差异显示逆转录PCR鉴定约氏疟原虫红前期发育相关基因及其特征分析  

Identification of Plasmodium yoelii development-related genes during exo-erythrocytic stage by differential display PCR and analysis on their characterizations

作　　者：张锡林[1] 宋蓓[1] 吕扬[1] 何谐[1] 

机构地区：[1]第三军医大学基础部病原生物学教研室,重庆400038

出　　处：《国际医学寄生虫病杂志》2009年第1期4-9,共6页International JOurnal of Medical Parasitic Diseases

基　　金：国家自然科学基金资助项目（30872366,30371261）

摘　　要：目的寻找约氏疟原虫红前期发育相关基因，进行序列分析并推测其功能。方法分别提取约氏疟原虫感染大鼠（IL）和正常大鼠（UL）肝脏总RNA，根据疟原虫基因A＋T含量高（〉70％）的特点，设计可选择性扩增约氏疟原虫基因的G＋C含量为40％的引物，采用差异显示RT—PCR（differential display RT—PCR，DD—PCR）技术进行扩增，筛选差异片段，经TA克隆后测序并进行序列分析。结果获得6个约氏疟原虫红前期发育相关基因PyHs4、PyHs5、PyHs6、PyHs7、PyHs8、PyHs9；其中PyHs4与约氏疟原虫乙酰葡糖胺磷酸变位酶（phosphoacetylglucosamine mutase，AGM，ID：PY02130）基因具有91％的相似性，PyHs5与约氏疟原虫红细胞膜蛋白3（erythrocyte membrance prontein3，PyEMP3，ID：PY05500）基因具有100％的同源性，PyHs6、PyHs7、PyHs8、PyHs9经Blast序列分析为疟原虫基因，但功能未知。结论应用DD-PCR技术成功扩增了约氏疟原虫红前期发育相关基因，为进一步进行重组表达、功能及疫苗研究奠定了基础。Objective To find the development-related genes of Plasmodium yoelii （P. yoelii） during the exo-erythrocytic stage by sequence analysis and predict their functions. Methods Total RNA was extracted from the liver of normal rats and infected rats followed by differential display RT-PCR（DD-PCR） with the primer containing 40% G ＋ C. The primer designed and synthesized accommodates the high adenine and thymidine rich genome of P. yoelii. The special bands of the PCR products were purified and cloned into plasmid T- vector for sequencing, and then identified by Blast to find the homology sequences. Results Six genes, named PyHs4,PyHs5 .PyHs6.PyHs7,PyHs8,PyHs9, were found in relation to the development of P. yoelii during exo-erythrocytic stage. According to Blastn analysis, in these genes, 91% sequences of PyHs4 were identical to phosphoacetylglucosamine mutase （AGM, PY02130） gene, and PyHs5 were exactly the same gene encoding erythrocyte memberance protein 3 （ EMP3, PY05500 ）. The sequences of PyHs6, PyHs7, PyHs8, PyHs9 were supposed to be hypothetical genes of malaria parasite with unknown functions. Conclusions The developmental genes of P. yoelii during exo-erythrocytic stage have been successfully amplified by DD-PCR technique thus to lay the foundation for the research on expression of the recombinant and development of new vaccine.

关 键 词：疟原虫 约氏 差异显示逆转录聚合酶链反应 红前期 差异基因 

分 类 号：R531.305[医药卫生—内科学] R734.2[医药卫生—临床医学]