品用于鼠疫的检测。
通过我们对芯片的杂交条件改进，取消标记样品的纯化过程，提高杂交温度从42℃至52℃和在低严谨洗脱之前先使用蒸馏水冲洗，可有效提高DNA芯片的杂交阳性信号，降低背景和非特异性干扰。提高了检测芯片的信噪比，使其特异性显著提高，获得可信的杂交结果。基因芯片结合RD技术，既快速又能在标记过程中实现样品扩增，发挥基因芯片高度灵敏、准确和简便快速的优点。同时平行检测多个基因，这对鼠疫这种烈性传染病的诊断提供新的科学方法。
进行Blast分析，确定出鼠疫菌最为特异的序列，用以设计成60mers
的寡核营酸探针。固定探针在用多聚一L一赖氨酸处理过的玻片上，制备
成寡核昔酸基因芯片，同样采用RD技术进行样品标记，用以检测。通
过我们对杂交条件的改进，能更好的降低非特异性杂交，结果显示，该
芯片具有很好的特异性。
同时，我们将0119。芯片用于比较鼠疫活疫苗菌株Ev和分离自我国
云南的野生株之间己知毒性基因的表达差异，采用随机引物标记方法，
在逆转录合成第一链cDNA过程中，分别掺入cy3/C ys一dCTP，并与芯
片杂交，经比较分析，我们发现分离自我国云南的野生株的cafl和caf]R
基因有表达下调的趋势，EV株存在卢宕脚基因缺失，在分析结果中，也
被验证。
为了确定野生株的‘叨和caflR基因的表达弱于EV株原因，我们
将分离自中国云南的两株菌1351和482连同对照的EV株，采用LA PCR
将覆盖Cafj操纵子的整个区域扩增，再以Sau3AI不完全消化，制备
克隆文库，用3730测序仪测定后，拼接成完整序列，经Blast分析，该
段序列在3株菌之间没有差异，与Genebank上的CO92和KIM株的这
段序列没有差别，显示这段序列对鼠疫菌是非常保守。
综上所述，基因芯片技术用于鼠疫耶尔森菌研究，不仅为鼠疫的快
速诊断提供了新的方法，在其表达谱分析中，也在野生株和EV株之间
确定了毒性基因的表达差异。通过对cafl操纵子序列的测定，明确了该
段序列的保守性。同时本文的工作对芯片的技术的改进提供了新的手
段，进一步拓展了基因芯片在传染性疾病诊断中的应用范围。
Yersinia pestis is the cause of the severe infectious disease, also named as plague, which has been responsible for three worldwide pandemics in the human history. Plague initially out-brook in the 6th century, which was collectively responsible for the loss of 200 million lives. Although plague pandemic has been controlled throughout the world, it is reported that there are still many endemic areas and some sporadic new endemic areas. The potential use of Yersinia pestis as an agent of biological weapon by bioterrorist means that plague still poses threat to human society. In order to fully control the plague pandemic's reoccurrence, the rapid diagnostic method and novel plague vaccine should be developed. It is indispensable to make a further diagnostic study to Yersinia pestis with DNA chip technology.
DNA chip technology was first described in the late 1990s. The novel technique was originated from the idea of making integrated computer chip for parallel detection of biological agents. Based on principle of the Southern blot, the DNA fragments or oligonucleotide probes were deposited on the surface of substrate in parallel based on addressable approaches. The high density DNA microarray is formed by these probes fixed on the substrate. The DNA or RNA could be extracted from samples of various origins and labeled with fluorescent dyes. The labeled materials could hybridize with the DNA array according to the strict complementary nature of the base pairs, and the probes specifically bind with fluorescent-labeled samples in a form of double helix. The information of the double helix molecules in the DNA chips is acquired through the laser excitation and scanning. In this manner, the genetic information of the biological samples can be studied with high quantity, high throughput and in parallel.
Yersinia pestis carries three plasmids associated with its pathogenicity: 9.5kb, 70kb and 110kb. The number of recognized ORF in plasmids
sequence shows that the coding density is more than 70%, and most of the pathogenic genes specific to Yersinia pestis are located on these three plasmids. DNA probes of restricted fragments of the plasmids were prepared by nested PCR for the preparation of the Yersinia pestis DNA chips. The DNA samples were labeled fluorescent dye via the RD-PCR technique invented by Wenli Ma and Wenling Zheng.
