人乳头瘤病毒16型E5蛋白真核载体的构建及表达

目的构建人乳头瘤病毒115型（HPV16）E5蛋白真核表达载体pcDNA3．1（＋）／HPV16E5，分析E5在HeLa细胞及BALB／c小鼠肌肉组织中的表达。方法设计HPV16E5基因引物，PCR扩增E5基因。经Bam-HI、EcoR1酶切后将其连接入pcDNA3．1（＋），PCR及双酶切筛选阳性克隆并测序。将构建的pcDNA3．1（＋）／HPV16E5转染HeLa细胞，利用RT—PCR测定HPV16E5mRNA表达。将6只BALB／c小鼠分为3组，分别取100mgpcDNA3．1（＋）／HPV16E5、100mgpcDNA3．1（＋）、100mLPBS经肌肉注射小鼠，2周1次，共4次；末次接种后第14天取小鼠股四头肌制成石蜡切片，免疫组织化学法检测E5蛋白在小鼠肌肉组织中的表达。结果PCR扩增得到252bpHPV16E5基因片段，DNA测序结果显示E5基因片段正确地连入pcDNA3．1（＋）。构建的pcDNA3．1（＋）／HPV16E5在HeLa细胞表达大小为252bp的HPV16E5mRNA。小鼠股四头肌细胞膜被染成棕黄色。结论构建的真核表达载体pcDNA3．1（＋）／HPV16E5能在HeLa细胞及小鼠肌肉组织中表达HPV16E5蛋白。

Construction and Expression of Eukaryotic Expression Vector of Human Papillomavirus Type 16 E5

Objective To construct the eukaryotic expression vector of human papillomavirus type16（HPV16） E5, and to analyze HPV16 E5 expression in HeLa cells and in mouse＇s quadriceps femoris. Methods The HPV16 E5 gene was amplified by polymerase chain reaction （PCR） with specific primers designed by PRIMERS. 0. After PCR products were digested with BamHI and EcoRI, the fragment was inserted into pcDNA3.1 （ ＋ ）. The inserted fragment was identified by PCR and restriction enzymatic digestion and sequencing. Eukaryotic expression vector pcDNA3.1 （ ＋ ）/HPV16 E5 was constructed. The plasmid of pcDNA3.1 （ ＋ ）/HPV16 E5 was transfected into HeLa cells,and HPV16 E5 mRNA was detected using RT-PCR. Six BALB/c mice were randomly divided into 3 groups：Group pcDNA3.1 （ ＋ ）/HPV16 ES, pcDNA3.1 （ ＋ ） and PBS. Every group contained 2 mice. The mice were injected intramuscularly with 100rag plasmid or 100ml PBS for four times every 2 weeks. The quadriceps femoris of the mice were made of paraffin section. The expressions of HPV16 E5 protein were detected using immunohistochemistry. Results DNA sequencing result showed that HPV16 E5 gene fragment（252bp） was amplified by PCR. The eukaryotic expression vector pcDNA3.1 （ ＋ ）/HPV16 E5 expressed HPV16 E5 mRNA in HeLa cells by RT-PCR. The cell raembranes of the quadriceps femoris were stained brown. Conclusions The eukaryotic expression vector pcDNA3.1 （ ＋ ）/HPV16 E5 was successfully constructed, HPV16 E5 mRNA expressed in HeLa cells, and E5 proteins expressed in the quadriceps femoris of the mice.