靶向TPX2基因shRNA慢病毒载体的构建及鉴定

Construction and identification of shRNA lentiviral vector targeting TPX2 gene

目的构建靶向TPX2基因的RNA干扰慢病毒载体,获得稳定感染的人宫颈癌He La细胞株,为宫颈癌细胞TPX2基因的相关研究奠定基础。方法以TPX2基因为靶点,设计并合成四组对应于目标shRNA的双链DNA发卡结构,分别与慢病毒载体Pglv2-U6-Puro连接经转化感受态细胞,阳性克隆经测序、病毒包装、滴度测定、感染细胞、嘌呤霉素筛选得到TPX2基因沉默稳转细胞株。实时荧光定量PCR和Western blot检测TPX2 mRNA和蛋白的沉默效果。流式细胞计量术检测其对细胞周期分布及凋亡的影响。结果测序结果证实5种慢病毒载体均包装成功,采用0.4μg/m L嘌呤霉素成功筛选出TPX2沉默细胞株。He La细胞感染4种载有TPX2-shRNA的重组慢病毒后,均发挥了沉默效应,尤以TPX2-shRNA-1沉默效果最佳,TPX2 mRNA相对表达量为0.21±0.07,低于对照组的1.08±0.07(P<0.01);TPX2蛋白质相对表达量为0.19±0.28,低于对照组的0.64±0.03(P<0.01);G2及S期细胞高于对照组(P<0.05);细胞凋亡率明显多于对照组(P<0.05)。结论获得了一种有效TPX2基因的干扰序列,成功构建了TPX2 shRNA慢病毒干扰载体,并筛选出TPX2基因沉默的He La细胞株,为进一步研究TPX2基因在宫颈癌中的作用奠定了基础。

Objective To construct the lentiviral RNA interference vector targeting TPX2 and to obtain the human cervical cancer HeLa cell strain stably infected by TPX2-shRNA for studying the relationship between hu- man cervical carcinoma and TPX2 gene. Methods By targeting TPX2 gene, four double-stranded DNA hairpin structures corresponding to shRNA were designed, synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids. Then these recombinant plasmids were transformed into DHSα competent cells. The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervi- cal carcinoma HeLa cell infected by these recombinant lentiviral was screened by Puromycin, then stable cell strain was obtained.The silencing effect of TPX2 in HeLa cell was detected by RT- fluorescent quantitative PCR and West- ern blot. Cell cycle and cell apoptosis wer detected by Flow cytometry. Results Sequencing results confirmed that 5 lentiviral was packaged successfully. The steady cell strain transfered TPX2-shRNA was screened with 0. 4 μg/mL puromycin. HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA- 1.In the group of TPX2-shRNA- 1, TPX2mRNA （0.21_＋0.07） and protein（ 0. 19±0.28） rela- tive expression level is lower than those in the control group （ 1.08±0.07 ） （P〈0.01） and（ 0.64±0.03 ） （P〈 0.01 ）respectively; G2 and S-phase cells are higher than those in the control group （P〈0.05）and the apoptosis rate was significantly more than those in the control group （P〈0.05）.Conclusions The effective TPX2 genetic interference sequence was obtained, lentiviral vectors carrying TPX2shRNA was successfully constructed, and the HeLa cell strain with TPX2 silenced was successfully screened,which lay the research foundation for the study of the role of TPX2 in cervical cancer.