Improvement of hybridization protocol, such as cancelled purification of labeled samples, increased reaction temperature from 42 to 52and added rinsing steps with distilled water prior to low stringency washing at room temperature, have enhanced the sensitivities of plague diagnostic microarray and effectively kept the lower cross-hybridization and background of hybridized arrays. The modified hybridization protocol improved further the signal-to-noise ratio of hybridized arrays, yielding high quality hybridization signals. DNA chip in combination with RD-PCR technique, by which the samples can be amplified during the labeling process. In addition its high efficiency, high sensitivity, accuracy and expediency are achieved, while multiple genes can be tested simultaneously. Evidently, the DNA chip technology provides a new and scientific approach for the diagnosis of the severe infectious disease.
Since the whole genome of Yersinia pestis has been sequenced, we chose the major known pathogenic genes and analyzed their specificity to Yersinia pestis with Blast program. Highly specific gene fragments were adopted to design probes with 60mers in length. All synthesized probes were printed on ploy-L-Lysine coated slides to prepare an oligo chip. RD-PCR technique was also used to label DNA sample as described above. The non-specific hybridization signals decreased with the improvement of hybridization efficiency. The results showed that oligo chip was able to obtain even higher signal-to-noise ratio.
Furthermore, Oligo chip was applied to compare the expression profiles of the known pathogenic genes specific to Yersinia pestis between EV strain
and wild strains isolated from Yunnan province, China
基因芯片技术是上世纪90年代兴起，源于计算机集成化芯片理念的一门新技术。实质就是利用Southern blot原理，以可寻址的方式在载体表面，有序地点阵排列大量DNA双链或Oligo探针。这些被固定在基质的探针上就形成了高密度DNA微阵列。样品DNA或RNA经过荧光标记，与阵列上的探针进行杂交反应，按照碱基互补配对原则，探针特异性地结合相应被荧光标记的分子。用激光激发荧光标记物，就可获得样品分子的信息，从而对基因序列及功能进行大规模、高通量、平行地研究。
鼠疫耶尔森菌含有3个公认与致病性有关的质粒，分别是9.5kb、70kb和110kb，已知的序列提示它们的开放阅读框编码密度很高在70％以上，并且集中了多数已知的鼠疫致病性基因，我们直接利用其限制性片段，采用我们建立的巢式PCR方法进行扩增，制备出供芯片打印的探针，并结合马文丽、郑文岭等建立的限制性显示(Restriction Display，简称为RD)技术标记样品用于鼠疫的检测。
通过我们对芯片的杂交条件改进，取消标记样品的纯化过程，提高杂交温度从42℃至52℃和在低严谨洗脱之前先使用蒸馏水冲洗，可有效提高DNA芯片的杂交阳性信号，降低背景和非特异性干扰。提高了检测芯片的信噪比，使其特异性显著提高，获得可信的杂交结果。基因芯片结合RD技术，既快速又能在标记过程中实现样品扩增，发挥基因芯片高度灵敏、准确和简便快速的优点。同时平行检测多个基因，这对鼠疫这种烈性传染病的诊断提供新的科学方法。
进行Blast分析，确定出鼠疫菌最为特异的序列，用以设计成60mers
的寡核营酸探针。固定探针在用多聚一L一赖氨酸处理过的玻片上，制备
成寡核昔酸基因芯片，同样采用RD技术进行样品标记，用以检测。通
过我们对杂交条件的改进，能更好的降低非特异性杂交，结果显示，该
芯片具有很好的特异性。
同时，我们将0119。芯片用于比较鼠疫活疫苗菌株Ev和分离自我国
云南的野生株之间己知毒性基因的表达差异，采用随机引物标记方法，
在逆转录合成第一链cDNA过程中，分别掺入cy3/C ys一dCTP，并与芯
片杂交，经比较分析，我们发现分离自我国云南的野生株的cafl和caf]R
基因有表达下调的趋势，EV株存在卢宕脚基因缺失，在分析结果中，也
被验证。
为了确定野生株的‘叨和caflR基因的表达弱于EV株原因，我们
将分离自中国云南的两株菌1351和482连同对照的EV株，采用LA PCR
将覆盖Cafj操纵子的整个区域扩增，再以Sau3AI不完全消化，制备
克隆文库，用3730测序仪测定后，拼接成完整序列，经Blast分析，该
段序列在3株菌之间没有差异，与Genebank上的CO92和KIM株的这
段序列没有差别，显示这段序列对鼠疫菌是非常保守。
综上所述，基因芯片技术用于鼠疫耶尔森菌研究，不仅为鼠疫的快
速诊断提供了新的方法，在其表达谱分析中，也在野生株和EV株之间
确定了毒性基因的表达差异。通过对cafl操纵子序列的测定，明确了该
段序列的保守性。同时本文的工作对芯片的技术的改进提供了新的手
段，进一步拓展了基因芯片在传染性疾病诊断中的应用范围。
Yersinia pestis is the cause of the severe infectious disease, also named as plague, which has been responsible for three worldwide pandemics in the human history. Plague initially out-brook in the 6th century, which was collectively responsible for the loss of 200 million lives. Although plague pandemic has been controlled throughout the world, it is reported that there are still many endemic areas and some sporadic new endemic areas. The potential use of Yersinia pestis as an agent of biological weapon by bioterrorist means that plague still poses threat to human society. In order to fully control the plague pandemic's reoccurrence, the rapid diagnostic method and novel plague vaccine should be developed. It is indispensable to make a further diagnostic study to Yersinia pestis with DNA chip technology.
