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目的 查找猬迭宫绦虫幼虫裂头蚴阶段特异性表达基因。 方法 裂头蚴和成虫组织用异硫氢酸胍一步法提取总 RNA,用 DNA酶去除总 RNA中污染的 DNA。使用 T1 2 MA、T1 2 MC、T1 2 MG和 T1 2 MT4种锚定引物反转录合成 c DNA,再用 1种随机引物与上述 4种锚定引物在含同位素的反应液中进行 PCR反应。将 PCR产物用变性聚丙烯酰胺凝胶电泳分离 ,经放射自显影后 ,从凝胶上选出裂头蚴与成虫不同的差异带 ,PCR扩增后经杂交试验鉴定出不同种类的基因片段 ;以筛选出的差异带作探针 ,分别与裂头蚴和成虫 RNA进行 Northern杂交证实裂头蚴阶段表达基因 ;将差异带测序后与 Gen Bank中的序列进行同源性比较。 结果 从凝胶中共选出 11条差异带。将回收的 11条带再经PCR扩增和杂交试验 ,从中选出 3种不同的基因片段。经 Northern杂交证实片段 1和片段 2为裂头蚴特异表达基因 ,而片段 3为裂头蚴和成虫共同表达的基因。3个基因片段测序后与 Gen Bank中的基因进行同源性分析 ,基因片断 1和 2无同源序列 ;基因片段 3与多种生物的 2 8S r RNA同源。 结论 通过 m RNA差异显示技术查找出 2个裂头蚴阶段特异性表达基因片段
Objective To search for the stage-specific expression of splenicciferae in rhesus metacercaria larvae. Methods Total RNA was extracted by one-step method using guanidine isothiocyanate from the sporozoite and adult tissues. DNase was used to remove the contaminated DNA from the total RNA. Reverse transcription of c DNA using 4 kinds of anchor primers of T1 2 MA, T1 2 MC, T1 2 MG and T1 2 MT was performed to perform PCR reaction in the isotope-containing reaction solution with one kind of random primers and the above four kinds of anchor primers . The PCR products were separated by denaturing polyacrylamide gel electrophoresis. After autoradiography, different bands were selected from the gel, and different types of genes were identified by hybridization after PCR amplification. The difference bands were used as probes. Northern blotting was carried out with the RNA of the sporozoite and adult respectively to confirm the expression of the gene in the stage of the sporozoite. The sequence of GenBank was compared with the sequence of GenBank. Results A total of 11 differential bands were selected from the gel. The 11 bands recovered were then subjected to PCR amplification and hybridization to select three different gene fragments. Northern blotting confirmed that fragment 1 and fragment 2 were specifically expressed by the sporozoite, whereas fragment 3 was a gene co-expressed by both the sporozoite and the adult. Three gene fragments were sequenced and analyzed in GenBank for homology analysis. Gene fragments 1 and 2 have no homologous sequences. Gene fragment 3 is homologous to 2 8S rRNA of various organisms. Conclusion Two mtDNA-specific gene fragments were found by m RNA differential display