白纹伊蚊β-肌动蛋白基因片段的克隆及其作为基因表达内参照的应用

Cloning and sequences of Aedes albopictus β-actin gene fragment and its application as an internal control

目的获取白纹伊蚊β-肌动蛋白基因序列并探讨其作为基因表达内参照的作用。方法根据昆虫β-肌动蛋白核苷酸序列的高度保守区设计引物,通过PCR的方法从白纹伊蚊C6/36细胞中扩增获得白纹伊蚊β-肌动蛋白基因片段,进一步通过RT-PCR的方法验证其在稳定转染空载体和40S核糖体蛋白S4(RPS4)基因的白纹伊蚊C6/36细胞中的表达。结果获得白纹伊蚊β-肌动蛋白基因片段,长911 bp,与其他几种蚊β-肌动蛋白基因对应序列的相似性在89%以上。在稳定转染空载体和RPS4基因的C6/36细胞中,均可稳定地扩增出目的基因。结论成功获得了白纹伊蚊β-肌动蛋白基因片段,并且该片段完全可以用作基因表达差异分析时的内参照基因。

Objective Objective To isolate and sequence Aedes albopictus β-actin gene and test its applicability as an internal control in gene expression studies. Methods Primers were designed based on the conserved nucleotide sequences of insect β-actin genes. A fragment of Ae. albopictus β-actin gene was amplified from the cDNA of C6/36 cells. The expression levels of Ae. albopictus β-actin in RPS4 gene stably transfected and control vector transfected C6/36 cells were both detected by RT-PCR. Results A fragment of 911 bp was isolated which shows over 89% identities with the corresponding sequences of other mosquito β-actin genes. The fragment was also successfully amplified from RPS4 gene transfected and control vector transfected C6/36 cells. Conclusion Ae. albopictus β-actin gene fragment was successfully cloned. As a housekeeping gene, it can be used as an internal control in gene expression studies.