DNA chip technology was first described in the late 1990s. The novel technique was originated from the idea of making integrated computer chip for parallel detection of biological agents. Based on principle of the Southern blot, the DNA fragments or oligonucleotide probes were deposited on the surface of substrate in parallel based on addressable approaches. The high density DNA microarray is formed by these probes fixed on the substrate. The DNA or RNA could be extracted from samples of various origins and labeled with fluorescent dyes. The labeled materials could hybridize with the DNA array according to the strict complementary nature of the base pairs, and the probes specifically bind with fluorescent-labeled samples in a form of double helix. The information of the double helix molecules in the DNA chips is acquired through the laser excitation and scanning. In this manner, the genetic information of the biological samples can be studied with high quantity, high throughput and in parallel.
Yersinia pestis carries three plasmids associated with its pathogenicity: 9.5kb, 70kb and 110kb. The number of recognized ORF in plasmids
sequence shows that the coding density is more than 70%, and most of the pathogenic genes specific to Yersinia pestis are located on these three plasmids. DNA probes of restricted fragments of the plasmids were prepared by nested PCR for the preparation of the Yersinia pestis DNA chips. The DNA samples were labeled fluorescent dye via the RD-PCR technique invented by Wenli Ma and Wenling Zheng.
Improvement of hybridization protocol, such as cancelled purification of labeled samples, increased reaction temperature from 42 to 52and added rinsing steps with distilled water prior to low stringency washing at room temperature, have enhanced the sensitivities of plague diagnostic microarray and effectively kept the lower cross-hybridization and background of hybridized arrays. The modified hybridization protocol improved further the signal-to-noise ratio of hybridized arrays, yielding high quality hybridization signals. DNA chip in combination with RD-PCR technique, by which the samples can be amplified during the labeling process. In addition its high efficiency, high sensitivity, accuracy and expediency are achieved, while multiple genes can be tested simultaneously. Evidently, the DNA chip technology provides a new and scientific approach for the diagnosis of the severe infectious disease.
Since the whole genome of Yersinia pestis has been sequenced, we chose the major known pathogenic genes and analyzed their specificity to Yersinia pestis with Blast program. Highly specific gene fragments were adopted to design probes with 60mers in length. All synthesized probes were printed on ploy-L-Lysine coated slides to prepare an oligo chip. RD-PCR technique was also used to label DNA sample as described above. The non-specific hybridization signals decreased with the improvement of hybridization efficiency. The results showed that oligo chip was able to obtain even higher signal-to-noise ratio.
