治疗性HPV疫苗的研制与动物实验研究
发布时间：2019-03-01 16:33
【摘要】：人类乳头瘤状病毒(Human papillomavirus, HPV)感染是宫颈癌的主要危险因子,近年来分子流行病学发现,HPV感染与口腔鳞癌的发生也有着密切的关系,HPV作为一个独立的危险因子,与口咽癌和口腔癌密切相关。25%的HNSCC可检测到HPV的DNA,尤其是HPV16,90%的HPV相关头颈部肿瘤由HPV16引起。但是HPV在不同地区的检出率及检出型别报道不一,本课题首先对HPV在武汉地区的OSCC患者中的发生率进行了流行病学的调查。 另外,尽管目前有预防性疫苗和各种筛查措施来预防HPV的感染,但HPV引起的疾病在全球依然很普遍,全球有三分之一的感染性肿瘤是由HPV引起。很多研究都证明业已存在的HPV预防性疫苗对尚未感染过该病毒的人群有很好的预防作用,但是不能清除已经存在的病毒感染,因此,HPV治疗性疫苗的发展对治疗HPV相关肿瘤有着重大意义。 本研究包含三个部分： 第一部分：HPV在武汉地区的口腔癌患者中的流行病学调查 收集从2009到2013年间诊断为OSCC的病例标本以及健康人群的第三磨牙拔除术中的牙龈组织,样本收集后立即储存于-80℃冰箱备用。用Qiagen试剂盒提取新鲜／冰冻组织中的总DNA,选取HPV通用引物GP5+/GP6+(5'-TTTGTTACTGTG GTAGATACYAC-3'/5'-GAAAAATAAACTGTAAATCATATTC-3'),进行PCR分析,阳性结果送往生工测序鉴定HPV型别。SPSS20.0软件分析统计结果。结果显示OSCC组的HPV感染率达到27.5%,而对照组的HPV感染率是2.9%。 第二部分：HPV治疗性疫苗的构建与检测 1、HPV疫苗的构建 为了降低HPV的转化活性,HPV16E7的第24位、26位和91位氨基酸C24E26和C91修改为了甘氨酸,HPV16E6的第63位和106为氨基酸C63和C106也修改为甘氨酸以破坏原有的锌指结构。把E7E6的基因序列直接连接,然后用人缘化密码子优化,得到的融合蛋白的基因序列在Invitrogen公司合成,并克隆大pVAX1载体上,命名为pE7E6。 然后E7E6基因片段通过两端的KpnI和NotI酶切位点克隆到防龋疫苗pGJAP/VAX1上,得到带有靶向序列CTLA-4的疫苗pCTLA4-E7E6。同样,未经过修改的对照质粒疫苗命名为pwCTLA4-E7E6和pwE7E6,为了增强对照,未经修改过氨基酸序列的野生型E7和野生型E6也分别克隆到载体pVAX1上得到pwE7和pwE6。所有质粒都经DNA测序鉴定其正确性。结论：靶向HPV疫苗pCTLA4-E7E6及非靶向对照疫苗pE7E6、野生型对照疫苗pwCTLA4-E7E6、 pwE7E6、pE7、pE6成功构建。 2、HPV疫苗的性能及机制检测 构建好的疫苗以对照疫苗分别用Lipofectamine转染试剂转染293细胞,48h后提取表达的蛋白,分别检测E7、E6、p53、Rb等蛋白的表达。同时,用ELISA试验检测转染上清中的蛋白浓度,然后用流式细胞术检测融合蛋白结合DC2.4细胞系的能力。 结论：新构建的融合CTLA-4及E7E6优化序列的质粒DNA,在体外细胞内能够正确表达相关蛋白,并且与p53、Rb等抑癌因子的结合力下降,无致癌危险。体外培养细胞上清中分泌的融合蛋白能够结合到DC2.4细胞系,证实其靶向性作用机制。 第三部分：HPV治疗性疫苗在小鼠体内外的实验研究 质粒pCTLA4-E7E6、pE7E6、pVAX1经真空干燥后重新溶解到生理盐水中以得到DNA的终浓度为1μg/μl. 1、治疗性免疫 选用6-8周龄的C57BL/6小鼠30只,随机分成4组：pCTLA4-E7E6、pE7E6、 pVAX1和PBS组。每只小鼠皮下注射2×105个TC-1细胞,在第10天的时候给予第一次免疫,免疫组小鼠在左后腿肌肉内注射100μL质粒,对照组注射100μLpVAX1或PBS。所有小鼠在第17天接受第二次免疫。每周记录肿瘤大小2-3次。第0、24、38天的时候收集小鼠上清做ELISA实验,分析小鼠体内抗体水平。在第38天,取脾细胞做CTL分析。结论：pCTLA4-E7E6诱导了比pE7E6更强的治疗性免疫效果。 2、预防性免疫 6-8周龄的C57BL/6小鼠32只随机分成4组：pCTLA4-E7E6、pE7E6、pVAX1和PBS组。在第0、14天进行免疫,第28天,每只小鼠注射6×104个TC-1细胞,第0、14、28、60天的时候收集小鼠上清做ELISA实验,分析小鼠体内抗体水平。在第80天,取脾细胞做CTL分析。结论：pCTLA4-E7E6诱导了比pE7E6更强的预防性免疫效果。
[Abstract]:Human papillomavirus (HPV) infection is the main risk factor of cervical cancer. In recent years, molecular epidemiology has found that HPV infection has a close relationship with the occurrence of oral squamous cell carcinoma. HPV is an independent risk factor. HPV-related DNA, especially HPV16,90% of HPV-associated head and neck tumors were detected by HPV16 in 25% of HNSCC. However, the detection rate and the detection type of HPV in different regions are different, and the incidence of HPV in the patients with OSCC in Wuhan is investigated. In addition, despite the current preventive vaccine and various screening measures to prevent the infection of HPV, the disease caused by HPV is still widespread in the world, and one-third of the world's infectious tumors are caused by HPV As a result, many studies have shown that the already existing HPV preventive vaccine has a good preventive effect on the population that has not yet been infected with the virus, but it is not possible to remove the already existing virus infection. Therefore, the development of the HPV therapeutic vaccine is of great interest to the treatment of HPV-related tumors. I. The present study contains three Part 1: The first part: HPV is in the patients