摘要
目的构建人β-防御素3(h BD3)与杀菌/通透性增加蛋白(BPI)的重组腺病毒载体p Adxsi-BPIBD3,并转染鼠胚胎间充质干细胞系(C3H10T1/2),观察融合蛋白的表达。方法酶切原始质粒,得到BPI-BD3片段,连接到p Shuttle-CMV得到p Shuttle-BPI-BD3,验证后将插入片段转移至p Adxsi载体构建重组腺病毒质粒,线性化后转染HEK293细胞,扩增纯化后,TCID50测定重组腺病毒滴液。p Adxsi-BPI-BD3及空载体p Adxsi(作为对照组)转染C3H10T1/2。RT-PCR、Western blot检测BPI-BD的表达;MTT法检测其对细胞增殖的影响。结果成功构建重组腺病毒载体p Adxsi-BPI-BD3,纯化后病毒滴度达1.2×1010pfu/ml。p AdxsiBPI-BD3转染C3H10T1/2后,RT-PCR、Western blot结果显示,BPI-BD3融合蛋白表达成功。MTT结果显示,转染的C3H10T1/2生长无明显影响(P>0.05)。结论成功构建重组腺病毒载体p Adxsi-BPI-BD3,且转染C3H10T1/2后可稳定表达BPI-BD3融合蛋白,为进一步研究应用抗菌肽BPI-BD3奠定基础。
[objective] To construct a recombinant adenovirus vector expressing human beta defensin-3 （hBD3） and bactericidal/permeability increasing protein （BPI）, and to establish a mesenchymal stem cell line expressing hBD3-BPI. [Methods] BPI-BD3 gene sequence was obtained from the original plasmid, and then subcloned into pShuttle-CMV vector which was subsequently digested with enzyme and inserted into pAdxsi vetor to package the recombinant adenovirus vector （pAdxsi-BPI-BD3）. After verification, pAdxsi-BPI-BD3 was amplified in HEK293 cells, purified and titrated using TCID50 assay. C3H10T1/2 cells were cultured, and then were infected with pAdxsi-BPI-BD3 or pAdxsi. BPI-BD3 fusion protein expression was verified by RT-PCR and Western blot. MTT was used for investigating the influence of the transfection on the proliferation of C3HIOT1/2 cells. [Results] The pAdsxi-BPI-BD3 was successfully constructed and amplified with titer of 1.2 ×10^10 pfu/ml. RT-PCR and Western blot analyses confirmed the expression of the fusion protein BPI-BD3 in C3H10T1/2/BD3-BPI. Growth curve evaluated by M＇IT demonstrated no significant differences among C3H10T1/2 cells transfected with pAdxsi-BPI-BD3, pAdxsi and no treatment control in 7 days （P 〉 0.05）. [Conclusions] Recombinant adenovirus vector pAdxsi-BPI-BD3 has been successfully constructed, and a BPI-BD3 gene-modified C3H10T1/2 cell line has been successfully established which lays a foundation for future study of its biological activities in vitro and in vivo.
出处
《中国现代医学杂志》
CAS
北大核心
2015年第20期17-22,共6页
China Journal of Modern Medicine
关键词
腺病毒载体
人β-防御素3
杀菌/通透性增加蛋白
adenovirus vector
human beta defensins-3
bactericidal/permeability increasing protein
