CAJ | 학술논문

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HPV58 mE6E7.将重组质粒转染NIH/3T3细胞,pIRES-neo-HPV58 mE6E7转染组为实验组,pIRES-neo-HPV58 E6E7转染组为阳性对照组,pIRES-neo空载转染组为阴性对照组.流式细胞术、免疫荧光和Western blot检测转染细胞HPV58 mE6E7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HPV58 mE6E7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γ干扰素的特异性CD8+T细胞数.结果将pGEM-T easy/HPV58 mE6E7和pMD-18T/HPV58 E6E7质粒分别测序,HPV58 E6E7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HPV58 E6E7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HPV58 E6E7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HPV58 mE6E7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HPV58 mE6E7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25 600,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.
목적 탐토인유두류병독(HPV) 58형E6E7융합기인돌변체적전화활성급항원성.방법 분별정점돌변HPV58형E6화E7기인중3개안기산적밀마자,거제돌변후적E6화E7기인적종지마병장기기인편단융합,돌변수식후적기인명명위HPV58 mE6E7.장중조질립전염NIH/3T3세포,pIRES-neo-HPV58 mE6E7전염조위실험조,pIRES-neo-HPV58 E6E7전염조위양성대조조,pIRES-neo공재전염조위음성대조조.류식세포술、면역형광화Western blot검측전염세포HPV58 mE6E7융합단백적표체,연경지집락형성화라서피하성류실험검측HPV58mE6E7융합단백적전화활성.이HPV58 mE6E7융합기인위파항원구건DNA역묘,면역소서후채용매련면역흡부시험검측면역서혈청중특이성항체,매련면역반점법검측면역서비림파세포중가분비γ간우소적특이성CD8+T세포수.결과 장pGEM-T easy/HPV58 mE6E7화pMD-18T/HPV58 E6E7질립분별측서,HPV58 E6E7융합기인여HPV58형E6화E7기인표준서렬적동원성체100％,병증실점돌변성공.은정전염HPV58mE6E7화HPV58 E6E7적NIH/3T3세포표체솔분별위70.3％화84.1％.실험조화양성대조조HPV58mE6E7화HPV58 E6E7융합단백적상대표체수평분별위2.1±1.7화3.8±1.4,음성대조조HPV58 mE6E7화HPV58E6E7융합단백정음성표체.실험조화음성대조조미견집락형성；양성대조조유31개집락형성,기중＞ 50개세포적집락10개.실험조화음성대조조세포접충라서후,재4주내균미성류；이양성대조조중유6지라서형성종류.이HPV58 mE6E7융합기인위파항원적DNA역묘구유량호적항원성,항체적도위25 600,특이성면역반점수위(218.8 ±34.4)개,명현고우대조조.결론 수식후적HPV58E6E7기인가융합표체,병재소제기전화활성적동시보류기항원성,가작위HPV58양성상관종류치료성DNA역묘적파기인.
Objective To develop a prophylactic and therapeutrc vaccine against numan 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 pIRES-neo-HPV58 mE6E7.Then NIH/ 3T3 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 cells 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 cells 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-γof 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 in the nude mice of experimental and negative control groups,but among the 10 mice of positive control group tumor was formed in 6 mice.Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine,the antibody titer was 25 600,and specific immunity spots were 218.8 ± 34.4,significantly higher than that in the control group.Conclusions The fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity.HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.