中国4个弓形虫虫株GRA6基因的比较分析

 通过对国内来源于不同宿主的ZS人株、SH人株、CN猪株、QH绵羊株4个弓形虫虫株，以及国际标准强毒株RH株的致密颗粒抗原（GRA6）基因进行PCR-RFLP分析，首次对中国弓形虫虫株进行了基因分型研究，旨在了解中国弓形虫基因型的分布情况，从而为进一步的分子遗传学研究以及弓形虫病的防制提供资料。PCR-RFLP分析显示两种带型：RH、SH、CN 3株弓形虫为一种带型，QH和ZS 2株弓形虫为另一种电泳带型。GRA6序列分析结果显示：RH、SH、CN 3株弓形虫的GRA6基因序列上的MseΙ酶切位点与国外报道的RH株一致，同属于基因型Ι，为强毒株；QH和ZS 2株弓形虫的MseΙ酶切位点与国外报道的弱毒株BEVERLEY株和ME49株一致，同属于基因型Ⅱ，与PCR-RFLP分析结果一致。除MseΙ酶切位点外，中国的几个虫株GRA6序列与GenBankTM注册的RH株及BEVERLEY和ME49两个弱毒株有个别碱基的差异。结果证明中国人源弓形虫包含了Ι、Ⅱ两种基因型，而动物源性弓形虫则因宿主和来源地不同亦有Ι、Ⅱ两个基因型。

Comparative Studies on GRA6 Gene of Toxoplasma gondii Isolates from China

Toxoplasma gondii is a parasitic protozoan infecting a variety of hosts with a worldwide distribution. The objective of the present project is to study the genotypes of T. gondii strains from different hosts and geographical locations in China employing the technique of PCR-linked restriction fragment length polymorphism (PCR-RFLP) utilizing the dense granule antigen GRA6 gene as genetic markers. Partial GRA6 sequence was amplified from strains ZS (human), SH (human), CN (pig), QH (sheep) and RH of T. gondii, the amplicons were digested with restriction enzyme Mse I, and then the digestion fragments were separated by agarose gel electrophoresis. Two different restriction profiles were observed among the five strains, with strains RH, SH and CN having identical banding patterns, whereas strains QH and ZS having a different banding pattern. Sequence analyses revealed that strains RH, SH and CN had almost identical GRA6 sequences and the same locations for two Mse I restriction sites, whereas strains QH and ZS had another type, almost identical sequences and the same locations for two Mse I restriction sites, consistent with results of PCR-RFLP analysis. Based on the results of PCR-RFLP and sequence comparison, strains RH, SH and CN were considered to represent genotype I of T. gondii, whereas strains QH and ZS represent genotype II of T. gondii, consistent with their virulence in mice. The results indicated that at least two different genotypes of T. gondii were present in China. These findings should have important implications for studying the molecular epidemiology, molecular ecology and population genetic structures of T. gondii strains from different hosts and geographic locations in China and elsewhere.