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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 antisense mRNAs encoding surface variant antigen (VSP) of Giardia lamblia (Giardia lamblia). Methods The Trizol method was used to extract the total RNA of Giardia monocytogenes and the cDNA was reverse transcribed by SMART method. Design and synthesize specific primers (one for each of the inner and outer primers) for the conserved region of 3 ’end of VSPs gene, amplify the cDNA fragments derived from sense and antisense mRNA by nested PCR combined with SMART primer. The PCR product was ligated to the pGEM-T vector and sequenced, and the sequencing results were aligned with known sequences in the GenBank library. Specific primers were designed according to the sequencing results, and nested PCR products were used as templates to detect the sense and antisense cDNAs. Results The results of nested PCR showed that both the mRNA of VSP and the cDNA of antisense mRNA were successfully amplified, and the products amplified by electrophoresis were located at 0.1-4 kb and 0.2-2 kb, respectively. A total of 34 Giardia VSP coding sequences were obtained by cloning, sequencing and sequence alignment. Among them, 5 were derived from the amplification product of sense mRNA and 29 from the antisense mRNA. The obtained VSP coding genes contained Giardia VSP mRNA N-terminal and C-terminal specific conserved regions. The specific primers designed according to the five sense mRNA sequences all obtain the amplification products in the sense mRNA, while only four of the antisense mRNAs give the amplification products. Three design-specific primers were selected from 29 antisense mRNA sequences for PCR, and both the sense and antisense mRNAs were amplified. Conclusions There are many positive mRNAs and antisense mRNAs of VSP gene in Giardia virus that exist simultaneously. The gene has the regulatory mechanism of post-transcriptional gene silencing.