炭疽芽孢杆菌MLVA分子分型研究
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摘要
炭疽病是由炭疽芽孢杆菌(Bacillus anthracis)引起的人兽共患传染病,曾多次被恐怖分子用来发动生物恐怖袭击。随着国际上生物战剂的研究发展和恐怖组织的活动频繁,炭疽越来越对人畜构成了威胁,因此建立快速、经济的分子分型方法,对预防和控制炭疽芽孢杆菌的大面积爆发和流行来说显得非常必要。
本研究对我国的炭疽芽孢杆菌进行了可变数目串联重复序列分析(Multiple Locus Variable- Number Tandem Repeats Analysis, MLVA)分析,初步探讨了可变数目串联重复(Variable Number Tandem Repeat, VNTR)位点在炭疽芽孢杆菌中的多态性,以及中国地区的主要流行菌株之间的遗传关系。
本文使用经典的苯酚氯仿方法,对全国各炭疽自然疫源地分离到的198株炭疽芽孢杆菌的基因组DNA进行了提取。利用Tandem Repeats Finder软件进行生物信息学分析后,最终选取了18个VNTR多态性位点。针对不同的VNTR多态性位点两端的基因组序列,设计了特异性荧光标记引物,PCR扩增后通过毛细管电泳检测。通过Genemarker软件确定PCR扩增片段长度,计算每个菌株不同串联重复位点拷贝数。采用Bionumerics软件分析,选用非加权组平均法(UPGMA)对炭疽芽孢杆菌进行聚类分析,研究炭疽芽孢杆菌菌株之间的遗传差异。
结果表明：使用18个串联重复位点进行UPGMA聚类分型,可鉴定出96个基因型。按遗传学距离归纳为2个集群A和B,共分为4个组Al,A2,B 1,B2。A1组可划分为两个亚组A1.a,Ai.b,B1组可划分为两个亚组B1.a，B1.b。B2组也可划分为两个亚组B2.a,B2.b。炭疽芽孢杆菌分布主要集中在A1.b亚群以及B1组和B2组中。A1.b亚群主要分布的基因型为4、18和31,Bl组主要分布的基因型为57、59、68、77和78,B2组主要分布的基因型为84和92。VNTR多态性位点多态性信息含量分析结果显示,Bams1、Bams34、A031、Bams3、VrrC1、Bams30、VrrC2、Bams31八个VNTR多态性位点具有较高的多态性,确定了一个新的VNTR多态性位点A031。选用这八个多态信息含量最高位点进行组合后对炭疽芽孢杆菌进行UPGMA聚类分析,可鉴定出66个基因型。
本论文的初步研究结果将对推动我国炭疽芽孢杆菌的分子分型,保障我国在生物防控领域的监控水平,起到积极的推动作用。
Anthrax is a zoonotic disease caused by Bacillus anthracis, which has been used as biological weapon by terrorists to launch bio-terrorist attacks. With the development of biological weapon and the activities of international terrorist organizations, anthrax is a new developing threat to humans and animals. Therefore it is necessary to establish a rapid, effective and economical method to prevent and control outbreak of B. anthracis diseases. In this research, we analyzed the B. anthracis isolated from China by Multiple Locus Variable- Number Tandem Repeats Analysis (MLVA) in order to reveal the polymorphism of different Variable Number Tandem Repeat (VNTR) locus and the genetic relationship among major epidemic strains in China.
The classic phenol chloroform extraction method was used to obtain the genome of 198 strains of B. anthracis isolated in China.18 VNTR loci were selected after biological information analysis by tandem repeats finder software. Specific fluorescent labeled primers were selected according to the information of 18 VNTR loci. The PCR products were detected by electrophoresis. The length of PCR product of all strains was analyzed by Genemarker software, and thereafter the copies of all strains were calculated. UPGMA cluster analysis method was analyzed to obtain the genetic differences among B. anthracis with the aid of bionumerics analysis software.
The results showed that all B. anthracis strains were classified into 96 genotypes by UPGMA cluster analysis with 18 VNTR loci. All strains were grouped into clusters A and B, and further into group Al, A2, B1 and B2. Al group strains was divided into two subgroups A1.a and A1.b, and B1 group into two subgroups B1.a and B1.b., and B2 group into B2.a and B2.b. The main genotypes of 198 strains were mainly distributed in A1.b, B1 and B2. Al.b subgroups were mainly distributed in the sequence type 4、18 and 31, B1 group was mainly distributed in genotype 57,59,68, 77 and 78, and B2 group was mainly distributed genotype type 84 and 92.
