治療性HPV疫苗的研制與動物實驗研究
發(fā)布時間：2019-03-01 16:33
【摘要】：人類乳頭瘤狀病毒(Human papillomavirus, HPV)感染是宮頸癌的主要危險因子,近年來分子流行病學(xué)發(fā)現(xiàn),HPV感染與口腔鱗癌的發(fā)生也有著密切的關(guān)系,HPV作為一個獨立的危險因子,與口咽癌和口腔癌密切相關(guān)。25%的HNSCC可檢測到HPV的DNA,尤其是HPV16,90%的HPV相關(guān)頭頸部腫瘤由HPV16引起。但是HPV在不同地區(qū)的檢出率及檢出型別報道不一,本課題首先對HPV在武漢地區(qū)的OSCC患者中的發(fā)生率進行了流行病學(xué)的調(diào)查。 另外,盡管目前有預(yù)防性疫苗和各種篩查措施來預(yù)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腫瘤是由HPV引起。很多研究都證明業(yè)已存在的HPV預(yù)防性疫苗對尚未感染過該病毒的人群有很好的預(yù)防作用,但是不能清除已經(jīng)存在的病毒感染,因此,HPV治療性疫苗的發(fā)展對治療HPV相關(guān)腫瘤有著重大意義。 本研究包含三個部分： 第一部分：HPV在武漢地區(qū)的口腔癌患者中的流行病學(xué)調(diào)查 收集從2009到2013年間診斷為OSCC的病例標(biāo)本以及健康人群的第三磨牙拔除術(shù)中的牙齦組織,樣本收集后立即儲存于-80℃冰箱備用。用Qiagen試劑盒提取新鮮／冰凍組織中的總DNA,選取HPV通用引物GP5+/GP6+(5'-TTTGTTACTGTG GTAGATACYAC-3'/5'-GAAAAATAAACTGTAAATCATATTC-3'),進行PCR分析,陽性結(jié)果送往生工測序鑒定HPV型別。SPSS20.0軟件分析統(tǒng)計結(jié)果。結(jié)果顯示OSCC組的HPV感染率達到27.5%,而對照組的HPV感染率是2.9%。 第二部分：HPV治療性疫苗的構(gòu)建與檢測 1、HPV疫苗的構(gòu)建 為了降低HPV的轉(zhuǎn)化活性,HPV16E7的第24位、26位和91位氨基酸C24E26和C91修改為了甘氨酸,HPV16E6的第63位和106為氨基酸C63和C106也修改為甘氨酸以破壞原有的鋅指結(jié)構(gòu)。把E7E6的基因序列直接連接,然后用人緣化密碼子優(yōu)化,得到的融合蛋白的基因序列在Invitrogen公司合成,并克隆大pVAX1載體上,命名為pE7E6。 然后E7E6基因片段通過兩端的KpnI和NotI酶切位點克隆到防齲疫苗pGJAP/VAX1上,得到帶有靶向序列CTLA-4的疫苗pCTLA4-E7E6。同樣,未經(jīng)過修改的對照質(zhì)粒疫苗命名為pwCTLA4-E7E6和pwE7E6,為了增強對照,未經(jīng)修改過氨基酸序列的野生型E7和野生型E6也分別克隆到載體pVAX1上得到pwE7和pwE6。所有質(zhì)粒都經(jīng)DNA測序鑒定其正確性。結(jié)論：靶向HPV疫苗pCTLA4-E7E6及非靶向?qū)φ找呙鏿E7E6、野生型對照疫苗pwCTLA4-E7E6、 pwE7E6、pE7、pE6成功構(gòu)建。 2、HPV疫苗的性能及機制檢測 構(gòu)建好的疫苗以對照疫苗分別用Lipofectamine轉(zhuǎn)染試劑轉(zhuǎn)染293細胞,48h后提取表達的蛋白,分別檢測E7、E6、p53、Rb等蛋白的表達。同時,用ELISA試驗檢測轉(zhuǎn)染上清中的蛋白濃度,然后用流式細胞術(shù)檢測融合蛋白結(jié)合DC2.4細胞系的能力。 結(jié)論：新構(gòu)建的融合CTLA-4及E7E6優(yōu)化序列的質(zhì)粒DNA,在體外細胞內(nèi)能夠正確表達相關(guān)蛋白,并且與p53、Rb等抑癌因子的結(jié)合力下降,無致癌危險。體外培養(yǎng)細胞上清中分泌的融合蛋白能夠結(jié)合到DC2.4細胞系,證實其靶向性作用機制。 第三部分：HPV治療性疫苗在小鼠體內(nèi)外的實驗研究 質(zhì)粒pCTLA4-E7E6、pE7E6、pVAX1經(jīng)真空干燥后重新溶解到生理鹽水中以得到DNA的終濃度為1μg/μl. 1、治療性免疫 選用6-8周齡的C57BL/6小鼠30只,隨機分成4組：pCTLA4-E7E6、pE7E6、 pVAX1和PBS組。每只小鼠皮下注射2×105個TC-1細胞,在第10天的時候給予第一次免疫,免疫組小鼠在左后腿肌肉內(nèi)注射100μL質(zhì)粒,對照組注射100μLpVAX1或PBS。所有小鼠在第17天接受第二次免疫。每周記錄腫瘤大小2-3次。第0、24、38天的時候收集小鼠上清做ELISA實驗,分析小鼠體內(nèi)抗體水平。在第38天,取脾細胞做CTL分析。結(jié)論：pCTLA4-E7E6誘導(dǎo)了比pE7E6更強的治療性免疫效果。 2、預(yù)防性免疫 6-8周齡的C57BL/6小鼠32只隨機分成4組：pCTLA4-E7E6、pE7E6、pVAX1和PBS組。在第0、14天進行免疫,第28天,每只小鼠注射6×104個TC-1細胞,第0、14、28、60天的時候收集小鼠上清做ELISA實驗,分析小鼠體內(nèi)抗體水平。在第80天,取脾細胞做CTL分析。結(jié)論：pCTLA4-E7E6誘導(dǎo)了比pE7E6更強的預(yù)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.8
本文編號：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.8
【參考文獻】
相關(guān)期刊論文 前1條
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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