CAJ | 학술논문

目的：构建并鉴定USP22基因ShRNA慢病毒载体,为进一步研究USP22基因在鼻咽癌中的作用机制奠定基础。方法针对USP22基因的编码序列设计并合成2条特异性干扰序列,序列两端含有限制性内切酶位点HpaⅠ和X hoⅠ。寡核苷酸链退火生成寡核苷酸双链,5'端磷酸化后将含有酶切位点的寡核苷酸双链克隆到pLL3.7慢病毒表达载体。连接产物经转化、培养,提取其质粒,提取出来的质粒经HpaⅠ和XhoⅠ酶切电泳鉴定,鉴定正确的质粒进行测序。构建成功的慢病毒表达载体pLL-USP22-shRNA与包装载体质粒混匀共转染于293T细胞。通过荧光显微镜下观察绿色荧光蛋白(GFP)情况,对病毒滴度和感染效率进行检测。结果成功构建慢病毒表达载体pLL-USP22-shRNA。与包装载体质粒共转染293T细胞后测定慢病毒滴度为4×107 TU/ml。结论本实验应用相关技术成功构建USP22 ShRNA慢病毒载体,为进一步研究USP22基因的生物学功能奠定了基础。
목적：구건병감정USP22기인ShRNA만병독재체,위진일보연구USP22기인재비인암중적작용궤제전정기출。방법침대USP22기인적편마서렬설계병합성2조특이성간우서렬,서렬량단함유한제성내절매위점HpaⅠ화X hoⅠ。과핵감산련퇴화생성과핵감산쌍련,5'단린산화후장함유매절위점적과핵감산쌍련극륭도pLL3.7만병독표체재체。련접산물경전화、배양,제취기질립,제취출래적질립경HpaⅠ화XhoⅠ매절전영감정,감정정학적질립진행측서。구건성공적만병독표체재체pLL-USP22-shRNA여포장재체질립혼균공전염우293T세포。통과형광현미경하관찰록색형광단백(GFP)정황,대병독적도화감염효솔진행검측。결과성공구건만병독표체재체pLL-USP22-shRNA。여포장재체질립공전염293T세포후측정만병독적도위4×107 TU/ml。결론본실험응용상관기술성공구건USP22 ShRNA만병독재체,위진일보연구USP22기인적생물학공능전정료기출。
Objective To construct and identify ShRNA lentivirus vector in gene USP22,and lay the foundation for fur-ther study of gene USP22 on mechanism of nasopharyngeal carcinoma. Methods Two special interference sequences targeting on coded sequence of gene USP22 were designed and synthesized.HpaⅠand XhoⅠas two sites of restriction enzyme were included at both ends of the sequence.Oligodeoxynucleotides was annealed to generate double strands of oligodeoxynucleotides.After 5’end phosphorylation,double strands of oligodeoxynucleotides containing restriction enzyme cutting site was cloned to pLentiLox3.7 (PLL3.7) lentiviral expression vector.The connection product was transformed and cultivated in order to extract plasmid.The extracted plasmid was identified by restriction enzyme digestion and electrophoresis,and sequencing was carried on only for correct plasmid after identification.The successfully-constructed lentiviral expression vector of pLL-USP22-shRNA and packaging plasmid were mixed and cotransfected into 293T cell. Conditions of green fluorescent protein (GFP) were observed under fluorescence microscope to determine virus titer and efficiency of infection. Results The sequencing showed that lentiviral expression vector of pLL-USP22-shRNA was successfully constructed.The lentiviral titer was 4í107 TU/ml after cotransfecting into 293T cell with packaging plasmid. Conclusion The lentivirus vector of USP22-ShRNA is successfully constructed by related techniques in the experi-ment,which lays the foundation for further study of biological function of gene USP22.