人乳头瘤病毒假病毒制备方法的优化以及高通量中和实验检测系统的建立

发布时间：2018-05-31 07:23

本文选题：人乳头瘤病毒 + 假病毒　；参考：《厦门大学》2008年硕士论文

【摘要】： 人乳头瘤病毒(HPV)能够引起包括宫颈癌在内的多种生殖道疾病,严重危害人类健康。HPV具有严格的宿主特异性和组织分化依赖性,难以通过常规组织培养方法扩增,也难以从病变组织中分离获得足够的病毒。因此建立简便、高效的HPV体外感染模型是发展HPV预防疫苗与治疗药物的迫切需要。近来研究发现HPV假病毒可用于在体外有效模拟HPV的感染,以此基础建立的中和实验方法具有较突出准确、便捷的优点,在HPV预防疫苗、中和抗体研究中显示出巨大的应用潜力。HPV假病毒中和实验方法的广泛使用需要进一步提高假病毒构建效率,同时能够满足高通量检测的需要。本实验室已应用磷酸钙多质粒共转染方法在293FT细胞中建立了构建HPV假病毒的方法,本研究以此为基础,进一步对HPV假病毒构建体系进行系统改进和优化,建立了更为高效且适合高通量检测的HPV中和实验方法。建立简便高效、高通量的假病毒中和实验检测方法对提高实验效率,满足大规模抗体检测的需要具有十分重要的意义。同时,建立多个型别的HPV假病毒中和实验模型为具有高保护力的多价疫苗的开发提供了全面的中和抗体评价体系。本研究首先探讨了在HPV假病毒构建体系中优化质粒转染比例以及磷酸钙转染条件对假病毒构建效率的影响。结果显示,在该体系中不同的HPV结构蛋白表达质粒转染比例对于假病毒构建效率有显著影响,HPV主要结构蛋白L1的表达水平是影响构建效率的主要因素,L2表达质粒的用量过高或过低均不利于假病毒的构建。报告质粒的用量对假病毒构建效率的影响相对较小。综合比较显示,在该体系中L1和L2表达质粒和报告质粒以合适比例(1:1/10:1/2)进行共转染可获得较理想的构建效率。同时通过优化磷酸钙转染试剂的用量和DNA在磷酸钙溶液中浓度获得了较优的转染条件。通过优化改进,本研究建立了更为高效的HPV假病毒大量制备方法,在HPV16、HPV18、HPV6、HPV11假病毒构建中的应用结果显示相比原方法可显著提高构建效率10～20倍。为HPV假病毒中和实验的规模化应用提供了有利条件。为适应高通量检测的需要,本研究通过改进报告基因及检测方法建立了新型的HPV假病毒中和实验体系。本研究选择半乳糖苷酶作为报告基因,构建了携带优化后的半乳糖苷酶表达元件的HPV假病毒,研究结果显示,假病毒感染后的细胞加入显色底物后可呈现显著的蓝色,并且该标记可为酶联免疫图像分析系统(Elispot)所检测。酶联免疫图像分析系统可对显色后的96孔细胞培养板进行连续快速的自动扫描并计数,满足了高通量检测的需要。本研究进一步对显色方法进行优化,提高了检测效率。通过综合应用优化的半乳糖苷酶报告基因表达元件与酶联免疫图像分析系统,本研究建立新型的可满足高通量检测要求的HPV假病毒中和实验方法。本研究应用该方法对四型HPV中和单抗的鉴定结果表明,半乳糖苷酶假病毒中和实验方法与目前应用的EGFP假病毒中和实验方法的检测灵敏度相当,同时新方法还具有可自动连续检测、操作简便、结果显示直观的优点,可更好地满足大规模中和抗体检测实验的需要。本研究应用该方法鉴定获得了2株HPV16中和单抗,3株HPV18中和单抗,以及HPV6和HPV11中和单抗各5株;同时对本中心生产的HPV16、HPV18、HPV6和HPV11四价VLP疫苗的抗体保护性进行了评价,为临床实验阶段所需疫苗的合适剂量提供了参考。 HPV58和HPV52是中国较为流行的高危HPV型别,但一直以来研究较少。建立HPV58和HPV52假病毒中和实验模型对于研究HPV58和HPV52以及预防疫苗的开发具有重要意义。我们将HPV58和HPV52的衣壳蛋白表达序列进行密码子人源化优化后,克隆到表达载体上,与报告质粒分别共转染293FT获得了感染性的HPV58和HPV52假病毒。将质粒通过尾静脉高压注射方式免疫小鼠,成功在小鼠体内诱导了高滴度的HPV58和HPV52的中和性抗体。Western blotting结果显示这两型假病毒在约55KD处均具有L1蛋白活性。多抗血清交叉中和实验表明HPV58和HPV52、HPV16和HPV58之间存在一定水平的抗体交叉保护,而HPV16和HPV52之间没有可检测的抗体交叉保护性。
[Abstract]:Human papillomavirus (HPV) can cause a variety of reproductive tract diseases, including cervical cancer, which seriously endangers human health and.HPV has strict host specificity and tissue differentiation dependence. It is difficult to expand by conventional tissue culture method, and it is difficult to isolate enough virus from the pathological tissue. Therefore, a simple and efficient HPV in vitro is established. The infection model is an urgent need for the development of HPV vaccine and therapeutic drugs. Recent studies have found that HPV pseudo virus can be used to effectively simulate HPV infection in vitro. The neutralization experiment based on this method has the advantages of prominent accuracy and convenience. It has shown great potential application potential of.HPV false disease in HPV vaccine and anti body research. The extensive use of toxic and experimental methods needs to further improve the efficiency of pseudo virus construction and meet the needs of high throughput detection. This laboratory has established a method of constructing HPV pseudo virus in 293FT cells by using calcium phosphate multi plasmids co transfection method. This study is based on this and further studies the construction of HPV pseudo virus. System improvement and optimization, a more efficient and high-throughput HPV neutralization test method has been established.
The establishment of a simple, efficient, high throughput test method for pseudo virus neutralization test is of great significance to improve the efficiency of the experiment and meet the needs of large-scale antibody detection. At the same time, the establishment of HPV pseudo virus neutralization experiment model of multiple types provides a comprehensive neutralization antibody evaluation system for the development of high protective polyvalent vaccines.
