Bak1腺病毒表达载体的构建及对人成骨肉瘤细胞的影响  

Construction of a recombinant Bak1-expressed adenovirus vector and its effect on human osteosarcoma cells line

作　　者：柴爽 黄红[2] 万雷[3] 王吉利 王伟[4] 黄宏兴[3] CHAI Shuang;HUANG Hong;WAN Lei;WANG Ji - li;WANG Wei;HUANG Hong - xing(Third Clinical Medical College, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, Guangdong, China)

机构地区：[1]广州中医药大学第三临床医学院,广东广州510006 [2]广州中医药大学护理学院,广东广州510006 [3]广州中医药大学附属骨伤科医院骨科,广东广州510240 [4]中国人民解放军广州疗养院理疗科,广东广州510515

出　　处：《广东医学》2018年第9期1332-1336,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81373653;81674004);广东省科技计划项目(编号:2016A020216024)

摘　　要：目的构建人Bcl2拮抗1(Bak1)基因重组表达腺病毒pAd-Bak1,并感染人成骨肉瘤细胞MG63建立过表达Bak1的细胞模型,检测其对细胞活性的影响。方法利用qPCR扩增Bak1全长cDNA,利用穿梭质粒pENTER构建载有Bak1基因的重组穿梭质粒pENTER-Bak1,通过基因测序鉴定载体序列。将重组载体PmeⅠ线性化、脱磷酸化后,转入含有腺病毒骨架质粒pAd-Amp的感受态BJ5183细胞中进行基因同源重组。将重组腺病毒质粒pAD-Bak1经PacⅠ酶切线性化后,用Lipofectamine 2000脂质体转染到HEK-293细胞中进行包装扩繁,荧光定量PCR法测定病毒滴度。感染MG63细胞,qPCR和Western blot检测Bak1的表达,MTT检测细胞活性。结果测序及酶切验证pENTER-Bak1构建成功。pAD-Bak1感染MG63细胞,qPCR检测到细胞中Bak1基因过表达(与NC对照组和空白对照组相比P<0.01、P<0.01),Western blot检测到细胞中Bak1蛋白过表达。MTT检测显示与NC对照组相比,细胞活性减弱(P<0.05)。结论成功构建了Bak1重组表达腺病毒,验证了其能够感染MG63细胞表达Bak1蛋白,证明了过表达Bak1可以减弱细胞的活性。为进一步研究Bak1的功能奠定了基础。Objective To construct a recombinant BCL2-antagonist/killer 1（ Bak1）-expressed adenovirus,thus to establish a cell model by infecting human osteosarcoma MG63 cells,and to study the effect of recombinant Bak1 adenovirus on MG63 cells. Methods The full-length cDNA of Bak1 was prepared through qPCR. Plasmid pENTER-Bak1 containing Bak1 gene was constructed by using adenovirus shuttle plasmid pENTER,and the sequence was identified by restriction enzyme digestion method. After linearized by Enzyme digestion of Pme I,it was recombined with BJ5183 cells which contained adenoviral back-bone plasmid pAd-Amp. The recombinant adenovirus vector was linearized by Pac I enzyme and transfected into HEK-293 cell. Bak1 recombinant adenovirus was extracted by repeated centrifugation and low temperature cracking. Real-time PCR was applied to measure the adenovirus titer. MG63 cells were infected with recombinant adenovirus. The expression of Bak1 was assessed with qPCR and Western blot. The cell activity was determined by MTT. Results It was proved by gene sequencing and enzyme digestion that pENTER-Bak1 was successfully constructed. The expression of Bak1 gene was confirmed by qPCR,and the expression of Bak1 protein was confirmed by Western blot. MTT showed that the recombinant expression of Bak1 adenovirus could weaken the viability of MG63 cells.Conclusion Bak1 recombinant adenovirus vector is successfully constructed,and the recombinant adenovirus vector could infect MG63 cells and express Bak1 protein. Bak1 adenovirus could weaken the viability of MG63 cells.

关 键 词：人Bcl2拮抗1(Bak1)蛋白 重组腺病毒载体 人成骨肉瘤细胞MG63 

分 类 号：R580[医药卫生—内分泌]