人乳頭瘤病毒（HPV）16，-18，-58型單克隆抗體的制備分析及初步應(yīng)用
發(fā)布時間：2018-07-29 09:51
【摘要】：在全球范圍內(nèi),宮頸癌是婦女第二位高發(fā)的惡性腫瘤。我國是宮頸癌的高發(fā)區(qū),在經(jīng)濟落后地區(qū),宮頸癌的危害更為嚴(yán)重。慢性高危型人乳頭瘤病毒(Human papillomavirus, HPV)的感染是宮頸癌的主要誘因。此外,低危型HPV中的HPV-6,-11可誘發(fā)90%的生殖器疣。在世界范圍內(nèi),高危型HPV-16、-18是位居前兩位的優(yōu)勢流行病毒株,誘發(fā)約70%的宮頸癌。在我國,除HPV-16,-18外(檢出率分別為58.7%和11%),高危型HPV-58的檢出率也很高(7.2%),居于第三位。因此,針對HPV-16,-18,-58疫苗的相關(guān)研究值得重視。 目前國外已有兩種HPV L1病毒樣顆粒(Virus-like particles, VLPs)疫苗上市,分別是Merck公司利用酵母表達體系生產(chǎn)的含四價疫苗(HPV-16、-18、-6、-11L1VLPs),及GSK公司利用桿狀病毒-昆蟲細胞表達生產(chǎn)的二價疫苗(HPV-16、-18L1VLPs)。臨床實驗結(jié)果顯示,兩種疫苗均具有很好的安全性、免疫原性及保護性,而且疫苗的免疫保護活性與疫苗接種血清中和抗體的水平具有很好的相關(guān)性。由于HPV感染嚴(yán)格限制在人的上皮組織、生活周期為上皮分化依賴的,體外難以大量培養(yǎng)獲得病毒顆粒,缺乏有效的動物模型,因此準(zhǔn)確評價疫苗接種人群后誘發(fā)的血清中和抗體水平存在很大的困難。另外,和大多數(shù)基因工程蛋白不同,HPV L1VLPs屬于高度組裝的重組蛋白,誘發(fā)的保護性抗體識別的表位大多是L1蛋白構(gòu)象依賴的。變性或錯誤折疊的L1蛋白不具有保護活性,其越接近天然病毒,保護活性越高,因此在疫苗研制過程中需要對體外表達獲得的L1重組蛋白的活性進行檢測。HPV中和單抗可用于檢測L1重組蛋白中誘發(fā)保護性抗體的活性位點。H16.V5和H18.J4分別是國外報道的HPV-16、-18型特異性構(gòu)象表位依賴中和單抗,均已被應(yīng)用于ELISA方法對HPV-16、-18L1VLPs疫苗生產(chǎn)過程中的有效成分的質(zhì)量控制；此外H16.V5和H18.J4還被用于建立競爭性免疫分析方法(Competitive Luminex immunoassay, cLIA),對免疫人群產(chǎn)生的HPV-16,-18型特異性中和抗體進行高通量評價,結(jié)果顯示基于H16.V5的cLIA方法靈敏,檢測結(jié)果與中和抗體實際水平一致；但基于H18.J4的cLIA方法的靈敏度較差,某些cLIA陰性血清中仍可檢測到HPV-18中和抗體,有待于進一步優(yōu)化；目前未見有包含HPV-58VLPs的疫苗問世,也未見有HPV-58單抗應(yīng)用于疫苗研究的報道。 對HPV感染機制的初步研究結(jié)果表明L1蛋白包含多個涉及病毒感染入胞的關(guān)鍵位點,目前這些位點的序列特征尚不清楚,對其進一步的研究需要多種不同類型單抗的輔助。本實驗室正在研發(fā)包含HPV-16,-18,-58型多價L1VLPs混合性疫苗,為了配合適合我國國情L1VLPs疫苗的研發(fā),本研究采用雜交瘤技術(shù),制備針對HPV-16,-18,-58的中和單抗,分析了抗體的中和活性即具有50%感染抑制率時的單抗?jié)舛?50%inhibitory concentrations, IC50)、單抗表位特征、單抗間及單抗與H16.V5、H18.J4標(biāo)準(zhǔn)株的識別表位是否一致等。在此基礎(chǔ)上建立了基于HPV-16中和單抗的雙抗夾心ELISA方法,對L1VLPs進行定量檢測。研究結(jié)果為HPV-16、-18.-58L4VLPs疫苗的研發(fā)及HPV感染入胞機制的深入研究打下了基礎(chǔ)。 獲得了10株HPV-16構(gòu)象表位依賴中和單抗,IC50范圍為0.03-1.72nM。所識別表位的性質(zhì)與H16.V5一樣依賴于病毒子粒；相加實驗顯示除XM16-1和XM16-5、XM16-3和XM16-6疑似分別識別相同表位外,其它單抗的識別表位均不同,且與H16.V5的表位不同；交叉中和活性分析顯示,除XM16-17可交叉中和HPV-18外,其余均為型特異性中和單抗。