Furthermore, Oligo chip was applied to compare the expression profiles of the known pathogenic genes specific to Yersinia pestis between EV strain
and wild strains isolated from Yunnan province, China
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64．吴清华，马文丽，石嵘等 基因芯片中60mer寡核苷酸SARS冠状病毒探针的制备广东医学2003 24(S1)：7-10
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66．祝骥，马文丽，李凌，等一种限制性cDNA文库的构建．遗传，2002；24(2)：174-6
67．李凌，马文丽，祝骥，等．应用RD-PCR技术制备HIV基因芯片探针．中国生物化学与分子生物学报，2002，18(1)：105-9
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76．奥斯伯F，布伦特R，金斯顿RE等著．精编分子生物学实验指南．颜子颖，王海林译，金东雁校 北京：科学出版社，1998
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64．吴清华，马文丽，石嵘等 基因芯片中60mer寡核苷酸SARS冠状病毒探针的制备广东医学2003 24(S1)：7-10
65. Hoyt PR, Tack L, Jones BH, et al. Automated high-throughput probe production for DNA microarray analysis. Biotechniques. 2003; 34(2):402-7
66．祝骥，马文丽，李凌，等一种限制性cDNA文库的构建．遗传，2002；24(2)：174-6
67．李凌，马文丽，祝骥，等．应用RD-PCR技术制备HIV基因芯片探针．中国生物化学与分子生物学报，2002，18(1)：105-9
68. Gerhold D, Rushrnore T, Caskey CT. DNA chips: promising toys have become powerful tools. Trends Biochem Sci. 1999; 24(5): 168-73.
69．黄海，马文丽，王洪敏，等．用巢式PCR方法制备基因芯片探针生命科学研究2003：7(3)：271-4
70. Hinnebusch J, Schman G. new method for plague surveillance using
polymerase chain reaction to detect yersinia pestis in fleas. J. chin Microbiol. 1993; 31(6): 1511
71. Neubacer H, Meyer, H, Prior J, et al. a combination of different polymerase chain reaction (PCR) assays for the presumptive identification of yersinia pestis. J. Vet Med. 2000; 47(8):573-8
72. Tsukano H, Itoh K, Suzukl S, et al. Detection and identification of yersinia pestis by polymerase chain reaction (PCR) using multiplex primers. Microbiol Immunol. 1996; 40(10): 773-6
73．萨姆布鲁克，弗里奇，曼尼阿蒂斯等著．分子克隆实验指南．金冬雁，黎孟枫等译．第二版．北京：科学出版社，1992
74. Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 1997; 25(17): 3389-402
75. Rakin A, Noelting C, Schropp P, et al. Integrative module of the high-pathogenicity island of Yersinia. Mol Microbiol. 2001,39(2): 407-415.
76．奥斯伯F，布伦特R，金斯顿RE等著．精编分子生物学实验指南．颜子颖，王海林译，金东雁校 北京：科学出版社，1998
77. Tomiuk S, Hofmann K. Microarray probe selection strategies. Brief Bioinform 2001; 2(4): 329-40
78. Chizhikov V, Wagner M, Ivshina A, et al. Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization. J Clin Microbiol. 2002; 40(7): 2398-407
79. Kane MD, Jatkoe TA, Stumpf CR, et al. Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Res. 2000; 28(22): 4552-7
80. Russell P, Eley SM, Hibbs SE, et al. A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a routine model. Vaccine. 1995; 13(16): 1551-6
81. Byrne WR, Welkos SL, Pitt ML, et al. Antibiotic treatment of experimental pneumonic plague in mice. Antimicrob Agents Chemother 1998: 42:675-81
82．赵寿元 主编 英汉遗传工程词典(增订版)上海：复旦大学出版社
1999: 297
83. Karlyshev AV, Oyston PC, Williams K, et al. Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes, Infect Immun. 2001; 69(12):7810-9.
84. Hinnebusch BJ, Rudolph AE, Cherepanov P, et al. Role of Yersinia Murine Toxin in Survival of Yersinia pestis in the Midgut of the Flea Vector. Science 2002; 296:733-5
85. Zavyalov VP, Chemovskaya TV, Chapman DA, et al. Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone CaflM of Yersinia pestis. Biochem J 1997; 324 : 571-8.
86. Titball RW, Howells AM, Oyston PC, et al. Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague. Infect Immun 1997;65: 1926-30.