恙虫病东方体山东分离株Sta56基因全序列扩增及酶切分析

Amplification and restriction fragment length polymorphism analysis on the complete sequence of Sta56 gene of Orientia tsutsugamushi isolated from Shandong area

目的明确山东恙虫病东方体(Ot)分离株Sta56基因全序列与其他已知的序列之间的遗传变异关系。方法对从山东省费县恙虫病患者、黑线姬鼠、小盾纤恙螨体内分离的3株Ot分离株Sta56基因全序列进行聚合酶链反应(PCR)扩增;选取4种内切酶HinfⅠ、HhaⅠI、HaeⅢ、PstⅠ对PCR扩增产物进行限制性片段长度多态性分析(RFLP);对代表株XDM2株Sta56基因全序列测序,运用Clustal X(5.0)和PHYLIP软件对测得的序列与GenBank中已知的Ot序列构建系统发育树,进行比较分析。结果患者分离株B-16、黑线姬鼠分离株FXS2和小盾纤恙螨分离株XDM2均扩增出接近1.6 kbp的目的条带。Ot山东分离株PCR扩增产物经HhaⅠ、HinfⅠ、HaeⅢ、PstⅠ酶切后图谱一致,但与国际参考株Gilliam、Karp、Kato株的RFLP图谱均不相同;虽与日本地方株Kawasaki株的酶切图谱有相似之处,但存在酶切位点的突变。序列同源性分析结果显示,山东地区代表株XDM2株Sta56基因全序列与Kawasaki型相应的序列同源性最高,为97%,氨基酸序列同源性为92%。结论经Sta56基因全序列分析,Ot山东分离株基因型虽与日本Kawasaki型相似,但也存在差异。

Objective To analyze the genetic differences of Orientia tsutsugamushi （Ot） Sta56 gene between Shandong isolates and other strains deposited in GenBank. Methods PCR and restriction fragment length polymorphism （RFLP） were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot- Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X （1.8） and PHYLIP software. Results The complete sequences （about 1.6 kbp） of Ot-Sta56 gene were amplified from B16 strain （isolated from patients）, FXS2 strain （isolated from A. agrarius ） and XDM2 strain. Four species of restriction endonucleases （ Hha Ⅰ , Hinf Ⅰ , Hae Ⅲ , Pst Ⅰ ） were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97 %, and deduced amino acid sequence was 92 %. Conclusion Data from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.