還獲得了10株線性表位依賴中和單抗,IC50范圍為1.78-4.17nM,其中有5株可交叉中和HPV-18和/或-58,如XM16-13、XM16-16、XM16-20可交叉中和HPV-18,XM16-15可交叉中和HPV-58,XM16-14可交叉中和HPV-18、-58。以構(gòu)象表位依賴單抗XM16-6為捕獲抗體、HRP標(biāo)記的線性表位依賴單抗XM16-12為酶標(biāo)抗體建立了雙抗夾心ELISA方法,測定范圍為0.05-1.5μg/mL,穩(wěn)定性好。 獲得了7株HPV-18型特異性構(gòu)象表位依賴中和單抗,IC50范圍為0.04-0.89nM。所識別表位性質(zhì)與H18.J4(識別表位依賴于完整VLPs)不同,均為子粒內(nèi)表位；相加實驗結(jié)果提示單抗間識別表位均不相同,也不同于H18.J4的識別表位。還獲得了6株線性表位依賴中和單抗,IC50范圍為0.44-2.34nM,其中3株(XM18-12、XM18-13、XM18-14)可交叉中和HPV-6,其余均為型特異性單抗。 獲得了7株HPV-58型特異性構(gòu)象表位依賴中和單抗,IC50范圍為0.045-1.0nM；其中3株單抗(XM58-6、XM58-7、XM58-8)識別表位依賴于完整VLPs,其余均識別子粒內(nèi)表位；相加實驗結(jié)果提示各單抗間識別表位均不同。還獲得了9株線性表位依賴中和單抗,IC50范圍為0.44-1.17nM；其中XM-21和XM58-14可交叉中和HPV-18,XM-24可交叉中和HPV-6,而XM58-13可交叉中和HPV-11,其余為型特異性單抗。建立了基于線性表位依賴中和單抗XM-22的間接ELIA方法,但該方法穩(wěn)定性欠佳。 結(jié)果表明：HPV-16、-18、-58L1VLPs構(gòu)象表位依賴的中和單抗多是型特異性單抗,型特異性中和活性強。絕大多數(shù)構(gòu)象表位依賴單抗與H16.V5一樣識別表位依賴于子粒,并發(fā)現(xiàn)H18.J4識別表位依賴于完整VLPs,提示研發(fā)獲得的各株子粒依賴單抗在疫苗質(zhì)控及免疫活性檢測中具有潛在的應(yīng)用價值；首次報道了3株識別表位依賴于完整VLPs的HPV-58型特異性中和單抗。線性表位依賴中和單抗中一半具有交叉中和活性,型特異性中和活性較弱。研究獲得的3株可交叉中和HPV-6的HPV-18單抗、4株可交叉中和HPV-18或-6或-11的HPV-58單抗尚未見有文獻報道,并獲得了5株可交叉中和HPV-18和／或HPV-58的HPV-16單抗。以HPV-16中和單抗為基礎(chǔ)建立的雙抗夾心ELISA方法在疫苗質(zhì)控中具有應(yīng)用價值。研究獲得的這些特性各異的單抗可望為病毒感染入胞機制的研究及HPV L1VLPs預(yù)防性疫苗的研究提供實驗材料。
[Abstract]:In the world, cervical cancer is the second high incidence of malignant tumor in women. China is a high incidence area of cervical cancer. In economically backward areas, cervical cancer is more serious. Chronic high-risk human papillomavirus (Human papillomavirus, HPV) infection is the main cause of cervical cancer. In addition, HPV-6 in low risk HPV, -11 can induce 90%. Genital warts. In the world, high risk HPV-16 and -18 are the top two dominant virus strains and induce about 70% of cervical cancer. In our country, in addition to HPV-16 and -18 (58.7% and 11% respectively), the detection rate of high risk HPV-58 is also high (7.2%) and resides in third. Therefore, the related research on HPV-16, -18, -58 vaccine should be paid attention to.
