单纯疱疹病毒胸苷激酶基因重组真核表达载体的构建及在烫伤大鼠皮肤组织中的表达

Construction of eukaryotic recombinant plasmid of herpes simplex virus thymidine kinase gene and its expression in skin tissue of scald rats

目的:构建单纯疱疹病毒胸苷激酶(HSV-tk)基因重组真核表达载体pcDNA3.1/HSV-tk,并观察其在烫伤大鼠皮肤中的表达。方法:实验于2003-07/12在解放军第一军医大学热带卫生学系实验室进行。在原核表达质粒pUChHyTk基础上,利用分子克隆技术,构建重组真核表达质粒pcDNA3.1/HSV-tk。取20只Wistar大鼠,全麻后背部浸入95℃水浴锅中,烫伤12s,造成30%Ⅲ度烫伤,烫伤后随机分为两组(n=10):①pcDNA3.1/tk组:烫伤后当天及第1,2,3,4周后于大鼠左后背部创面边缘(距创面边缘0.5cm)皮下注射脂质体和重组质粒的混合物192μL眼3.6μgpcDNA3.1/tk(2μL)+10μL脂质体+180μL生理盐水演。②pcDNA3.1组:注射时间和方法同前,脂质体和质粒混合物为3.0μgpcDNA3.1(2μL)+10μL脂质体+180μL生理盐水。伤后5周断头处死大鼠,取注射点附近皮肤,用反转录-聚合酶链反应法检测HSV-tk基因的表达。结果:20只大鼠全部进入结果分析。大鼠重组质粒经酶切鉴定与预期结果一致,DNA测序结果表明tk基因已正确插入到pcDNA3.1质粒,说明了重组真核表达载体pcDNA3.1/HSV-tk已成功构建。反转录-聚合酶链反应结果显示脂质体介导的HSV-tk基因转移可在创面成纤维细胞中出现阳性表达。结论:应用分子克隆技术成功地构建了重组真核表达载体pcDNA3.1/HSV-tk,并在烫伤大鼠皮肤中得到了阳性表达。

AIM： To construct recombinant eukaryotic expression vector of pcDNA3.1/HSV-tk of herpes simplex virus thymidine kinase （HSV-tk）, and observe the gene expression of HSV-tk in wound of scald rat. METHODS： The experiment was carried out in the laboratory of Institute of Tropic Hygiene of First Military Medical University from July to December 2003. The pcDNA3.1/HSV-tk gene from prokaryotic plasmids was constructed with molecular cloning technique based on the pUChHyTk. Twenty healthy Wistar rats were selected and randomly divided into 2 groups （n=10）, after dipping into bath pot at 95℃ for 12 s to make 30% Ⅲ degree scald： ①pcDNA3.1/tk group： Mixture of liposomes and recombinant plasmid 192 μL [3.6 μg pcDNA3.1/tk （2μL） ＋10μL liposomes＋ 180μL saline] were injected with subcutaneous injection in left dosal wound （0.5 cm from the edge of wound） at the day of scald, 1^st, 2^nd, 3^rd and 4^th weeks later. ②pcDNA3.1 group： The injected time and method were the same to before. The mixture of liposomes and plasmid was 3.0μg peDNA3.1 （2μL） ＋10μL liposomes ＋180μL saline. Five days after scald the rats were killed, and skin around injected point was got. The expression of HSV-tk gene was detected with reverse transcription polymerase chain reaction （RT-PCR）. RESULTS： A total of 20 rats were involved in the result analysis. Recombinant plasmid of rats after assessing with enzyme was the same to expected result. The result of DNA sequencing revealed that tk gene had inserted into pcDNA3.1 plasmid correctly. It was indicated that pcDNA3.1/HSV-tk had been established successfully. The results of RT-PCR suggested that HSV-tk gene transported by liposome expressed positively in fibroblast in the burn wound. CONCLUSION： Eukaryotic recombinant plasmid of pcDNA3.1/HSV-tk was constructed successfully by molecular cloning method. Positive expression of HSV-tk gene could be detected in scald skin of rats.