HCV结构蛋白腺病毒表达载体的构建及T细胞疫苗对HCV清除作用的研究
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摘要
HCV感染是当前危害人类健康的重要传染病。其体外培养尚未成功，至今尚无满意的感染模型，这极大的限制了HCV的研究及丙型肝炎的防治工作。构建并包装表达HCV结构基因的复制缺陷型腺病毒载体，在丙型肝炎的防治和HCV的研究中具有重要意义。
HCV的感染呈世界范围性，其致病常引起持续感染，且HCV的慢性感染与肝硬化及肝癌明显相关。至今尚无理想的预防和治疗措施。所以，寻找新的抗病毒途径，预防和清除HCV持续感染很有必要。T细胞疫苗作为一种防治HCV感染的新途径，正在受到人们的重视。探讨防治HCV感染的T细胞疫苗的制备方法及其在体内外对HCV的清除作用，对HCV感染的研究及防治也具有现实意义。
本研究利用分子克隆技术，构建了三种HCV结构蛋白腺病毒载体穿梭质粒，并以此为基础包装、筛选和鉴定了三种HCV结构蛋白腺病毒表达载体，选择并合成了HCV结构基因区5条CTL表位多肽，并以此诱导了5种HCV特异性CTL，并对其进行了体内外的实验测定。此外，我们还探讨了诱导前免疫致敏对T细胞疫苗活性的影响，并比较了HCV腺病毒载体和质粒载体在此免疫致敏功能方面的差异。我们还将T细胞疫苗与DNA疫苗在体内对HCV移植瘤的抑制作用进行了比较，探讨它们对HCV感染的保
第四军医大学博上学位论文
护和治疗作用的差异。
结论如下：
1．构建了3种HCV结构基因腺病毒载休穿梭质粒pAd．HCV（、
pAd．HCV－Cm手 pAd．HCV－CE EZ，经双酶切、PCR和坝序鉴定，它们 分
另IJ插入了 HCV C、C＋EI和 C＋EI＋EZ基因。
＊ 〕将构建的 3种穿梭质粒与腺病毒基囚纲质粒 pJNll7共转染 293细胞，
通过土斑技术包装、筛选了三种 ＊＊V 纪构蛋日腺病毒天达载体
pAdHC＼＊C、pAdHCV－CEI耳 pAd．HCV－CEIEZ，经鉴定具有较好的感染
性，能较好的表达插入的HCV结构蛋白或融合结构蛋臼，与质粒DNA载
体相比，腺病毒载体具有更好的免疫效果，可望发展成为抗HCV感染的腺
病毒载体疫苗。
3．选择并合成了可与 H工‘分于结合的 5条 HCV结构基回区 CTL表位
多肽，建立了体夕「特异性CTL的诱导、增）rt的力法，诱导产生了了5种HCV
特异性CTL。
4．在诱导T细胞疫苗之前，用＊＊V腺炳毒载体或质粒载体讨小鼠进
厅免疫致敏，可以明显提高T细胞疫苗的活性；其中腺病毒载体的免疫致
敏效果要明显好于质粒载体。
5．将诱导的5种＊＊V特异性C＊L，在体外进了丁特异性杀伤实验，证
一
实，出n、P3和 P4三条多肽诱导的脾纠＊泡含较高比例的 CD。＋纠1巾d，叫－产
上较好的CTL杀伤活性，可以用作T细胞疫苗进fi体内抗HCV实验。
6．建立了HCV结构基囚体外细胞转染系SPyo－NCV，爿。以此细胞系
建立了皮下移植瘤 HCV小鼠模型，经免疫组化叶大，SPZ／0－HCV细胞及其
移植瘤组织内均有HCV结构蛋白的表达。
7．将诱导的solkHCV特异性CTL，以T纠1）地疫苗的形式免疫皮下移
仙册NCV小鼠栓型，证丈，山PI、n和N三条多肌诱导的T纠1胞疫析对
6
父四军医大学阵土学位沦文
＋1（V｛＄趴W人叫卜的抑制作川。
8．将3种丁细胞疫苗与表达＊＊V结构基因的质粒＊＊A疫苗在＊＊v
移他瘤小鼠捎型内进行治疗对比，发现djJ者明显忧于后者，提示，T细胞疫
祈在NCV感染的防治研究中可能具有较奸的发展汕景。
HCV infection has been one of the most important damaging infectious diseases world-wide. Without a reliable cell culture system in vitro and successful infection model, study on HCV and prevention of hepatitis C have been extremely limited. Construction and packing of replication-defective adenovirus vectors expressing HCV structural proteins will play an important role in research of HCV and vaccination.