At present, there are two kinds of HPV L1 virus like particles (Virus-like particles, VLPs) vaccines listed in foreign countries, which are the four valent vaccines (HPV-16, -18, -6, -11L1VLPs) produced by Merck company in yeast expression system, and GSK company using baculovirus insect cells to express the production of vaccine (HPV-16, two). The results of clinical experiments show that two The vaccine has good safety, immunogenicity and protection, and the immune protection activity of the vaccine has a good correlation with the level of antibody in the vaccinated serum. Because HPV infection is strictly limited in human epithelial tissue and the life cycle is dependent on the epithelial differentiation, it is difficult to cultivate virus particles in vitro. Effective animal models are so difficult to accurately evaluate the level of serum neutralization antibodies induced by vaccinated populations. In addition, unlike most genetic engineering proteins, HPV L1VLPs is a highly assembled recombinant protein, and much of the epitopes of protective antibody recognition are dependent on the conformation of L1 proteins. The L1 protein does not have protective activity, the closer to the natural virus, the higher the protective activity, so in the process of developing the vaccine, the activity of the recombinant protein of the recombinant protein, which is obtained in vitro, is needed to detect the activity of the.HPV neutralization monoclonal antibody which can be used to detect the protective antibody of the recombinant protein in the L1 recombinant protein,.H16.V5 and H18.J4, respectively, the foreign reported HPV -16, type -18 specific conformation epitopes and monoclonal antibodies have been applied to the quality control of the effective components of the HPV-16, -18L1VLPs vaccine production by the ELISA method; in addition, H16.V5 and H18.J4 are also used to establish a competitive immunoassay (Competitive Luminex immunoassay, cLIA) and HPV-16 The high flux evaluation of the heterosexual and neutralizing antibodies showed that the H16.V5 based cLIA method was sensitive and the detection results were consistent with the actual level of neutralizing antibody; but the sensitivity of the cLIA method based on H18.J4 was poor, and the HPV-18 neutralization antibody in some cLIA negative sera was still to be further optimized; there was no HPV-58VLPs containing the HPV-58VLPs at present. The vaccine has been published, and no HPV-58 monoclonal antibody has been used in vaccine research.
A preliminary study of the mechanism of HPV infection indicates that L1 protein contains a number of key sites involved in the infection of the virus, and the sequence characteristics of these sites are not yet clear. The further study of these sites requires the assistance of a variety of different types of monoclonal antibodies. This laboratory is developing a mixed HPV-16, -18, -58 polyvalent L1VLPs mixed vaccine. It is suitable for the research and development of L1VLPs vaccine in our country. This study uses hybridoma technique to prepare neutralization monoclonal antibody against HPV-16, -18 and -58. The neutralization activity of antibody, that is, the concentration of monoclonal antibody (50%inhibitory concentrations, IC50) with 50% infection inhibition rate (50%inhibitory concentrations, IC50), the epitope characteristic of the monoclonal antibody, the monoclonal antibody and the monoclonal antibody and H16.V5, and the identification of the H18.J4 standard strain On the basis of this, a double anti sandwich ELISA method based on HPV-16 neutralization monoclonal antibody was established for quantitative detection of L1VLPs. The results of the study lay the foundation for the development of HPV-16, the development of -18.-58L4VLPs vaccine and the in-depth study of the mechanism of HPV infection.