The analysis results of VNTR polymorphism information content showed that eight VNTR Locus, namely, Bams1, Bams34, A031, Bams3, VrrCl, Bams30, VrrC2 and Bams31 presented high polymorphism, thereof A031 was a novel VNTR site. Combined the eight VNTR locus with highest polymorphic information content for UPGMA cluster analysis, B. anthracis was grouped into 66 genotypes.
In conclusion, the preliminary results in this thesis will promote the development of genotyping technology of Bacillus anthracis, thus ensure the level of monitoring and prevention of bio-terrorist in China.
本研究对我国的炭疽芽孢杆菌进行了可变数目串联重复序列分析(Multiple Locus Variable- Number Tandem Repeats Analysis, MLVA)分析,初步探讨了可变数目串联重复(Variable Number Tandem Repeat, VNTR)位点在炭疽芽孢杆菌中的多态性,以及中国地区的主要流行菌株之间的遗传关系。
本文使用经典的苯酚氯仿方法,对全国各炭疽自然疫源地分离到的198株炭疽芽孢杆菌的基因组DNA进行了提取。利用Tandem Repeats Finder软件进行生物信息学分析后,最终选取了18个VNTR多态性位点。针对不同的VNTR多态性位点两端的基因组序列,设计了特异性荧光标记引物,PCR扩增后通过毛细管电泳检测。通过Genemarker软件确定PCR扩增片段长度,计算每个菌株不同串联重复位点拷贝数。采用Bionumerics软件分析,选用非加权组平均法(UPGMA)对炭疽芽孢杆菌进行聚类分析,研究炭疽芽孢杆菌菌株之间的遗传差异。
结果表明：使用18个串联重复位点进行UPGMA聚类分型,可鉴定出96个基因型。按遗传学距离归纳为2个集群A和B,共分为4个组Al,A2,B 1,B2。A1组可划分为两个亚组A1.a,Ai.b,B1组可划分为两个亚组B1.a，B1.b。B2组也可划分为两个亚组B2.a,B2.b。炭疽芽孢杆菌分布主要集中在A1.b亚群以及B1组和B2组中。A1.b亚群主要分布的基因型为4、18和31,Bl组主要分布的基因型为57、59、68、77和78,B2组主要分布的基因型为84和92。VNTR多态性位点多态性信息含量分析结果显示,Bams1、Bams34、A031、Bams3、VrrC1、Bams30、VrrC2、Bams31八个VNTR多态性位点具有较高的多态性,确定了一个新的VNTR多态性位点A031。选用这八个多态信息含量最高位点进行组合后对炭疽芽孢杆菌进行UPGMA聚类分析,可鉴定出66个基因型。
本论文的初步研究结果将对推动我国炭疽芽孢杆菌的分子分型,保障我国在生物防控领域的监控水平,起到积极的推动作用。
Anthrax is a zoonotic disease caused by Bacillus anthracis, which has been used as biological weapon by terrorists to launch bio-terrorist attacks. With the development of biological weapon and the activities of international terrorist organizations, anthrax is a new developing threat to humans and animals. Therefore it is necessary to establish a rapid, effective and economical method to prevent and control outbreak of B. anthracis diseases. In this research, we analyzed the B. anthracis isolated from China by Multiple Locus Variable- Number Tandem Repeats Analysis (MLVA) in order to reveal the polymorphism of different Variable Number Tandem Repeat (VNTR) locus and the genetic relationship among major epidemic strains in China.
The classic phenol chloroform extraction method was used to obtain the genome of 198 strains of B. anthracis isolated in China.18 VNTR loci were selected after biological information analysis by tandem repeats finder software. Specific fluorescent labeled primers were selected according to the information of 18 VNTR loci. The PCR products were detected by electrophoresis. The length of PCR product of all strains was analyzed by Genemarker software, and thereafter the copies of all strains were calculated. UPGMA cluster analysis method was analyzed to obtain the genetic differences among B. anthracis with the aid of bionumerics analysis software.
The results showed that all B. anthracis strains were classified into 96 genotypes by UPGMA cluster analysis with 18 VNTR loci. All strains were grouped into clusters A and B, and further into group Al, A2, B1 and B2. Al group strains was divided into two subgroups A1.a and A1.b, and B1 group into two subgroups B1.a and B1.b., and B2 group into B2.a and B2.b. The main genotypes of 198 strains were mainly distributed in A1.b, B1 and B2. Al.b subgroups were mainly distributed in the sequence type 4、18 and 31, B1 group was mainly distributed in genotype 57,59,68, 77 and 78, and B2 group was mainly distributed genotype type 84 and 92.