HCV infection often result in persistent infection. Moreover, it has been known that chronic HCV infection is associated with high risk of liver cirrhosis and hepatocellular carcinoma but without perfect prevention and therapeutic procedure. Exploration of new way to vaccination and clearance of HCV will be a great challenge. T-cell vaccine is being noticed as a new way of HCV prevention and clearance, and research on preparation of T-cell vaccine and its clearance against HCV will contribute to it.
We constructed 3 kinds of shuttle plasmid encoding HCV structural proteins by means of molecular cloning technique; based on it, 3 kinds of
adenovirus vectors of HCV structural proteins were packed, screened and identified. We also selected and synthesized 5 kinds of CTL epitope peptides of HCV structural gene which induced 5 kinds of corresponding HCV specific CTL, which were evaluated by experiments in vivo and in vitro. Furthermore, we discussed the influence of immune-sensibilization before induction to the activity of T-cell vaccine, compared the immune-sensibilization difference between HCV-adenovirus vectors and plasmid vectors. We also compared T-cell vaccine with DNA vaccine on in vivo inhibition to HCV transplanting tumor to evaluate the difference effects of protection from HCV infection. Conclusion:
1 The 3 constructed adenovirus vectors shuttle plasmid of HCV structural gene. pAd.HCV-C. pAd.HCV-CEl and pAd.HCV-CE!E2. were confirmed to be inserted the sequences of HCV C, C+E1 and C+E1+E2 by double endonucleases, PCR and sequencing.
2 After co-transfection with the 3 kinds of shuttle plasmid and adenovirus genome plasmid (p.IM17) into 293 cell. 3 HCV structural protein expressing adenovirus vectors. pAd.HCV-C, pAd.HCV-CEl and pAd.HCV-CE!E2 were packed and screened by virus plaques technique, identified to be good infectivity and expressivity of inserted HCV structural proteins or fusional structural. In contrast to plasmid DNA vector, adenovirus vectors appeared to be better immunizational effects and with the hope of being developed to be vaccine against HCV.
3 We also selected and synthesized 5 CTL epitopes peptides of HCV structural gene which were capable of combining with H-2d molecule, established methods of in vitro elicitation and proliferation of specific CTL and
obtained 5 kinds of specific CTLs.
4 We observed that T cell vaccine activity could be greatly improved through immune-sensibilization to mice with HCV adenovirus vectors or plasmid vector before T-cell vaccine induction, and adenovirus vectors proved to be superior to plasmid vector.
5 In vitro specific killing experiments showed that spleen cells induced by PI, P3 and P4, which contained higher proportion of CDg+ cells and generated better CTL activity, could be introduced to in vivo experiment against HCV
6 We successfully setup a HCV structural gene in vitro cell-transfection system SP2/0-HCV, through which also setup a murine model with subcutaneous transplanting tumor of HCV. Determined by immunohistochemical technique, there were HCV structural proteins in both SP2/0-HCV and its transplanting tumor tissue.
7 When the murine model were immunized by the 5 kinds of elicited HCV-specific CTLs as a role of T-cell vaccine, significant inhibition to HCV transplanting tumor were observed in groups of PL P3. and P4.
