中国弓形虫虫株529bp重复序列的PCR扩增、克隆及分析

 【目的】首次对中国来源于不同宿主、不同地域的9个弓形虫虫株(ZS1人株、PY猪株、GY猪株、ZC猪株、NT猪株、ZS人株、SH人株、CN猪株、QHO绵羊株)以及国际标准强毒RH株之间在529 bp重复序列的变异进行研究，从而为进一步的分子诊断和分子遗传学研究以及弓形虫病的防制奠定基础。【方法】抽提基因组DNA后，用PCR方法对10个虫株的529 bp重复序列进行扩增；扩增产物经纯化后克隆于pGEM-T Easy质粒载体，再经菌落PCR及酶切鉴定阳性克隆，然后对阳性克隆进行测序及序列分析。【结果】10个弓形虫虫株的529 bp重复序列都不完全相同，它们之间的核苷酸序列变异范围为0.8%～2.9%。变异主要位于第32～55位碱基之间,这些碱基变异与虫株的宿主来源、地域来源及毒力之间没有相关性。【结论】529 bp重复序列可作为遗传标记，用于弓形虫与其它寄生虫的种间鉴定，但不适合用于研究弓形虫的种内遗传变异。

Amplification, Cloning and Sequence Analysis of a Repetitive 529bp DNA Fragment from Toxoplasma gondii Strains from China

Abstract: 【Objective】The objective of the present study was to examine sequence variation in the repetitive 529 bp DNA fragment among 9 T. gondii strains from different hosts and geographical locations in China, and to compare the sequences with that of the RH reference strain. 【Method】 The 529 bp fragment was amplified by PCR from genomic DNA of the 10 T. gondii strains, and the amplicons were purified, cloned into pGEM-T Easy vector and the recombinant plasmids were identified by colony PCR and digestion with endonuclease EcoR I, and then sequenced. 【Results】 Sequence variation in the repetitive 529 bp DNA fragment ranged between 0.8-2.9% among the 10 T. gondii strains, with sequence positions of 32-55 nucleotides being the most variable region. But the sequence variation was not related to virulence. 【Conclusion】The findings of the present study showed that the intraspecific variation in the repetitive DNA fragment was low and this repetitive DNA fragment provides an ideal genetic marker for the differentiation of T. gondii from other organisms, but it is not suitable for the studies of genetic variability within T. gondii.