with oral cancer in Wuhan The epidemiological survey collected case specimens diagnosed as OSCC from 2009 to 2013, as well as the third molar extraction of the healthy population Gingival tissue during operation, immediately after sample collection- The total DNA in fresh/ frozen tissue was extracted with Qiagen kit, and the HPV general primers GP5 +/ GP6 + (5 '-TTGTTACTGTG GTAGATACYAC-3'/5 '-GAAAAATAAACTGTAAATCATATTC-3') were selected for PCR analysis. The positive results were sent to the raw workers for sequencing to identify the HPV type. The SPSS10.0 software analyzed the statistical results. The results showed that the HPV infection rate in the OSCC group 27.5%, while HPV in the control group The dye rate is 2.9%. The second part: HPV treatment Construction and detection of therapeutic vaccine 1. Construction of HPV vaccine to reduce the conversion activity of HPV, the 24th, 26th and 91 amino acids C24E of HPV16E7 26 and C91 are modified for glycine, and the 63 and 106 amino acids C63 and C106 of HPV16 E6 are also modified to be glycine to destroy the original zinc finger structure. The gene sequence of E7E6 is directly connected, and then the gene sequence of the fusion protein obtained by the human limbal codon is optimized, and the obtained fusion protein is synthesized by the Invitrogen company. and clone large pVAX The vector was named pE7E6. The E7E6 gene fragment was then cloned into the anti-caries vaccine pGJAP/ VAX1 by the KpnI and NotI cleavage sites at both ends to give the vaccine pCTLA4-E7E6 with the targeting sequence CTLA-4. Also, the unmodified control plasmid vaccine was named pwCTLA4-E7E. In order to enhance the control, the wild-type E7 and the wild-type E6 of the unmodified amino acid sequence were also cloned into the vector pVAX1 to obtain pwE7 and pwE6, respectively. All the plasmids were identified by DNA sequencing. Conclusion: The target HPV vaccine pCTLA4-E7E6 and the non-targeted control vaccine pE7E6, the wild-type control vaccine pwCTLA4- E7E6, pWE 7. The successful construction of E6, pE7 and pE6 and 2, the performance and the mechanism of the HPV vaccine detect the constructed vaccine to transfect the 293 cells with the Lipofectamine transfection reagent, respectively, and extract the expressed protein after 48 hours to respectively detect the E7; and the expression of the protein such as E6, p53, Rb and the like is detected by an ELISA test, The ability of the fusion protein to bind to the DC2.4 cell line was tested. Conclusion: The plasmid DNA of the newly constructed fusion CTLA-4 and E7E6 optimized sequence is in vivo. The relevant protein can be expressed correctly in the external cell, and the binding force of the cancer-inhibiting factors such as p53, Rb and the like is reduced, and the cancer-free risk can be avoided. The fusion protein secreted in the supernatant of the in-vitro culture cell can be The targeting mechanism of DC2.4 cell line was confirmed. Part III: The HPV therapeutic vaccine is external to the mouse The plasmid pCTLA4-E7E6, pE7E6 and pVAX1 were dried by vacuum. dissolved in physiological saline 30 of C57BL/6 mice were randomly divided into 4 groups: pCTLA4-E7E6, pE7E6 and pV. AX1 and PBS group. Each mouse was subcutaneously injected