8 Comparison were performed between the 3 T-cell vaccine and plasmid DNA vaccine expressing HCV structural gene on therapeutic effects in HCV transplanting tumor murine model. The result showed that the former was obviously superior the
HCV的感染呈世界范围性，其致病常引起持续感染，且HCV的慢性感染与肝硬化及肝癌明显相关。至今尚无理想的预防和治疗措施。所以，寻找新的抗病毒途径，预防和清除HCV持续感染很有必要。T细胞疫苗作为一种防治HCV感染的新途径，正在受到人们的重视。探讨防治HCV感染的T细胞疫苗的制备方法及其在体内外对HCV的清除作用，对HCV感染的研究及防治也具有现实意义。
本研究利用分子克隆技术，构建了三种HCV结构蛋白腺病毒载体穿梭质粒，并以此为基础包装、筛选和鉴定了三种HCV结构蛋白腺病毒表达载体，选择并合成了HCV结构基因区5条CTL表位多肽，并以此诱导了5种HCV特异性CTL，并对其进行了体内外的实验测定。此外，我们还探讨了诱导前免疫致敏对T细胞疫苗活性的影响，并比较了HCV腺病毒载体和质粒载体在此免疫致敏功能方面的差异。我们还将T细胞疫苗与DNA疫苗在体内对HCV移植瘤的抑制作用进行了比较，探讨它们对HCV感染的保
第四军医大学博上学位论文
护和治疗作用的差异。
结论如下：
1．构建了3种HCV结构基因腺病毒载休穿梭质粒pAd．HCV（、
pAd．HCV－Cm手 pAd．HCV－CE EZ，经双酶切、PCR和坝序鉴定，它们 分
另IJ插入了 HCV C、C＋EI和 C＋EI＋EZ基因。
＊ 〕将构建的 3种穿梭质粒与腺病毒基囚纲质粒 pJNll7共转染 293细胞，
通过土斑技术包装、筛选了三种 ＊＊V 纪构蛋日腺病毒天达载体
pAdHC＼＊C、pAdHCV－CEI耳 pAd．HCV－CEIEZ，经鉴定具有较好的感染
性，能较好的表达插入的HCV结构蛋白或融合结构蛋臼，与质粒DNA载
体相比，腺病毒载体具有更好的免疫效果，可望发展成为抗HCV感染的腺
病毒载体疫苗。
3．选择并合成了可与 H工‘分于结合的 5条 HCV结构基回区 CTL表位
多肽，建立了体夕「特异性CTL的诱导、增）rt的力法，诱导产生了了5种HCV
特异性CTL。
4．在诱导T细胞疫苗之前，用＊＊V腺炳毒载体或质粒载体讨小鼠进
厅免疫致敏，可以明显提高T细胞疫苗的活性；其中腺病毒载体的免疫致
敏效果要明显好于质粒载体。
5．将诱导的5种＊＊V特异性C＊L，在体外进了丁特异性杀伤实验，证
一
实，出n、P3和 P4三条多肽诱导的脾纠＊泡含较高比例的 CD。＋纠1巾d，叫－产
上较好的CTL杀伤活性，可以用作T细胞疫苗进fi体内抗HCV实验。
6．建立了HCV结构基囚体外细胞转染系SPyo－NCV，爿。以此细胞系
建立了皮下移植瘤 HCV小鼠模型，经免疫组化叶大，SPZ／0－HCV细胞及其
移植瘤组织内均有HCV结构蛋白的表达。
7．将诱导的solkHCV特异性CTL，以T纠1）地疫苗的形式免疫皮下移
仙册NCV小鼠栓型，证丈，山PI、n和N三条多肌诱导的T纠1胞疫析对
6
父四军医大学阵土学位沦文
＋1（V｛＄趴W人叫卜的抑制作川。
8．将3种丁细胞疫苗与表达＊＊V结构基因的质粒＊＊A疫苗在＊＊v
移他瘤小鼠捎型内进行治疗对比，发现djJ者明显忧于后者，提示，T细胞疫
祈在NCV感染的防治研究中可能具有较奸的发展汕景。
HCV infection has been one of the most important damaging infectious diseases world-wide. Without a reliable cell culture system in vitro and successful infection model, study on HCV and prevention of hepatitis C have been extremely limited. Construction and packing of replication-defective adenovirus vectors expressing HCV structural proteins will play an important role in research of HCV and vaccination.
