人乳头瘤病毒（HPV）16，-18，-58型单克隆抗体的制备分析及初步应用
发布时间：2018-07-29 09:51
【摘要】：在全球范围内,宫颈癌是妇女第二位高发的恶性肿瘤。我国是宫颈癌的高发区,在经济落后地区,宫颈癌的危害更为严重。慢性高危型人乳头瘤病毒(Human papillomavirus, HPV)的感染是宫颈癌的主要诱因。此外,低危型HPV中的HPV-6,-11可诱发90%的生殖器疣。在世界范围内,高危型HPV-16、-18是位居前两位的优势流行病毒株,诱发约70%的宫颈癌。在我国,除HPV-16,-18外(检出率分别为58.7%和11%),高危型HPV-58的检出率也很高(7.2%),居于第三位。因此,针对HPV-16,-18,-58疫苗的相关研究值得重视。 目前国外已有两种HPV L1病毒样颗粒(Virus-like particles, VLPs)疫苗上市,分别是Merck公司利用酵母表达体系生产的含四价疫苗(HPV-16、-18、-6、-11L1VLPs),及GSK公司利用杆状病毒-昆虫细胞表达生产的二价疫苗(HPV-16、-18L1VLPs)。临床实验结果显示,两种疫苗均具有很好的安全性、免疫原性及保护性,而且疫苗的免疫保护活性与疫苗接种血清中和抗体的水平具有很好的相关性。由于HPV感染严格限制在人的上皮组织、生活周期为上皮分化依赖的,体外难以大量培养获得病毒颗粒,缺乏有效的动物模型,因此准确评价疫苗接种人群后诱发的血清中和抗体水平存在很大的困难。另外,和大多数基因工程蛋白不同,HPV L1VLPs属于高度组装的重组蛋白,诱发的保护性抗体识别的表位大多是L1蛋白构象依赖的。变性或错误折叠的L1蛋白不具有保护活性,其越接近天然病毒,保护活性越高,因此在疫苗研制过程中需要对体外表达获得的L1重组蛋白的活性进行检测。HPV中和单抗可用于检测L1重组蛋白中诱发保护性抗体的活性位点。H16.V5和H18.J4分别是国外报道的HPV-16、-18型特异性构象表位依赖中和单抗,均已被应用于ELISA方法对HPV-16、-18L1VLPs疫苗生产过程中的有效成分的质量控制；此外H16.V5和H18.J4还被用于建立竞争性免疫分析方法(Competitive Luminex immunoassay, cLIA),对免疫人群产生的HPV-16,-18型特异性中和抗体进行高通量评价,结果显示基于H16.V5的cLIA方法灵敏,检测结果与中和抗体实际水平一致；但基于H18.J4的cLIA方法的灵敏度较差,某些cLIA阴性血清中仍可检测到HPV-18中和抗体,有待于进一步优化；目前未见有包含HPV-58VLPs的疫苗问世,也未见有HPV-58单抗应用于疫苗研究的报道。 对HPV感染机制的初步研究结果表明L1蛋白包含多个涉及病毒感染入胞的关键位点,目前这些位点的序列特征尚不清楚,对其进一步的研究需要多种不同类型单抗的辅助。本实验室正在研发包含HPV-16,-18,-58型多价L1VLPs混合性疫苗,为了配合适合我国国情L1VLPs疫苗的研发,本研究采用杂交瘤技术,制备针对HPV-16,-18,-58的中和单抗,分析了抗体的中和活性即具有50%感染抑制率时的单抗浓度(50%inhibitory concentrations, IC50)、单抗表位特征、单抗间及单抗与H16.V5、H18.J4标准株的识别表位是否一致等。在此基础上建立了基于HPV-16中和单抗的双抗夹心ELISA方法,对L1VLPs进行定量检测。研究结果为HPV-16、-18.-58L4VLPs疫苗的研发及HPV感染入胞机制的深入研究打下了基础。 获得了10株HPV-16构象表位依赖中和单抗,IC50范围为0.03-1.72nM。