人乳头瘤病毒的基因分型及HPV16亚型L1、E6和E7基因的克隆和序列分析
发布时间：2018-08-12 11:20
【摘要】： 背景 人乳头瘤病毒是一类特异感染人皮肤、粘膜的双链环状DNA病毒，属乳多空病毒科多瘤病毒亚科，其分子量大约为8000bp。目前已有100多种HPV亚型被鉴定，其中约40余种与人类生殖器皮肤黏膜病变有关。根据其对生殖系统的致癌性，可将其分为低危型和高危型，前者包括HPV6，11，40，42，43，44，54，61，70，72，81等，以HPV6，11最为常见，主要引起尖锐湿疣等良性病变；后者包括HPV16，18，31，33，35，39，45，51，52，56，58，59，，68，72，82等，其中以HPV16，18最为常见，常引起恶性病变，如宫颈癌。由于HPV的致病性与其亚型密切相关，所以对其进行检测和分型对于临床诊断具有重要意义。 另外，对HPV16亚型HPV病毒E6，E7，L1基因突变的研究表明特定位点的突变会使病毒更易诱导产生癌变及增大再次感染或从宿主免疫系统逃逸的机会。如HPV16型E6蛋白83位的亮氨酸残基变为缬氨酸(碱基突变T350G)的突变株在瑞典妇女宫颈癌组织中出现的比例远大于野生型，一些研究认为在部分人群中，该突变与组织癌变进程相关。不同的变异导致的氨基酸的变化可影响宿主对病毒的免疫应答，进而与细胞恶性转化的危险性相关。因此对宫颈癌组织中HPV16亚型基因组突变的研究对宫颈癌疫苗研制策略及HPV致癌机理的研究也具有重要意义。 目的 1．探讨各种妇科疾病与人乳头瘤病毒感染的关系，建立不同妇科疾病和不同年龄段妇女人乳头瘤病毒感染的流行病学资料库。 2．了解中国西南地区HPV16亚型基因的一级结构特点，为临床诊断、治疗由HPV16亚型感染引起的疾病特别是宫颈癌提供理论依据。 方法 1．应用反向点杂交技术分别对疾病组1512例病人标本和正常组500例标本进行HPV检测，所得数据用SPSS13.0软件包进行统计分析。 2．收集宫颈癌组织标本，针对HPV16亚型分别设计L1、E6和E7基因特异性引物，用PCR方法从宫颈癌组织中获得各基因的全长序列，并构建克隆载体pMD18-T，进行序列分析，同时分别以L1、E6和E7基因序列进行进化分析。 结果 1．正常组500例标本中，阳性标本101例，感染率为20.2％；疾病组1512例标本中，阳性标本523例，感染率为34.6％，其中双重感染82例，占阳性标本的15.7％，多重感染34例，占阳性标本的6.5％，而高危感染340例，感染率为22.5％，占阳性标本的65.0％，其中单纯HPV16亚型感染155例，占所有阳性标本的29.6％；不同年龄段、不同性生活史、不同妇科疾病以及不同首次性生活年龄的HPV感染检出率都有显著性差异。 2．与德国标准株相比，四个标本中总共发现20个突变位点，其中8个为四个标本所共有：131(G-A)(Gly-Arg)、178(T-G)(Asp-Glu)、350(G-T)(Val-Leu)、647(A-G)(Asn-Asp)、846(T-C)(同义突变)、L1基因第966(C-T)(同义突变)、L1基因第1302(C-T)(同义突变)和L1基因第1434(A-G)(同义突变)。 结论 1．正常组和疾病组的HPV感染率有显著性差异(x~2=36.37，P＜0.01)。在HPV感染的的普查当中可以有重点的选择高危人群，这样更加有针对性，从而大大降低普查成本。 2．在所检测的23种亚型种，高危型HPV16亚型所占比例最高，提示我们HPV16亚型对于宫颈疾病的发生具有重要的作用，需要我们对其进行深入的研究。 3．与标准序列比较，中国成渝地区HPV16亚型L1、E6和E7基因存在一定范围的变异，提示我们，在预防治疗成渝地区妇女宫颈癌病人以及研制开发针对成渝地区预防宫颈癌的HPV疫苗时，需要注意这些位点的变异，特别是一些引起氨基酸变异的位点。
[Abstract]:background
Human papillomavirus (HPV) is a kind of double-stranded circular DNA virus that specifically infects human skin and mucosa. It belongs to the polyomavirus subfamily of Lactopoviridae. The molecular weight of HPV is about 8000 bp. More than 100 HPV subtypes have been identified, of which about 40 are related to the pathological changes of human genital skin and mucosa. The former includes HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and HPV6, 11 is the most common, mainly causing benign lesions such as condyloma acuminatum; the latter includes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 72, 82, of which HPV16, 18 are the most common, often causing malignant lesions, such as cervical cancer. Type is closely related, so detection and typing are important for clinical diagnosis.