HCV infection often result in persistent infection. Moreover, it has been known that chronic HCV infection is associated with high risk of liver cirrhosis and hepatocellular carcinoma but without perfect prevention and therapeutic procedure. Exploration of new way to vaccination and clearance of HCV will be a great challenge. T-cell vaccine is being noticed as a new way of HCV prevention and clearance, and research on preparation of T-cell vaccine and its clearance against HCV will contribute to it.
We constructed 3 kinds of shuttle plasmid encoding HCV structural proteins by means of molecular cloning technique; based on it, 3 kinds of
adenovirus vectors of HCV structural proteins were packed, screened and identified. We also selected and synthesized 5 kinds of CTL epitope peptides of HCV structural gene which induced 5 kinds of corresponding HCV specific CTL, which were evaluated by experiments in vivo and in vitro. Furthermore, we discussed the influence of immune-sensibilization before induction to the activity of T-cell vaccine, compared the immune-sensibilization difference between HCV-adenovirus vectors and plasmid vectors. We also compared T-cell vaccine with DNA vaccine on in vivo inhibition to HCV transplanting tumor to evaluate the difference effects of protection from HCV infection. Conclusion:
1 The 3 constructed adenovirus vectors shuttle plasmid of HCV structural gene. pAd.HCV-C. pAd.HCV-CEl and pAd.HCV-CE!E2. were confirmed to be inserted the sequences of HCV C, C+E1 and C+E1+E2 by double endonucleases, PCR and sequencing.
2 After co-transfection with the 3 kinds of shuttle plasmid and adenovirus genome plasmid (p.IM17) into 293 cell. 3 HCV structural protein expressing adenovirus vectors. pAd.HCV-C, pAd.HCV-CEl and pAd.HCV-CE!E2 were packed and screened by virus plaques technique, identified to be good infectivity and expressivity of inserted HCV structural proteins or fusional structural. In contrast to plasmid DNA vector, adenovirus vectors appeared to be better immunizational effects and with the hope of being developed to be vaccine against HCV.
3 We also selected and synthesized 5 CTL epitopes peptides of HCV structural gene which were capable of combining with H-2d molecule, established methods of in vitro elicitation and proliferation of specific CTL and
obtained 5 kinds of specific CTLs.
4 We observed that T cell vaccine activity could be greatly improved through immune-sensibilization to mice with HCV adenovirus vectors or plasmid vector before T-cell vaccine induction, and adenovirus vectors proved to be superior to plasmid vector.
5 In vitro specific killing experiments showed that spleen cells induced by PI, P3 and P4, which contained higher proportion of CDg+ cells and generated better CTL activity, could be introduced to in vivo experiment against HCV
6 We successfully setup a HCV structural gene in vitro cell-transfection system SP2/0-HCV, through which also setup a murine model with subcutaneous transplanting tumor of HCV. Determined by immunohistochemical technique, there were HCV structural proteins in both SP2/0-HCV and its transplanting tumor tissue.
7 When the murine model were immunized by the 5 kinds of elicited HCV-specific CTLs as a role of T-cell vaccine, significant inhibition to HCV transplanting tumor were observed in groups of PL P3. and P4.
