表达降钙素基因相关肽的单纯疱疹病毒Ⅰ型扩增子载体的构建、包装和扩增  

The construction and package of herpes simplex virus type I amplicon vector with CGRP gene transferred

作　　者：吴军[1] 张海鸥[1] 陈玉丙[2] 周芬莉[1] 胡俊[1] 李珂静[1] 

机构地区：[1]北京大学深圳医院神经内科,广东深圳518036 [2]吉林大学第一临床医院血液科,吉林长春130021

出　　处：《中国实验诊断学》2006年第9期1033-1035,共3页Chinese Journal of Laboratory Diagnosis

基　　金：本课题受深圳市科技局科研基金资助(编号:200304123)

摘　　要：目的对单纯疱疹病毒Ⅰ型扩增子质料装载降钙素基因相关肽,并进行包装和扩增。方法分别钓取pac基因和γ134.5基因,克隆入pMD18-T载体中。pSV载体经HindⅢ和BamHⅠ双酶切,获得lacZ片段,自身环化形成pUPO-γ134.5-lacZ。合成CGRP基因序列,将此基因替换pUPO-γ134.5-lacZ质粒中的lacZ基因,插入γ134.5基因位点中,即pUPO-Δγ134.5-CGRP。再用单纯疱疹病毒温敏株辅助在BHK细胞中包装、扩增,通过检测lacZ的表达反映扩增子病毒滴度。结果酶切鉴定证实重组子构建成功。重组扩增子质粒的包装、扩增由BHK细胞的形态变化和X-gal原位检测载体lacZ的表达证实。扩增子假型病毒滴度达3.5×105TU/ml。结论本研究构建、包装、扩增的携带降钙素基因相关肽的假型病毒有较高感染性,这种扩增子病毒可作为实验研究的有力工具。Objective Neurotropie Herpes Simplex Virus type Ⅰ （HSV-Ⅰ） amplicon vector was adopted to have the CGRP gene transfer. The new recombinant amplicon vector has strong analgesic effect and be a helpful method of gene therapy in SAIl. Methods The pac gene fragment and γ134.5 gene fragment were amplified,and then the ligation of pMD18-T vector.CGRP gene was syntheses and replace the laeZ gene in the vector of pUPOγ134.5-lacZ. HSV-tsk was used to help the recombinant plasmid paekagod and amplified in BHK cells. The amplicon virus titer was examined by X-gal staining. Results the construction of recombinant plasmid was confirmed by digestion with restriction enzyme. The BHK cells infected by amplicon virus had obvious morphological changes. The amplicon virus fiters were determined to be at 3.5 ×10^5 TU/ml. Conclusion we have constructed a recombinant amplicon vector. The packaged and amplified amplicon virus has highly infective activity. The amplicon virus may be a useful tool for gene therapy of SAH.

关 键 词：HSV-Ⅰ降钙素基因相关肽 基因表达与调控 载体 

分 类 号：R346[医药卫生—基础医学]