广东地区梅毒螺旋体临床株的分离传代培养及非同义单核苷酸多态性检测

目的 分离培养广东地区梅毒螺旋体（Tp）临床株（GD1）并探讨其与Nichols标准株非同义单核苷酸多态性（nsSNPs）的差异。方法 从广东地区1例硬下疳患者皮损中分离出GD1，在兔睾丸中连续传代培养。暗视野显微镜、TP0548基因PCR、测序和基因分型多重验证传代情况。泛影葡胺梯度离心法分离兔组织并浓缩GD1。测序验证TP0443和TP0584基因nsSNPs位点。结果 本研究分离培养出的GD1株为TP0548 f亚型。与美国Nichols株比较，GD1株存在nsSNPs突变，位于TP0443的突变导致编码的第120位氨基酸由苏氨酸变为丙氨酸，TP0584的突变导致编码的第314位氨基酸由丙氨酸变为苏氨酸。结论 首次成功地从广东地区梅毒患者皮损中分离培养出GD1株Tp，并从病原学水平证实GD1存在nsSNPs突变。

Isolation,passage and culture of one clinical strain of Treponema pallidum in Guangdong province and the detection of its nonsynonymous single nucleotide polymorphisms

Ke Wujian, Huang Tao, Zhang Jun, Lyu Ping, Xue Yaohua, Zhang Xiaohui, Wang Liuyuan, Liu Yahui, Zheng HepingDermatology Hospital of Southern Medical University, Guangzhou 510009, ChinaCorresponding author: Zheng Heping, Email: zhhpf@hotmail.com【Abstract】 Objective To isolate and culture a clinical strain （GD1） of Treponema pallidum （Tp） in Guangdong province, and to investigate the difference in nonsynonymous single nucleotide polymor-phisms （nsSNPs） between the GD1 strain and Tp Nichols strain. Methods The GD1 strain was isolated from the hard chancre in a patient with primary syphilis in Guangdong province, and continuously subcultured in the testes of New Zealand white rabbit. The serial subcultivation of GD1 was multi-verified by dark-field microscopy, polymerase chain reaction （PCR） for TP0548 gene, DNA sequencing and genotyping. Meglumine diatrizoate density gradient centrifugation was performed to isolate rabbit tissues and concentrate GD1, and DNA sequencing was used to verify the nsSNPs in the TP0443 and TP0584 genes. Results The GD1 strain was successfully isolated from the lesions of the patient with syphilis, and classified as a subtype f of TP0548. Compared with the American Tp （Nichols strain）, there were nsSNP mutations in the GD1 strain. One mutation was located in the TP0443 gene, leading to the the substitution of threonine by alanine at amino acid position 120, and another one was located in the TP0584 gene, which caused a change from alanine to threonine at amino acid position 314. Conclusion The GD1 strain was successfully isolated firstly from the lesions in a patient with syphilis in Guangdong province, and nsSNP mutations were confirmed in the GD1 strain on the etiology.