8 Comparison were performed between the 3 T-cell vaccine and plasmid DNA vaccine expressing HCV structural gene on therapeutic effects in HCV transplanting tumor murine model. The result showed that the former was obviously superior the
引文
1 Manuel Caruso, Khiem PN, Kwong YL,et al.Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.Proc Natl Acad Sci USA,1996,93:11302-11306
2 Jon Cohen. The scientific challenge of hepatitis C. Science, 1999;285:26-30
3 Dai WJ, Jiang HC. Advances in gene therapy of liver cirrhosis: a review. World J Gastroentero, 2001;7:1-8
4 Smith AE. Viral vector in gene therapy. Annu Rev Microbiol,1995,49:807-838
5 Berkner KL. Expression of heterologous sequences in adenoviral vectors. Curr Top Microbiol Immunol,1992,158:39-66
6 Grakoui A,Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol,1993;67:1385-1395
7 J 萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯主编．金冬雁，黎孟枫等译．分子克隆实验指南．第二版．北京：科学出版社，1992
8 姜泊，张亚历，周殿元主编．分子生物学常用实验方法．第一版，北京：
人民军医出版社，1996
9 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCV C基因腺病毒表达载体骨架质粒pAd.HCV-C的构建、鉴定及表达．世界华人消化杂志，2001；9(6)635-639
10 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCV结构基因腺病毒表达载体骨架质粒pAd.HCV-S的构建、鉴定及表达．医学研究生学报，2001；14(5)377-381
11 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCVC区，E1区基因腺病毒表达载体骨架质粒pAd.HCV-CE1的构建、鉴定及表达．解放军医学杂志，2001；26(10)727-729
12 侯云德．。动物病毒载体与基因治疗的现状和前景。生物工程进展，1994；14(2)：6-10
13 Richard C,Mulligan.The basic Science of gene therapy.Science,1993,260:926
14 刘林，罗成基，袁良平等。复制缺陷型鼠IL-3重组腺病毒的构建和鉴定。免疫学杂志，1999；15(4)：242-244
15 Jon Cohen. The scientific challenge of hepatitis C. Science, 1999;285:26-30
16 Suzanne L, Elliott Stephanie Pye, Thuy Le, et al. Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems. Vaccine, 1999; 17:2009-2019
17 Bona CA, Cssares S, Brumeanu TD. Towards development of T-cell vaccines.Immunol Today,1998;19(3):126-133
2 Jon Cohen. The scientific challenge of hepatitis C. Science, 1999;285:26-30
3 Dai WJ, Jiang HC. Advances in gene therapy of liver cirrhosis: a review. World J Gastroentero, 2001;7:1-8
4 Smith AE. Viral vector in gene therapy. Annu Rev Microbiol,1995,49:807-838
5 Berkner KL. Expression of heterologous sequences in adenoviral vectors. Curr Top Microbiol Immunol,1992,158:39-66
6 Grakoui A,Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol,1993;67:1385-1395
7 J 萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯主编．金冬雁，黎孟枫等译．分子克隆实验指南．第二版．北京：科学出版社，1992
8 姜泊，张亚历，周殿元主编．分子生物学常用实验方法．第一版，北京：
人民军医出版社，1996
9 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCV C基因腺病毒表达载体骨架质粒pAd.HCV-C的构建、鉴定及表达．世界华人消化杂志，2001；9(6)635-639
10 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCV结构基因腺病毒表达载体骨架质粒pAd.HCV-S的构建、鉴定及表达．医学研究生学报，2001；14(5)377-381
11 郝春秋，周永兴，冯志华，李谨革，贾战生，王平忠．HCVC区，E1区基因腺病毒表达载体骨架质粒pAd.HCV-CE1的构建、鉴定及表达．解放军医学杂志，2001；26(10)727-729
12 侯云德．。动物病毒载体与基因治疗的现状和前景。生物工程进展，1994；14(2)：6-10
13 Richard C,Mulligan.The basic Science of gene therapy.Science,1993,260:926
14 刘林，罗成基，袁良平等。复制缺陷型鼠IL-3重组腺病毒的构建和鉴定。免疫学杂志，1999；15(4)：242-244
15 Jon Cohen. The scientific challenge of hepatitis C. Science, 1999;285:26-30
16 Suzanne L, Elliott Stephanie Pye, Thuy Le, et al. Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems. Vaccine, 1999; 17:2009-2019
17 Bona CA, Cssares S, Brumeanu TD. Towards development of T-cell vaccines.Immunol Today,1998;19(3):126-133