10 HPV-16 conformation epitopes and monoclonal antibodies were obtained. The properties of the epitopes identified by the IC50 range 0.03-1.72nM. were as dependent on the virus particles as H16.V5; the addition experiments showed that except XM16-1 and XM16-5, XM16-3 and XM16-6 identified the same epitopes respectively, and the other mAbs were different from the epitopes of the H16.V5; Neutralization activity analysis showed that except XM16-17 and HPV-18, the rest were type specific neutralization McAbs, and 10 linear epitope dependent neutralization McAbs were also obtained, and IC50 range was 1.78-4.17nM, of which 5 could cross neutralize HPV-18 and / or -58, such as XM16-13, XM16-16, XM16-20 intersecting and HPV-18, XM16-15 crossover neutralization HPV-58. The conformational epitopes dependent monoclonal antibody XM16-6 was used as a capture antibody in intersecting neutralization and HPV-18, and the linear epitope dependent monoclonal antibody XM16-12 of HRP was used to establish a double anti sandwich ELISA method for the enzyme labeled antibody. The determination range was 0.05-1.5 u g/mL, and the stability was good.
7 HPV-18 specific conformation epitopes and monoclonal antibodies were obtained. The epitopes identified by the IC50 range 0.04-0.89nM. were different from the H18.J4 (the recognition epitope depended on the complete VLPs), both were in the grain epitopes, and the results suggested that the epitopes of the McAb were different and different from the H18.J4 recognition epitopes, and 6 linear tables were also obtained. The median IC50 was 0.44-2.34nM, and 3 strains (XM18-12, XM18-13, XM18-14) were able to cross neutralize HPV-6 and the rest were type specific mAbs.
7 HPV-58 type specific conformation dependent epitopes and monoclonal antibodies were obtained, and IC50 range was 0.045-1.0nM; the identification epitopes of 3 monoclonal antibodies (XM58-6, XM58-7, XM58-8) were dependent on the complete VLPs, and the rest identified the epitopes in the grains; the addition of the experimental results suggested that the recognition epitopes of the McAbs were all different. 9 linear epitopes and McAbs, IC, IC were also obtained. The 50 range is 0.44-1.17nM; in which XM-21 and XM58-14 can cross neutralize HPV-18, XM-24 can cross neutralize HPV-6, and XM58-13 can cross neutralize HPV-11, and the rest are type specific monoclonal antibodies. The indirect ELIA method based on linear epitope dependence and monoclonal antibody XM-22 is established, but the method is not stable.
