人乳头瘤病毒58型E6E7融合基因突变体的转化活性及抗原性  

Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant

作　　者：王鹤[1] 于继云[2] 李力[1] 

机构地区：[1]广西医科大学附属肿瘤医院妇瘤科,南宁530021 [2]军事医学科学院基础医学研究所转化医学研究室

出　　处：《中华肿瘤杂志》2013年第7期491-496,共6页Chinese Journal of Oncology

基　　金：基金项目：广西卫生厅重点课题（重200970）

摘　　要：目的探讨人乳头瘤病毒（HPV）58型E6E7融合基因突变体的转化活性及抗原性。方法分别定点突变HPV58型E6和E7基因中3个氨基酸的密码子，去除突变后的E6和E7基因的终止码并将其基因片段融合，突变修饰后的基因命名为HPV58mE6E7。将重组质粒转染NIH／3T3细胞，plRES—neo-HPV58mE6E7转染组为实验组，plRES-neo-HPV58E6E7转染组为阳性对照组，plRES—neo空载转染组为阴性对照组。流式细胞术、免疫荧光和Westernblot检测转染细胞HPV58mE6E7融合蛋白的表达，软琼脂集落形成和裸鼠皮下成瘤实验检测HPV58mE6E7融合蛋白的转化活性。以HPV58mE6E7融合基因为靶抗原构建DNA疫苗，免疫小鼠后采用酶联免疫吸附试验检测免疫鼠血清中特异性抗体，酶联免疫斑点法检测免疫鼠脾淋巴细胞中可分泌1干扰素的特异性CD8’T细胞数。结果将pGEM—Teasy／HPV58mE6E7和pMD一18T／HPV58E6E7质粒分别测序，HPV58E6E7融合基因与HPV58型E6和E7基因标准序列的同源性达100％，并证实点突变成功。稳定转染HPV58mE6E7和HPV58E6E7的NIH／3T3细胞表达率分别为70．3％和84．1％。实验组和阳性对照组HPV58mE6E7和HPV58E6E7融合蛋白的相对表达水平分别为2．1±1．7和3．8±1．4，阴性对照组HPV58mE6E7和HPV58E6E7融合蛋白呈阴性表达。实验组和阴性对照组未见集落形成；阳性对照组有31个集落形成，其中〉50个细胞的集落10个。实验组和阴性对照组细胞接种裸鼠后，在4周内均未成瘤；而阳性对照组中有6只裸鼠形成肿瘤。以HPV58mE6E7融合基因为靶抗原的DNA疫苗具有良好的抗原性，抗体滴度为25600，特异性免疫斑点数为（218．8±34．4）个，明显高于对照组。结论修饰后的HPV58E6E7基因可融合表达，并在消除其转化活性的同时保留其抗原性，可作为HPV58阳性相关肿瘤治疗性DNA疫苗的靶基因。Objective To develop a prophylactic and therapeutic vaccine against human papillomavirus （ HPV ） type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity. Methods The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid plRES-neo-HPV58 mE6ET. Then NIH/ 333 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7- transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected ceils were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental ceils was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8 ＋ T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay. Results Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1% , respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1±1.7 and 3.8±1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks i

关 键 词：人乳头瘤病毒58 基因 E6 基因 E7 生物转化 疫苗 DNA 抗体 

分 类 号：R737.33[医药卫生—肿瘤]