蓝氏贾第鞭毛虫表面变异抗原反义mRNA的鉴定  

Identification of antisense mRNA of a variant surface antigen of Giardia lamblia

作　　者：郑文彧[1,2] 冯宪敏[1] 鞠晓红[1] 李瑶[1] 王月华[1] 

机构地区：[1]吉林医药学院病原生物学教研室,吉林吉林132013 [2]吉林市中心医院

出　　处：《中国病原生物学杂志》2014年第8期713-716,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81301450);吉林省科技厅国际合作项目(No.20130413035GH)

摘　　要：目的鉴定编码蓝氏贾第鞭毛虫（简称贾第虫）表面变异抗原（VSP）的正义和反义mRNA。方法采用Trizol法抽提贾第虫单克隆滋养体总RNA,SMART法逆转录合成cDNA。设计合成针对VSPs基因3′端保守区域的特异性引物（内、外引物各1条）,结合SMART引物采用巢式PCR分别扩增得到来源于正义和反义mRNA的cDNA片段。将PCR产物与pGEM-T载体连接并测序,测序结果与GenBank库中的已知序列进行比对。根据测序结果设计特异性引物,以巢式PCR产物为模板,进行正义和反义cDNA的检测。结果巢式PCR显示,VSP正义mRNA和反义mRNA的cDNA均被成功扩增,电泳鉴定扩到产物分别位于0.1~4kb和0.2~2kb。经克隆、测序和序列比对,共获得34个贾第虫VSP编码序列,其中5个来源于正义mRNA的扩增产物,29个来源于反义mRNA的扩增产物,所得VSP编码基因均含有贾第虫VSP mRNA N端变异区和C端特异性保守区。根据5个正义mRNA序列设计的特异性引物在正义mRNA均得到扩增产物,而在反义mRNA仅有4个得到扩增产物。从29个反义mRNA序列中挑选3个设计特异性引物进行PCR,正义mRNA和反义mRNA均得到扩增产物。结论贾第虫VSP基因转录的多个正义mRNA和反义mRNA序列同时存在,该基因存在转录后基因沉默调节机制。Objective To identify the sense and anti-sense mRNA of the variable surface proteins（VSPs）of Giardia lamblia. Methods The total RNA was extracted from cultured monoclonal trophozoites of a G.lamblia C2 isolate with Trizol,and total cDNA was reverse-transcribed from the RNA using the SMART method.Sense and anti-sense VSPs were amplified from total cDNA using nested PCR with primers designed from the 3’-conserved region of VSP and the known 5’terminal or 3’terminal region of cDNA.PCR products and pGEM-T were ligated and the ligation product was sequenced.The resulting sequence was compared to known sequences in GenBank.Specific primers were designed in accordance with the results of sequencing to identify the sense and anti-sense mRNA of VSPs. Results Sequencing indicated that cDNA was successfully amplified from the sense and antisense VSP,and the amplified products ranged from0.1to 4kb in length and from 0.2to 2kb in length,respectively.In total,34 sequences coding for G.lamblia VSPs were obtained;5were from sense mRNA of VSPs and 29 were from antisense mRNA.All 34 sequences contained highly conserved 3’termini and N-termini that varied in length and composition.Most of the sense and antisense mRNA of VSPs was identified except for that of one VSP（snsRNA4）that only exists in sense form when amplified with the primers designed in accordance with the 5sense mRNAs.Three of 29 randomly selected antisense sequences were also identified in both sense and antisense form. Conclusion The coexistence of sense and antisense mRNA of VSPs of G.lamblia as this study noted further confirms the post-transcriptional regulation of VSP expression.

关 键 词：蓝氏贾第鞭毛虫 变异特异性表面蛋白 反义mRNA 正义mRNA 转录后基因沉默机制 

分 类 号：R382.3[医药卫生—医学寄生虫学]