扩展莫尼茨绦虫丝氨酸蛋白酶抑制剂基因serpin部分片段克隆及不同体节mRNA转录水平分析

旨在初步了解丝氨酸蛋白酶抑制剂基因在扩展莫尼茨绦虫生长发育中的作用。采用分子克隆获得扩展莫尼茨绦虫丝氨酸蛋白酶抑制剂serpin基因部分片段,应用MEGA软件对其进行进化分析。同时以beta-Tubulin基因作为内参基因,采用SYBR Green real-time RT-PCR技术检测该基因在扩展莫尼茨绦虫4个不同发育阶段即头节、幼节、成节、孕节中的表达差异,最终克隆获得749bp的serpin基因片段。进化分析表明,扩展莫尼茨绦虫serpin基因与多方棘球蚴serpin基因有较近的亲缘关系。标准曲线分析显示,serpin和beta-Tubulin基因的Ct值与阳性质粒的浓度均呈良好的线性关系,相关系数均大于0.99,熔解曲线分析表明,产物为特异的单峰,具有较高的特异性。同时试验结果显示,serpin基因在虫体各发育阶段中的表达丰度存在差异,从高到低依次为孕节、头节、成节、幼节。由此初步推测,此基因可能在扩展莫尼茨绦虫虫卵发育为六钩蚴的过程中发挥一定作用。

Cloning of Partial serpin Gene and Its mRNA Transcription Analysis in Different Segments of Moniezia expansa

In order to explore the function of serpin gene in development of Moniezia expansa. This study cloned the partial sequence of serpin gene and developed a SYBR Green Real-time fluorescent quantitative PCR assay for detection of serpin mRNA in different segments of Moniezia expansa with beta-Tubulin as an interna control. In this study,749 bp nucleotide fragment of serpin gene was obtained from Moniezia expansa. The encoded protein sequence was similar to serpin of Echinococcus multilocularis using BLASTx searches. The results showed a good linear relationships (R>0.99) between the Ct value and the concentration of positive plasmid for each gene. The melting curve analysis showed the product was specific to a single peak,with high specificity. Real-time PCR results showed that the expression abundance of serpin gene was from high to low in gravid segment,scolex,mature segment and immature segment in order. In conclusion,the author speculate that the serpin gene may play a key role in the process of the oncosphere hatching from the eggs in Moniezia expansa.