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目的构建广州管圆线虫天冬氨酰蛋白酶(Asp-1)基因的原核表达系统,研究该基因在各虫期的转录水平。方法构建重组质粒pET-30a(+)-Asp-1,并转化至大肠杆菌BL21(DE3)表达载体,经IPTG诱导表达后,Ni-IDA亲和层析纯化表达产物。通过以β-肌动蛋白为内参、以各期虫cDNA为模板的实时荧光定量PCR,研究该基因在各虫期的表达水平。结果获得纯化的重组蛋白,相对分子质量约为43000,与生物信息学分析预测结果一致。qRT-PCR显示该基因在三期幼虫中表达丰度最高,四/五期幼虫、成虫雄虫、雌虫依次降低。结论 Asp-1基因的表达丰度在三期幼虫中最高的实验数据与三期幼虫感染性最强的事实相符,提示天冬氨酰蛋白酶有可能是幼虫入侵宿主的关键酶,为后续广州管圆线虫虫体侵入宿主的机制以及宿主保护机制研究打下基础。
Objective To construct a prokaryotic expression system of Asp-1 gene of Aphis gossypius, and to study the transcriptional level of Asp-1 at each stage. Methods The recombinant plasmid pET-30a (+) - Asp-1 was constructed and transformed into Escherichia coli BL21 (DE3) expression vector. After induced by IPTG, the expressed product was purified by Ni-IDA affinity chromatography. By using β-actin as internal reference, the expression of each gene was analyzed by real-time quantitative PCR using cDNA of each stage as template. Results The purified recombinant protein was obtained. The relative molecular mass was about 43000, which was consistent with the prediction by bioinformatics analysis. qRT-PCR showed that the gene was most abundant in the third stage larvae, while the fourth and fifth larvae, the adult male and the female decreased in turn. Conclusion The highest level of Asp-1 gene expression in the third stage larvae is consistent with the fact that the third stage larvae are the most infective, indicating that aspartyl protease may be the key enzyme in larvae invasion. The mechanism of invasion of C. elegans into the host and the host protection mechanism lay the foundation.