所识别表位的性质与H16.V5一样依赖于病毒子粒；相加实验显示除XM16-1和XM16-5、XM16-3和XM16-6疑似分别识别相同表位外,其它单抗的识别表位均不同,且与H16.V5的表位不同；交叉中和活性分析显示,除XM16-17可交叉中和HPV-18外,其余均为型特异性中和单抗。还获得了10株线性表位依赖中和单抗,IC50范围为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。以构象表位依赖单抗XM16-6为捕获抗体、HRP标记的线性表位依赖单抗XM16-12为酶标抗体建立了双抗夹心ELISA方法,测定范围为0.05-1.5μg/mL,稳定性好。 获得了7株HPV-18型特异性构象表位依赖中和单抗,IC50范围为0.04-0.89nM。所识别表位性质与H18.J4(识别表位依赖于完整VLPs)不同,均为子粒内表位；相加实验结果提示单抗间识别表位均不相同,也不同于H18.J4的识别表位。还获得了6株线性表位依赖中和单抗,IC50范围为0.44-2.34nM,其中3株(XM18-12、XM18-13、XM18-14)可交叉中和HPV-6,其余均为型特异性单抗。 获得了7株HPV-58型特异性构象表位依赖中和单抗,IC50范围为0.045-1.0nM；其中3株单抗(XM58-6、XM58-7、XM58-8)识别表位依赖于完整VLPs,其余均识别子粒内表位；相加实验结果提示各单抗间识别表位均不同。还获得了9株线性表位依赖中和单抗,IC50范围为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,其余为型特异性单抗。建立了基于线性表位依赖中和单抗XM-22的间接ELIA方法,但该方法稳定性欠佳。 结果表明：HPV-16、-18、-58L1VLPs构象表位依赖的中和单抗多是型特异性单抗,型特异性中和活性强。绝大多数构象表位依赖单抗与H16.V5一样识别表位依赖于子粒,并发现H18.J4识别表位依赖于完整VLPs,提示研发获得的各株子粒依赖单抗在疫苗质控及免疫活性检测中具有潜在的应用价值；首次报道了3株识别表位依赖于完整VLPs的HPV-58型特异性中和单抗。线性表位依赖中和单抗中一半具有交叉中和活性,型特异性中和活性较弱。研究获得的3株可交叉中和HPV-6的HPV-18单抗、4株可交叉中和HPV-18或-6或-11的HPV-58单抗尚未见有文献报道,并获得了5株可交叉中和HPV-18和／或HPV-58的HPV-16单抗。以HPV-16中和单抗为基础建立的双抗夹心ELISA方法在疫苗质控中具有应用价值。研究获得的这些特性各异的单抗可望为病毒感染入胞机制的研究及HPV L1VLPs预防性疫苗的研究提供实验材料。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 邹明发,林嘉友,刘尔翔,吴安然;IDENTIFICATION OF ANTI-HUMAN IgG MONOCLONAL ANTIBODIES AND MONOCLONAL ANTIBODIES WITH CONFINED SUBCLASS REACTIVITY[J];Science in China,Ser.B;1984年08期
2 许雪梅;;人乳头瘤病毒及宫颈癌疫苗的研究——解读2008年诺贝尔生理学或医学奖[J];生物化学与生物物理进展;2008年10期
3 苏洁;王斌;鲁晓晴;赵巍;闫志勇;钱冬萌;丁守怡;宋旭霞;杨丽;;D2蛋白酶双抗体夹心ELISA定量检测方法的建立[J];细胞与分子免疫学杂志;2010年03期
相关硕士学位论文 前1条
1 王金霞;双抗夹心ELISA定量检测血液中rHu IFN-ω的研究[D];重庆大学;2007年
，本文编号：2152301
本文链接：https://www.wllwen.com/xiyixuelunwen/2152301.html
最近更新
教材专著