The results showed that HPV-16, -18, and -58L1VLPs conformation epitopes depended on the type specific monoclonal antibody, and the type specific neutralization activity was strong. Most conformation epitopes dependent on the H16.V5 like epitopes dependent on the grain, and the H18.J4 recognition epitope depended on the complete VLPs. The seedling quality control and immunological activity detection have potential application value; 3 specific HPV-58 specific neutralization monoclonal antibodies, which are dependent on the complete VLPs, are reported for the first time. Half of the linear epitopes and McAbs have cross neutralization activity, and the type specific neutralization activity is weak. 3 strains of cross neutralization and HPV-6 have been obtained. 4 The HPV-58 McAbs which can cross neutralize HPV-18 or -6 or -11 have not been reported, and 5 HPV-16 McAbs which can be cross neutralized with HPV-18 and / or HPV-58 are obtained. The double resistance sandwich ELISA method based on HPV-16 neutralization monoclonal antibody is of value in the quality control of vaccine. The mechanism of infection and the study of HPV L1VLPs preventive vaccine provide experimental materials.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
本文編號：2152301
[Abstract]:In the world, cervical cancer is the second high incidence of malignant tumor in women. China is a high incidence area of cervical cancer. In economically backward areas, cervical cancer is more serious. Chronic high-risk human papillomavirus (Human papillomavirus, HPV) infection is the main cause of cervical cancer. In addition, HPV-6 in low risk HPV, -11 can induce 90%. Genital warts. In the world, high risk HPV-16 and -18 are the top two dominant virus strains and induce about 70% of cervical cancer. In our country, in addition to HPV-16 and -18 (58.7% and 11% respectively), the detection rate of high risk HPV-58 is also high (7.2%) and resides in third. Therefore, the related research on HPV-16, -18, -58 vaccine should be paid attention to.
At present, there are two kinds of HPV L1 virus like particles (Virus-like particles, VLPs) vaccines listed in foreign countries, which are the four valent vaccines (HPV-16, -18, -6, -11L1VLPs) produced by Merck company in yeast expression system, and GSK company using baculovirus insect cells to express the production of vaccine (HPV-16, two). The results of clinical experiments show that two The vaccine has good safety, immunogenicity and protection, and the immune protection activity of the vaccine has a good correlation with the level of antibody in the vaccinated serum. Because HPV infection is strictly limited in human epithelial tissue and the life cycle is dependent on the epithelial differentiation, it is difficult to cultivate virus particles in vitro. Effective animal models are so difficult to accurately evaluate the level of serum neutralization antibodies induced by vaccinated populations. In addition, unlike most genetic engineering proteins, HPV L1VLPs is a highly assembled recombinant protein, and much of the epitopes of protective antibody recognition are dependent on the conformation of L1 proteins. The L1 protein does not have protective activity, the closer to the natural virus, the higher the protective activity, so in the process of developing the vaccine, the activity of the recombinant protein of the recombinant protein, which is obtained in vitro, is needed to detect the activity of the.HPV neutralization monoclonal antibody which can be used to detect the protective antibody of the recombinant protein in the L1 recombinant protein,.H16.V5 and H18.J4, respectively, the foreign reported HPV -16, type -18 specific conformation epitopes and monoclonal antibodies have been applied to the quality control of the effective components of the HPV-16, -18L1VLPs vaccine production by the ELISA method; in addition, H16.V5 and H18.J4 are also used to establish a competitive immunoassay (Competitive Luminex immunoassay, cLIA) and HPV-16 The high flux evaluation of the heterosexual and neutralizing antibodies showed that the H16.V5 based cLIA method was sensitive and the detection results were consistent with the actual level of neutralizing antibody; but the sensitivity of the cLIA method based on H18.J4 was poor, and the HPV-18 neutralization antibody in some cLIA negative sera was still to be further optimized; there was no HPV-58VLPs containing the HPV-58VLPs at present. The vaccine has been published, and no HPV-58 monoclonal antibody has been used in vaccine research.
A preliminary study of the mechanism of HPV infection indicates that L1 protein contains a number of key sites involved in the infection of the virus, and the sequence characteristics of these sites are not yet clear. The further study of these sites requires the assistance of a variety of different types of monoclonal antibodies. This laboratory is developing a mixed HPV-16, -18, -58 polyvalent L1VLPs mixed vaccine. It is suitable for the research and development of L1VLPs vaccine in our country. This study uses hybridoma technique to prepare neutralization monoclonal antibody against HPV-16, -18 and -58. The neutralization activity of antibody, that is, the concentration of monoclonal antibody (50%inhibitory concentrations, IC50) with 50% infection inhibition rate (50%inhibitory concentrations, IC50), the epitope characteristic of the monoclonal antibody, the monoclonal antibody and the monoclonal antibody and H16.V5, and the identification of the H18.J4 standard strain On the basis of this, a double anti sandwich ELISA method based on HPV-16 neutralization monoclonal antibody was established for quantitative detection of L1VLPs. The results of the study lay the foundation for the development of HPV-16, the development of -18.-58L4VLPs vaccine and the in-depth study of the mechanism of HPV infection.
