PTG重组腺病毒过表达载体的构建  

Construction of recombinant adenoviral vector overexpressing PTG

作　　者：王晨玺 邓霞[1] 赵志聪 蔡珍生 张盼盼 李莲 李昊翔 赵丽 王东[1] 杨玲[1] 袁国跃[1] Wang Chenxi;Deng Xia;Zhao Zhicong;Cai Zhensheng;Zhang Panpan;Li Lian;Li Haoxiang;Zhao Li;Wang Dong;Yang Ling;Yuan Guoyue(Dept of Endocrinology,The Affiliated Hospital of Jiangsu University,Zhenjiang 212001)

机构地区：[1]江苏大学附属医院内分泌科,镇江212001

出　　处：《安徽医科大学学报》2022年第4期558-563,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81870548);江苏省自然科学基金(编号:BK20191222)。

摘　　要：目的构建及鉴定携带小鼠PTG基因(NM_016854)的过表达重组腺病毒载体,为深入研究PTG的功能奠定基础。方法化学合成小鼠PTG基因的编码序列,经聚合酶链式反应(PCR)扩增、酶切,后插入GV314载体(CMV-MCS-3FLAG-SV40-EGFP),得到重组穿梭质粒pGV314-PTG;进一步BamHⅠ/AgeⅠ双酶切,产物交换入线性化表达载体pDC315,构建重组腺病毒Ad-PTG,将其转染HEK293T细胞包装成重组病毒颗粒,经在HEK293T细胞反复扩增数代后,进行纯化及滴度检测,最后利用PCR、Western blot及测序验证重组腺病毒。结果经PCR、Western blot及测序后,结果显示成功构建pGV314-CMV-MCS-3FLAG-SV40-EGFP-PTG过表达腺病毒载体(Ad-PTG),终点稀释法测得病毒滴度为4×10^(10)PFU/ml,Western blot和荧光定量PCR显示PTG的蛋白和mRNA表达水平显著升高。结论成功构建携带小鼠PTG基因的重组过表达腺病毒载体,且能有效上调肝细胞中PTG基因的表达。Objective To construct and identify an overexpression recombinant adenovirus vector carrying the mouse PTG gene(NM_016854),and to lay a foundation for in-depth study of the function of PTG.Methods The coding sequence of the mouse PTG gene was chemically synthesized,amplified by polymerase chain reaction(PCR),digested with restriction enzymes,and inserted into the GV314 vector(CMV-MCS-3FLAG-SV40-EGFP)to obtain the recombinant shuttle plasmid pGV314-PTG.BamHⅠ/AgeⅠ double enzyme digestion was further carried out,and the product was transferred into linearized expression vector pDC315 to construct recombinant adenovirus Ad-PTG,which was transfected into HEK293T cells and packaged into recombinant virus particles.After repeated amplification of several generations of HEK293T cells,the recombinant adenovirus was purified and titer detected.Finally,PCR,Western blot and sequencing were used to verify the recombinant adenovirus.Results After PCR,Western blot and sequencing,the results showed that the pGV314-CMV-MCS-3FLAG-SV40-EGFP-PTG overexpression adenovirus vector(Ad-PTG)was successfully constructed,and the virus titer measured by end-point dilution method was 4×10^(10)PFU/ml,Western blot and RT-qPCR showed that the protein and mRNA expression levels of PTG increased significantly.Conclusion The recombinant adenovirus vector carrying mouse PTG gene is successfully constructed,and the expression of PTG gene in hepatocytes is effectively up regulated.

关 键 词：PTG 重组腺病毒载体 同源重组 2型糖尿病 

分 类 号：Q78[生物学—分子生物学] R589.9[医药卫生—内分泌]