In addition, studies of mutations in HPV16 subtype E6, E7, L1 genes have shown that mutations at specific sites make the virus more likely to induce canceration and increase the chances of reinfection or escape from the host immune system. Some studies have suggested that the mutation is associated with the progression of tissue canceration in some populations. Changes in amino acids caused by different mutations may affect the host's immune response to the virus, which in turn is associated with the risk of malignant transformation of cells. The research is of great significance for the research of cervical cancer vaccine development strategy and the mechanism of HPV carcinogenesis.
objective
1. To explore the relationship between various gynecological diseases and human papillomavirus infection, and to establish an epidemiological database of human papillomavirus infection in different gynecological diseases and women of different ages.
2. To understand the primary structural characteristics of HPV16 subtype gene in southwest China, and to provide theoretical basis for clinical diagnosis and treatment of diseases caused by HPV16 subtype infection, especially cervical cancer.
Method
1. Reverse dot blot hybridization was used to detect HPV in 1512 cases of disease group and 500 cases of normal group. The data were analyzed by SPSS 13.0 software package.
2. Collect specimens of cervical cancer tissues, design L1, E6 and E7 gene specific primers for HPV16 subtypes, obtain the full-length sequence of each gene from cervical cancer tissues by PCR method, and construct a clone vector pMD18-T for sequence analysis. At the same time, use L1, E6 and E7 gene sequences for evolutionary analysis.
Result
1. Of the 500 specimens in the normal group, 101 were positive, the infection rate was 20.2%. Of the 1512 specimens in the disease group, 523 were positive, the infection rate was 34.6%. 82 were double infection, accounting for 15.7% of the positive specimens, 34 were multiple infection, accounting for 6.5% of the positive specimens, and 340 were high-risk infection, accounting for 22.5%, 65.0% of the positive specimens. There were 155 cases of HPV 16 subtype infection, accounting for 29.6% of all positive specimens. The detection rates of HPV infection in different age groups, different sexual life history, different gynecological diseases and different first sexual life age were significantly different.
2. Compared with the German standard strain, 20 mutation sites were found in four specimens, 8 of which were common to four: 131 (G-A) (Gly-Arg), 178 (T-G) (Asp-Glu), 350 (G-T) (Val-Leu), 647 (A-G) (Asn-Asp), 846 (T-C) (synonymous mutation), 966 (C-T) (synonymous mutation), 1302 (C-T) (synonymous mutation) and 1434 (A-G) (synonymous mutation) of L1 gene. Synonymous mutation).
conclusion
1. There is a significant difference in the HPV infection rate between the normal group and the disease group (x~2=36.37, P<0.01). In the HPV infection census, we can focus on selecting high-risk groups, which is more targeted, thus greatly reducing the census cost.
2. Among the 23 subtypes tested, the high-risk HPV16 subtype accounted for the highest proportion, suggesting that HPV16 subtype plays an important role in the occurrence of cervical diseases, which needs further study.
3. Compared with the standard sequence, there are some variations in the L1, E6 and E7 genes of HPV16 subtypes in Chengdu-Chongqing region of China, suggesting that attention should be paid to the variations of these loci, especially some amino acid variations, in the prevention and treatment of women with cervical cancer in Chengdu-Chongqing region and in the development of HPV vaccine against cervical cancer in Chengdu-Chongqing region. Loci.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
本文编号：2178894
[Abstract]:background
Human papillomavirus (HPV) is a kind of double-stranded circular DNA virus that specifically infects human skin and mucosa. It belongs to the polyomavirus subfamily of Lactopoviridae. The molecular weight of HPV is about 8000 bp. More than 100 HPV subtypes have been identified, of which about 40 are related to the pathological changes of human genital skin and mucosa. The former includes HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and HPV6, 11 is the most common, mainly causing benign lesions such as condyloma acuminatum; the latter includes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 72, 82, of which HPV16, 18 are the most common, often causing malignant lesions, such as cervical cancer. Type is closely related, so detection and typing are important for clinical diagnosis.