The analysis results of VNTR polymorphism information content showed that eight VNTR Locus, namely, Bams1, Bams34, A031, Bams3, VrrCl, Bams30, VrrC2 and Bams31 presented high polymorphism, thereof A031 was a novel VNTR site. Combined the eight VNTR locus with highest polymorphic information content for UPGMA cluster analysis, B. anthracis was grouped into 66 genotypes.
In conclusion, the preliminary results in this thesis will promote the development of genotyping technology of Bacillus anthracis, thus ensure the level of monitoring and prevention of bio-terrorist in China.
引文
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2. Little SF, Webster WM, Fisher DE. Monoclonal antibodies directed against protective antigen of Bacillus anthracis enhance lethal toxin activity in vivo[J]. FEMS Immunol Med Microbiol 2011;62:11-22
3. Gill SC, Rubino CM, Bassett J, et al. Pharmacokinetic-pharmacodynamic assessment of faropenem in a lethal murine Bacillus anthracis inhalation postexposure prophylaxis model[J]. Antimicrob Agents Chemother 2010;54:1678-1683
4. Swartz MN. Recognition and management of anthrax--an update[J]. N Engl J Med 2001;345:1621-1626
5. Cui X, Su J, Li Y, et al. Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model [J]. Intensive Care Med 2010:36:148-156
6. Bolhuis A, Tjalsma H, Stephenson K, et al. Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants[J]. J Biol Chem 1999;274:15865-15868
7. Walsh JJ, Pesik N, Quinn CP, et al. A case of naturally acquired inhalation anthrax:clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor[J]. Clin Infect Dis 2007;44:968-971
8. Fouet A, Mock M. Regulatory networks for virulence and persistence of Bacillus anthracis[J]. Curr Opin Microbiol 2006;9:160-166
9. Sleytr UB. Regular arrays of macromolecules on bacterial cell walls:structure, chemistry, assembly, and function[J]. Int Rev Cytol 1978;53:1-62
10. Smith H. Discovery of the anthrax toxin:the beginning of in vivo studies on pathogenic bacteria[J]. Trends Microbiol 2000;8:199-200
11. Brossier F, Mock M. Toxins of Bacillus anthracis[J]. Toxicon 2001;39:1747-1755
12. Bradley K.A, Mogridge J, Mourez M, Collier RJ, Young JA. Identification of the cellular receptor for anthrax toxin[J]. Nature 2001;414:225-229
13. Pittman PR, Leitman SF, Oro JG, et al. Protective antigen and toxin neutralization antibody patterns in anthrax vaccinees undergoing serial plasmapheresis[J]. Clin Diagn Lab Immunol 2005:12:713-721
14. Park S, Leppla SH. Optimized production and purification of Bacillus anthracis lethal factor[J]. Protein Expr Purif 2000; 18:293-302
15. Makino S, Uchida I, Terakado N, Sasakawa C, Yoshikawa M. Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis[J]. J Bacteriol 1989; 171:722-730
16. Uchida I, Makino S, Sekizaki T, Terakado N. Cross-talk to the genes for Bacillus anthracis capsule synthesis by atxA, the gene encoding the trans-activator of anthrax toxin synthesis[J]. Mol Microbiol 1997;23:1229-1240
17. Muscillo M, La Rosa G, Sali M, et al. Validation of a pXO2-A PCR assay to explore diversity among Italian isolates of Bacillus anthracis strains closely related to the live, attenuated Carbosap vaccine[J]. J Clin Microbiol 2005;43:4758-4765
18. Drum CL, Yan SZ, Bard J, et al. Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin[J]. Nature 2002;415:396-402
19. Marston CK, Hoffmaster AR, Wilson KE, et al. Effects of long-term storage on plasmid stability in Bacillus anthracis[J]. Appl Environ Microbiol 2005;71:7778-7780
20. Mock M, Mignot T. Anthrax toxins and the host:a story of intimacy[J]. Cell Microbiol 2003;5:15-23
21. Holty JE, Kim RY, Bravata DM. Anthrax:a systematic review of atypical presentations[J]. Ann Emerg Med 2006;48:200-211
22. Blackburn JK, McNyset KM, Curtis A, Hugh-Jones ME. Modeling the geographic distribution of Bacillus anthracis, the causative agent of anthrax disease, for the contiguous United States using predictive ecological [corrected] niche modeling[J]. Am J Trop Med Hyg 2007;77:1103-1110
23. Mongoh MN, Dyer NW, Stoltenow CL, Khaitsa ML. Risk factors associated with anthrax outbreak in animals in North Dakota,2005:a retrospective case-control study[J]. Public Health Rep 2008;123:352-359
24. Jernigan JA, Stephens DS, Ashford DA, et al. Bioterrorism-related inhalational anthrax:the first 10 cases reported in the United States[J]. Emerg Infect Dis 2001;7:933-944
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