with 2 to 105 TC-1 cells, the first immunization was given at the time of day 10, and the immune group mice were injected with 100. m u.L of plasmid in the left hind leg muscle, and the control group was injected with 100. m u.L of pVAX1 or PBS. All mice received a second dose on day 17. The tumor size 2-was recorded weekly. The supernatant of the mice was collected at 0,24 and 38 days as an enzyme-linked immunosorbent assay (ELISA) to analyze the level of the antibody in the mice. CTLA4-E 7E6 induced a stronger therapeutic immune response than pE7E6.2. The C57BL/6 mice at 6-8 weeks of age were randomly divided into 4 groups: pCTLA4- E7E6, pE7E6, pVAX1, and PBS group were immunized on day 0 and 14, and 6-104 TC-1 cells were injected into each mouse at day 0,14,28 and 60, and the antibody level in mice was analyzed by ELISA.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.8
本文编号：2432605
[Abstract]:Human papillomavirus (HPV) infection is the main risk factor of cervical cancer. In recent years, molecular epidemiology has found that HPV infection has a close relationship with the occurrence of oral squamous cell carcinoma. HPV is an independent risk factor. HPV-related DNA, especially HPV16,90% of HPV-associated head and neck tumors were detected by HPV16 in 25% of HNSCC. However, the detection rate and the detection type of HPV in different regions are different, and the incidence of HPV in the patients with OSCC in Wuhan is investigated. In addition, despite the current preventive vaccine and various screening measures to prevent the infection of HPV, the disease caused by HPV is still widespread in the world, and one-third of the world's infectious tumors are caused by HPV As a result, many studies have shown that the already existing HPV preventive vaccine has a good preventive effect on the population that has not yet been infected with the virus, but it is not possible to remove the already existing virus infection. Therefore, the development of the HPV therapeutic vaccine is of great interest to the treatment of HPV-related tumors. I. The present study contains three Part 1: The first part: HPV is in the patients with oral cancer in Wuhan The epidemiological survey collected case specimens diagnosed as OSCC from 2009 to 2013, as well as the third molar extraction of the healthy population Gingival tissue during operation, immediately after sample collection- The total DNA in fresh/ frozen tissue was extracted with Qiagen kit, and the HPV general primers GP5 +/ GP6 + (5 '-TTGTTACTGTG GTAGATACYAC-3'/5 '-GAAAAATAAACTGTAAATCATATTC-3') were selected for PCR analysis. The positive results were sent to the raw workers for sequencing to identify the HPV type. The SPSS10.0 software analyzed the statistical results. The results showed that the HPV infection rate in the OSCC group 27.5%, while HPV in the control group The dye rate is 2.9%. The second part: HPV treatment Construction and detection of therapeutic vaccine 1. Construction of HPV vaccine to reduce the conversion activity of HPV, the 24th, 26th and 91 amino acids C24E of HPV16E7 26 and C91 are modified for glycine, and the 63 and 106 amino acids C63 and C106 of HPV16 E6 are also modified to be glycine to destroy the original zinc finger