Construction of recombinant lentiviral expression vector carrying K9 of Kaposi′s sarcoma-associated herpesvirus and preliminary exploration of K9 encoded protein
SHANG Yuan-cui, LU Chun, QIN Di
(Department of Microbiology, Nanjing Medical University, Nanjing Jiangsu 211166, China）
Abstract：Objective： To construct the recombinant plasmid carrying K9 gene of Kaposi′s sarcoma- associated herpesvirus(KSHV), and to explore the effect of K9 encoded protein vIRF1 on the proliferation of human umbilical vein endothelial cells(HUVECs). Methods： According to the sequence of K9, forward and reverse PCR primers with XhoⅠ and BamHⅠrestriction endonucleases sites were designed. Then the expression plasmid pCI-K9-Flag was used as template to amplify K9. Subsequently, PCR product digested by restriction endonucleases was inserted into pHAGE-CMV-MCS-IzsGreen vector (abbreviated as pHAGE) to construct recombinant plasmid pHAGE-K9. Then pHAGE-K9, package plasmid psPAX2 and envelope plasmid pMD2.G were cotransfected into the 293 T cells to produce lentivirus. The viral titer was determined by gradient dilution. Next, pHAGE-K9 lentivirus infected HUVECs was observed and the expression of vIRF1 protein was detected by Western blotting. Cells expressing vIRF1 were sorted by flow cytometry. Finally, the effect of vIRF1 on the proliferation of HUVECs was measured by CCK-8 assay. Results： Based on sequencing of recombinant plasmid corresponded with K9 gene, it was validated that recombinant plasmid was successful constructed. Western blotting showed lentivirus-infected HUVECs expressed vIRF1 successfully. The result of CCK-8 showed that compared with control group, proliferation of HUVECs stablely expressing vIRF1 was significantly increased (P<0.01,n=5). Conclusion： KSHV K9 encoded protein, vIRF1, enhanced the proliferation of HUVECs.
收稿日期: 2016-11-07
基金资助:
国家自然科学基金资助项目（81571984）
通讯作者:
秦娣（通讯作者），副教授，硕士生导师，E-mail:diqin@njmu.edu.cn
作者简介: 尚元翠(1991—)，女，硕士研究生
引用本文:
尚元翠, 卢春, 秦娣. 卡波氏肉瘤病毒K9基因重组慢病毒表达载体的构建及其编码蛋白功能初探[J]. 江苏大学学报:医学版, 2016, 26(06): 461-.
SHANG Yuan-Cui, Lu-Chun, Qin-Di. Construction of recombinant lentiviral expression vector carrying K9 of Kaposi′s sarcoma-associated herpesvirus and preliminary exploration of K9 encoded protein. Journal of Jiangsu University(Medicine Edition), 2016, 26(06): 461-.
2016, Vol. 26 