In this study, the effect of optimized plasmid transfection ratio and calcium phosphate transfection conditions on the efficiency of pseudo virus construction in the HPV pseudo virus construction system was investigated. The results showed that the transfection ratio of different HPV structure protein plasmids in the system had significant influence on the efficiency of pseudo virus construction, and the expression level of the main structural protein L1 of HPV was The main factors affecting the construction efficiency, the high or low dosage of the L2 expression plasmid are not conducive to the construction of the pseudo virus. The effect of the dosage of the reported plasmid on the construction efficiency of the pseudo virus is relatively small. The comprehensive comparison shows that the L1 and L2 expression plasmids and the reported plasmids can be co transfected with a suitable ratio (1:1/10:1/2) in the system. At the same time, better transfection conditions were obtained by optimizing the dosage of calcium phosphate transfection reagent and the concentration of DNA in calcium phosphate solution. Through optimization and improvement, a more efficient method for preparing HPV pseudo virus was established. The application results in the construction of HPV16, HPV18, HPV6, HPV11 pseudo virus showed that the original method was significant compared with the original method. The construction efficiency is increased 10~20 times, which provides favorable conditions for the large-scale application of HPV virus neutralization test.
In order to meet the needs of high throughput detection, a new type of HPV pseudo virus neutralization experiment system was established by improving the reporter gene and detection methods. This study selected galactosidase as a reporter gene and constructed a HPV pseudo virus carrying the optimized galactosidase expression element. The results showed that the cells after the false virus infection were added to the virus. The chromogenic substrate can present a significant blue color, and the marker can be detected by the enzyme linked immunoimage analysis system (Elispot). The enzyme linked immunoimage analysis system can continuously and quickly automatically scan and count the 96 pore cell culture plates after the color display, and meet the needs of high throughput test. This study further studies the color rendering method. In this study, a new HPV pseudo virus neutralization experiment was established to meet the requirements of high throughput detection. This method was applied to the identification of four type HPV neutralization McAbs and the results of this study showed that galactoside was used for the identification of galactoside. The enzyme pseudo virus neutralization test method is similar to the detection sensitivity of the EGFP pseudo virus neutralization test method, and the new method also has the advantages of automatic continuous detection, simple operation and direct display, which can better meet the needs of large-scale neutralization antibody test. 2 HPV1 strains were identified by this method. 6 neutralization mAb, 3 HPV18 neutralization McAbs, and 5 strains of HPV6 and HPV 11 and McAb respectively. The antibody protection of HPV16, HPV18, HPV6 and HPV11 tetravalent VLP vaccines produced in this center was evaluated to provide reference for the appropriate dosage of the vaccine needed in the clinical trial stage.
HPV58 and HPV52 are the most prevalent high risk HPV types in China, but have been less studied. The establishment of HPV58 and HPV52 pseudo virus neutralization experimental models is of great significance for the study of HPV58 and HPV52 and the development of vaccine prevention. We have optimized the expression sequence of the capsid protein of HPV58 and HPV52 and cloned the expression load. In vivo, the infected HPV58 and HPV52 pseudo virus were obtained by CO transfection of 293FT with the reported plasmid. The plasmid was immunized with the tail vein high pressure injection in mice. The high titer HPV58 and HPV52 neutralizing antibody.Western blotting results were successfully induced in mice. The results showed that the two types of false viruses had L1 protein activity at about 55KD. The cross neutralization test of polyclonal sera showed that there was a certain level of cross protection between HPV58 and HPV52, HPV16 and HPV58, and there was no detectable cross protection between HPV16 and HPV52.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373;R737.33

【共引文献】