人乳头瘤病毒18型E7基因和次级淋巴组织趋化因子基因疫苗的制备  

Construction of human papillomavirus 18 E7/secondary lymphoid chemokine combined gene vaccine

作　　者：曾凡慧[1,2] 朱平[2] 张昌菊[2] 雷小敏[3] 柳长柏[1] 

机构地区：[1]三峡大学分子生物学研究所,湖北宜昌443000 [2]三峡大学医学院,湖北宜昌443000 [3]三峡大学第二附属临床医学院仁和医院,湖北宜昌443000

出　　处：《中国新药与临床杂志》2009年第11期856-859,共4页Chinese Journal of New Drugs and Clinical Remedies

基　　金：湖北省教育厅指导项目(13200613001)

摘　　要：目的以减毒的鼠伤寒沙门菌为载体,制备人乳头瘤病毒(HPV)18型E7基因与次级淋巴组织趋化因子(SLC)基因疫苗。方法PCR方法扩增HPV 18 E7和SLC基因片段,将其连接至pcDNA3.1(-)载体中,构建重组质粒pcDNA3.1(-)-HPV 18 E7和pcDNA3.1(-)-SLC。酶切及测序鉴定重组质粒。重组质粒转染至小鼠黑色素瘤高转移细胞株B16F10中,RT-PCR和Western blotting方法检测目的基因和蛋白的表达。重组质粒电转化导入减毒鼠伤寒沙门菌SL3261,酶切鉴定重组质粒的正确导入。结果pcDNA3.1(-)-HPV 18 E7和pcDNA3.1(-)-SLC经相应酶切及测序证实其大小和序列与理论结果一致。HPV 18 E7和SLC在B16F10细胞中正确表达,RT-PCR和Western blotting所得结果及导入沙门菌SL3261的重组质粒酶切所得片段大小均在理论值范围内。结论成功制备以沙门菌SL3261为载体的HPV 18 E7和SLC基因疫苗。AIM To prepare gene vaccine constructed with human papillomavims （HPV） 18 E7 and secondary lymphoid chemokine （SLC）, while taking attenuated Salmonella typhimurium SL3261 as the vaccine vector. METHODS HPV18 E7 and SLC genes were amplified by PCR method. Recombined plasmid pcDNA3.1 （-）-HPV 18 E7 and pcDNA3.1（-）-SLC were constructed by adding HPV18 E7 and SLC genes to pcDNA3.1（-） vector, respectively. The recombined plasmids were identified by enzyme digestion and sequencing. After transfecting the recombined plasmids to B16F10 cells, the expressions of corresponding genes and protein were examined by RT-PCR and Western blotting. To prepare oral vaccine, pcDNA3.1（-）-HPV 18 E7 and pcDNA3.1 （-）-SLC were electrotransformed into attenuated Salmonella typhimurium SL3261. Then, the accurate introduction of the recombined plasmids was identified by enzyme digestion. RESULTS The digestion and sequencing result of the recombination plasmids confirmed the success construction. HPV 18 E7 and SLC were expressed correctly in B16F10 cells. The results of RT-PCR and Western blotting and the enzyme digestion fragment of recombined plasmid which electrotransformed into Solmonella typhimurium SL3261, were within the theoretical value. CONCLUSION HPV 18 E7 and SLC gene vaccines with attenuated Salmonella typhimurium SL3261 as vector are successfully constructed.

关 键 词：人乳头瘤病毒18 趋化因子 CC 疫苗 DNA 

分 类 号：R373.9[医药卫生—病原生物学]