In addition, studies of mutations in HPV16 subtype E6, E7, L1 genes have shown that mutations at specific sites make the virus more likely to induce canceration and increase the chances of reinfection or escape from the host immune system. Some studies have suggested that the mutation is associated with the progression of tissue canceration in some populations. Changes in amino acids caused by different mutations may affect the host's immune response to the virus, which in turn is associated with the risk of malignant transformation of cells. The research is of great significance for the research of cervical cancer vaccine development strategy and the mechanism of HPV carcinogenesis.
objective
1. To explore the relationship between various gynecological diseases and human papillomavirus infection, and to establish an epidemiological database of human papillomavirus infection in different gynecological diseases and women of different ages.
2. To understand the primary structural characteristics of HPV16 subtype gene in southwest China, and to provide theoretical basis for clinical diagnosis and treatment of diseases caused by HPV16 subtype infection, especially cervical cancer.
Method
1. Reverse dot blot hybridization was used to detect HPV in 1512 cases of disease group and 500 cases of normal group. The data were analyzed by SPSS 13.0 software package.
2. Collect specimens of cervical cancer tissues, design L1, E6 and E7 gene specific primers for HPV16 subtypes, obtain the full-length sequence of each gene from cervical cancer tissues by PCR method, and construct a clone vector pMD18-T for sequence analysis. At the same time, use L1, E6 and E7 gene sequences for evolutionary analysis.
Result
1. Of the 500 specimens in the normal group, 101 were positive, the infection rate was 20.2%. Of the 1512 specimens in the disease group, 523 were positive, the infection rate was 34.6%. 82 were double infection, accounting for 15.7% of the positive specimens, 34 were multiple infection, accounting for 6.5% of the positive specimens, and 340 were high-risk infection, accounting for 22.5%, 65.0% of the positive specimens. There were 155 cases of HPV 16 subtype infection, accounting for 29.6% of all positive specimens. The detection rates of HPV infection in different age groups, different sexual life history, different gynecological diseases and different first sexual life age were significantly different.
2. Compared with the German standard strain, 20 mutation sites were found in four specimens, 8 of which were common to four: 131 (G-A) (Gly-Arg), 178 (T-G) (Asp-Glu), 350 (G-T) (Val-Leu), 647 (A-G) (Asn-Asp), 846 (T-C) (synonymous mutation), 966 (C-T) (synonymous mutation), 1302 (C-T) (synonymous mutation) and 1434 (A-G) (synonymous mutation) of L1 gene. Synonymous mutation).
conclusion
1. There is a significant difference in the HPV infection rate between the normal group and the disease group (x~2=36.37, P<0.01). In the HPV infection census, we can focus on selecting high-risk groups, which is more targeted, thus greatly reducing the census cost.
2. Among the 23 subtypes tested, the high-risk HPV16 subtype accounted for the highest proportion, suggesting that HPV16 subtype plays an important role in the occurrence of cervical diseases, which needs further study.
3. Compared with the standard sequence, there are some variations in the L1, E6 and E7 genes of HPV16 subtypes in Chengdu-Chongqing region of China, suggesting that attention should be paid to the variations of these loci, especially some amino acid variations, in the prevention and treatment of women with cervical cancer in Chengdu-Chongqing region and in the development of HPV vaccine against cervical cancer in Chengdu-Chongqing region. Loci.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
【参考文献】
相关期刊论文 前6条
1 曹建彪;李世荣;朱秋屏;谷淑燕;李雅君;晨智敏;张红光;;人乳头瘤病毒与结直肠癌相关性的研究[J];世界华人消化杂志;2000年01期
2 马群风;刘锟;王云杰;程庆书;;食管癌术后胸胃出血伴近端胃坏死1例[J];世界华人消化杂志;2000年01期
3 谭晓燕,赵静,刘小宁,吴丽容,冯碧清,黄春英;珠海市正常人群妇女宫颈人乳头状瘤病毒感染情况分析[J];中国妇幼保健;2005年14期
4 李昭,张士伟,刘静,孙保存,赵秀兰,李正言,刘淑英;女性生殖道人乳头瘤病毒感染与宫颈癌前病变和宫颈癌的关系[J];中国肿瘤临床;1999年02期
5 王金花;子宫颈刮片中人乳头瘤病毒的基因分型[J];中华实验和临床病毒学杂志;1999年01期
6 乔友林,章文华,李凌,潘秦镜,杨玲,吴令英,戍寿德,李爱玲,张询,任生达,Belinson J;子宫颈癌筛查方法的横断面比较研究[J];中国医学科学院学报;2002年01期
本文编号：2178894
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2178894.html
最近更新
教材专著
