Transient over-expression of human papillomavirus type 16 E6 protein down-regulate the secretion of TNF-αor IL-1β LPS-induced from macrophages

In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β in lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈 0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （ P 〉 0.05）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.

尖锐湿疣患者人乳头瘤病毒6、11型的检测及L1基因的序列分析

目的 检测尖锐湿疣患者组织中人乳头瘤病毒 (HPV)6型和 11型的感染率，分析 L1晚期基因序列及其蛋白质的氨基酸序列，为基因工程疫苗的研究提供依据。方法 采用 PCR方法测定北京和锦州两地区 34例尖锐湿疣患者病损组织中 HPV6型及 11型的感染率 ,并扩增 L1晚期基因，构建重组测序质粒，双脱氧法测序观察其变异状况。结果 HPV11型感染率为 73.5％ (25/34)， 6型感染率为 44.1％ (15/34),其中两型混合感染率为 17.6％ (6/34)。 11型 L1基因 7～ 8个碱基变异，推导的蛋白质一级结构中可能有 1～ 3个氨基酸变异， 6型 L1基因仅有 3个碱基发生同义突变。结论 检测的 34例患者中以 HPV11型感染为主，兼有 6型和两型的混合感染。 11型 L1基因及蛋白有一定变异， 6型 L1基因仅发生少数碱基同义突变。

人乳头瘤病毒6型L1基因的克隆和表达及鉴定

克隆人乳头瘤病毒6型（HPV-6）保护性抗原L1基因。构建L1基因表达载体。从临床诊断尖锐湿疵的病理组织标本中提取HPV-6的DNA，采用高保真PCR扩增其保护性抗原L1基因，T-A克隆后测定核苷酸序列。构建pET32a的L1表达载体，在E．coli BL21DE3宿主菌中用不同浓度的IPTG诱导表达。采用SDS-PAGE鉴定表达产物。所克隆的L1基因与报道的相应核苷酸序列同源性为99．20％～99．93％，氨基酸序列同源性高达99．80％～100％。SDS-PAGE结果显示，在构建的载体中成功表达预计大小分子量的融合蛋白，成功构建了HPV-6的保护性基因L1的表达系统。