表达脑啡肽基因的单纯疱疹病毒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 foreign enkephalin gene transferred

作　　者：王锷[1] 郭曲练[2] 曹晖[3] 吴小兵[3] 毕好生[1] 白念岳[2] 程智刚[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉科,武汉市现在430030 [2]中南大学湘雅医院麻醉科 [3]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室

出　　处：《中华麻醉学杂志》2001年第12期734-736,共3页Chinese Journal of Anesthesiology

基　　金：国家863基因治疗重大关键技术项目资助课题(863-Z20-05-02)

摘　　要：目的 对单纯疱疹病毒Ⅰ型扩增子质粒装载脑啡肽基因，并进行包装和扩增后，评价其在疼痛的转基因治疗上的作用。方法 将ｐＣＭＶＨＰＰＥ质粒中的人前脑啡肽原基因插入单纯疱疹病毒Ⅰ型扩增子质粒ｐＨＳＶＩＲＥＳｌａｃＺ，再用单纯疱疹病毒温敏株辅助在ＢＨＫ－２１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－２１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１×１０６ＴＵ／ｍｌ。结论 本研究构建、包装、扩增的携带外源脑啡肽基因的假型病毒有较高感染性，这种扩增子病毒可作为疼痛基因治疗的实验研究的有力工具。Objective Neurotropic Herpes Simplex Virus type I (HSV- I ) amplicon vector was adopted to have the enkephalin gene (HPPE) transferred. The new recombinant amplicon vector has strong analgesic effect and be a helpful method of gene therapy in the pain treatment.Methods The HPPE gene fragment was cut down from pCMVHPPE plasmid and recovered. Then the ligation of HPPE fragment and HSV-1 amplicon vector pHSVIRESlacZ was done to construct a recombinat plasmid pHSVIRES-HPPE-lacZ. HSV-tsk was used to help the recombinat plasmid packaged and amplified in BHK-21cells. The amplicon virus liter was examined by X-gal staining. Results The construction of recombinat pHSVIRES-HPPE-lacZ was confirmed by digestion with restriction enzyme. The BHK-21 cells infected by amplicon virus had obvious morphological changes and beta-galactosidase expression. The amplicon virus tilers were determined lo be at 1 x 105-1 x 106TU/ml.Conclusions We have constructed a recombinant amplicon vector pHSVIRES-HPPE-lacZ. The package d and amplified amplicon virus has highly infeclive aclivily. This amplicon virus may be an useful lool for gene therapy of pain.

关 键 词：疱疹病毒Ⅰ型 脑啡肽类 基因治疗 扩增子病毒 疼痛 镇痛 阿片肽 

分 类 号：R614[医药卫生—麻醉学]