10 HPV-16 conformation epitopes and monoclonal antibodies were obtained. The properties of the epitopes identified by the IC50 range 0.03-1.72nM. were as dependent on the virus particles as H16.V5; the addition experiments showed that except XM16-1 and XM16-5, XM16-3 and XM16-6 identified the same epitopes respectively, and the other mAbs were different from the epitopes of the H16.V5; Neutralization activity analysis showed that except XM16-17 and HPV-18, the rest were type specific neutralization McAbs, and 10 linear epitope dependent neutralization McAbs were also obtained, and IC50 range was 1.78-4.17nM, of which 5 could cross neutralize HPV-18 and / or -58, such as XM16-13, XM16-16, XM16-20 intersecting and HPV-18, XM16-15 crossover neutralization HPV-58. The conformational epitopes dependent monoclonal antibody XM16-6 was used as a capture antibody in intersecting neutralization and HPV-18, and the linear epitope dependent monoclonal antibody XM16-12 of HRP was used to establish a double anti sandwich ELISA method for the enzyme labeled antibody. The determination range was 0.05-1.5 u g/mL, and the stability was good.
7 HPV-18 specific conformation epitopes and monoclonal antibodies were obtained. The epitopes identified by the IC50 range 0.04-0.89nM. were different from the H18.J4 (the recognition epitope depended on the complete VLPs), both were in the grain epitopes, and the results suggested that the epitopes of the McAb were different and different from the H18.J4 recognition epitopes, and 6 linear tables were also obtained. The median IC50 was 0.44-2.34nM, and 3 strains (XM18-12, XM18-13, XM18-14) were able to cross neutralize HPV-6 and the rest were type specific mAbs.
7 HPV-58 type specific conformation dependent epitopes and monoclonal antibodies were obtained, and IC50 range was 0.045-1.0nM; the identification epitopes of 3 monoclonal antibodies (XM58-6, XM58-7, XM58-8) were dependent on the complete VLPs, and the rest identified the epitopes in the grains; the addition of the experimental results suggested that the recognition epitopes of the McAbs were all different. 9 linear epitopes and McAbs, IC, IC were also obtained. The 50 range is 0.44-1.17nM; in which XM-21 and XM58-14 can cross neutralize HPV-18, XM-24 can cross neutralize HPV-6, and XM58-13 can cross neutralize HPV-11, and the rest are type specific monoclonal antibodies. The indirect ELIA method based on linear epitope dependence and monoclonal antibody XM-22 is established, but the method is not stable.
The results showed that HPV-16, -18, and -58L1VLPs conformation epitopes depended on the type specific monoclonal antibody, and the type specific neutralization activity was strong. Most conformation epitopes dependent on the H16.V5 like epitopes dependent on the grain, and the H18.J4 recognition epitope depended on the complete VLPs. The seedling quality control and immunological activity detection have potential application value; 3 specific HPV-58 specific neutralization monoclonal antibodies, which are dependent on the complete VLPs, are reported for the first time. Half of the linear epitopes and McAbs have cross neutralization activity, and the type specific neutralization activity is weak. 3 strains of cross neutralization and HPV-6 have been obtained. 4 The HPV-58 McAbs which can cross neutralize HPV-18 or -6 or -11 have not been reported, and 5 HPV-16 McAbs which can be cross neutralized with HPV-18 and / or HPV-58 are obtained. The double resistance sandwich ELISA method based on HPV-16 neutralization monoclonal antibody is of value in the quality control of vaccine. The mechanism of infection and the study of HPV L1VLPs preventive vaccine provide experimental materials.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
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