structure. The gene sequence of E7E6 is directly connected, and then the gene sequence of the fusion protein obtained by the human limbal codon is optimized, and the obtained fusion protein is synthesized by the Invitrogen company. and clone large pVAX The vector was named pE7E6. The E7E6 gene fragment was then cloned into the anti-caries vaccine pGJAP/ VAX1 by the KpnI and NotI cleavage sites at both ends to give the vaccine pCTLA4-E7E6 with the targeting sequence CTLA-4. Also, the unmodified control plasmid vaccine was named pwCTLA4-E7E. In order to enhance the control, the wild-type E7 and the wild-type E6 of the unmodified amino acid sequence were also cloned into the vector pVAX1 to obtain pwE7 and pwE6, respectively. All the plasmids were identified by DNA sequencing. Conclusion: The target HPV vaccine pCTLA4-E7E6 and the non-targeted control vaccine pE7E6, the wild-type control vaccine pwCTLA4- E7E6, pWE 7. The successful construction of E6, pE7 and pE6 and 2, the performance and the mechanism of the HPV vaccine detect the constructed vaccine to transfect the 293 cells with the Lipofectamine transfection reagent, respectively, and extract the expressed protein after 48 hours to respectively detect the E7; and the expression of the protein such as E6, p53, Rb and the like is detected by an ELISA test, The ability of the fusion protein to bind to the DC2.4 cell line was tested. Conclusion: The plasmid DNA of the newly constructed fusion CTLA-4 and E7E6 optimized sequence is in vivo. The relevant protein can be expressed correctly in the external cell, and the binding force of the cancer-inhibiting factors such as p53, Rb and the like is reduced, and the cancer-free risk can be avoided. The fusion protein secreted in the supernatant of the in-vitro culture cell can be The targeting mechanism of DC2.4 cell line was confirmed. Part III: The HPV therapeutic vaccine is external to the mouse The plasmid pCTLA4-E7E6, pE7E6 and pVAX1 were dried by vacuum. dissolved in physiological saline 30 of C57BL/6 mice were randomly divided into 4 groups: pCTLA4-E7E6, pE7E6 and pV. AX1 and PBS group. Each mouse was subcutaneously injected with 2 to 105 TC-1 cells, the first immunization was given at the time of day 10, and the immune group mice were injected with 100. m u.L of plasmid in the left hind leg muscle, and the control group was injected with 100. m u.L of pVAX1 or PBS. All mice received a second dose on day 17. The tumor size 2-was recorded weekly. The supernatant of the mice was collected at 0,24 and 38 days as an enzyme-linked immunosorbent assay (ELISA) to analyze the level of the antibody in the mice. CTLA4-E 7E6 induced a stronger therapeutic immune response than pE7E6.2. The C57BL/6 mice at 6-8 weeks of age were randomly divided into 4 groups: pCTLA4- E7E6, pE7E6, pVAX1, and PBS group were immunized on day 0 and 14, and 6-104 TC-1 cells were injected into each mouse at day 0,14,28 and 60, and the antibody level in mice was analyzed by ELISA.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.8
【参考文献】
相关期刊论文 前1条
1 ;Human Papillomavirus as an Independent Predictor in Oral Squamous Cell Cancer[J];International Journal of Oral Science;2009年03期
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