人乳頭瘤病毒的基因分型及HPV16亞型L1、E6和E7基因的克隆和序列分析
發(fā)布時間：2018-08-12 11:20
【摘要】： 背景 人乳頭瘤病毒是一類特異感染人皮膚、粘膜的雙鏈環(huán)狀DNA病毒，屬乳多空病毒科多瘤病毒亞科，其分子量大約為8000bp。目前已有100多種HPV亞型被鑒定，其中約40余種與人類生殖器皮膚黏膜病變有關。根據(jù)其對生殖系統(tǒng)的致癌性，可將其分為低危型和高危型，前者包括HPV6，11，40，42，43，44，54，61，70，72，81等，以HPV6，11最為常見，主要引起尖銳濕疣等良性病變；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最為常見，常引起惡性病變，如宮頸癌。由于HPV的致病性與其亞型密切相關，所以對其進行檢測和分型對于臨床診斷具有重要意義。 另外，對HPV16亞型HPV病毒E6，E7，L1基因突變的研究表明特定位點的突變會使病毒更易誘導產(chǎn)生癌變及增大再次感染或從宿主免疫系統(tǒng)逃逸的機會。如HPV16型E6蛋白83位的亮氨酸殘基變?yōu)槔i氨酸(堿基突變T350G)的突變株在瑞典婦女宮頸癌組織中出現(xiàn)的比例遠大于野生型，一些研究認為在部分人群中，該突變與組織癌變進程相關。不同的變異導致的氨基酸的變化可影響宿主對病毒的免疫應答，進而與細胞惡性轉化的危險性相關。因此對宮頸癌組織中HPV16亞型基因組突變的研究對宮頸癌疫苗研制策略及HPV致癌機理的研究也具有重要意義。 目的 1．探討各種婦科疾病與人乳頭瘤病毒感染的關系，建立不同婦科疾病和不同年齡段婦女人乳頭瘤病毒感染的流行病學資料庫。 2．了解中國西南地區(qū)HPV16亞型基因的一級結構特點，為臨床診斷、治療由HPV16亞型感染引起的疾病特別是宮頸癌提供理論依據(jù)。 方法 1．應用反向點雜交技術分別對疾病組1512例病人標本和正常組500例標本進行HPV檢測，所得數(shù)據(jù)用SPSS13.0軟件包進行統(tǒng)計分析。 2．收集宮頸癌組織標本，針對HPV16亞型分別設計L1、E6和E7基因特異性引物，用PCR方法從宮頸癌組織中獲得各基因的全長序列，并構建克隆載體pMD18-T，進行序列分析，同時分別以L1、E6和E7基因序列進行進化分析。 結果 1．正常組500例標本中，陽性標本101例，感染率為20.2％；疾病組1512例標本中，陽性標本523例，感染率為34.6％，其中雙重感染82例，占陽性標本的15.7％，多重感染34例，占陽性標本的6.5％，而高危感染340例，感染率為22.5％，占陽性標本的65.0％，其中單純HPV16亞型感染155例，占所有陽性標本的29.6％；不同年齡段、不同性生活史、不同婦科疾病以及不同首次性生活年齡的HPV感染檢出率都有顯著性差異。 2．與德國標準株相比，四個標本中總共發(fā)現(xiàn)20個突變位點，其中8個為四個標本所共有：131(G-A)(Gly-Arg)、178(T-G)(Asp-Glu)、350(G-T)(Val-Leu)、647(A-G)(Asn-Asp)、846(T-C)(同義突變)、L1基因第966(C-T)(同義突變)、L1基因第1302(C-T)(同義突變)和L1基因第1434(A-G)(同義突變)。 結論 1．正常組和疾病組的HPV感染率有顯著性差異(x~2=36.37，P＜0.01)。在HPV感染的的普查當中可以有重點的選擇高危人群，這樣更加有針對性，從而大大降低普查成本。 2．在所檢測的23種亞型種，高危型HPV16亞型所占比例最高，提示我們HPV16亞型對于宮頸疾病的發(fā)生具有重要的作用，需要我們對其進行深入的研究。 3．與標準序列比較，中國成渝地區(qū)HPV16亞型L1、E6和E7基因存在一定范圍的變異，提示我們，在預防治療成渝地區(qū)婦女宮頸癌病人以及研制開發(fā)針對成渝地區(qū)預防宮頸癌的HPV疫苗時，需要注意這些位點的變異，特別是一些引起氨基酸變異的位點。
[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.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373
本文編號：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.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373
【參考文獻】
相關期刊論文 前6條
1 曹建彪;李世榮;朱秋屏;谷淑燕;李雅君;晨智敏;張紅光;;人乳頭瘤病毒與結直腸癌相關性的研究[J];世界華人消化雜志;2000年01期
2 馬群風;劉錕;王云杰;程慶書;;食管癌術后胸胃出血伴近端胃壞死1例[J];世界華人消化雜志;2000年01期
3 譚曉燕,趙靜,劉小寧,吳麗容,馮碧清,黃春英;珠海市正常人群婦女宮頸人乳頭狀瘤病毒感染情況分析[J];中國婦幼保健;2005年14期
4 李昭,張士偉,劉靜,孫保存,趙秀蘭,李正言,劉淑英;女性生殖道人乳頭瘤病毒感染與宮頸癌前病變和宮頸癌的關系[J];中國腫瘤臨床;1999年02期
5 王金花;子宮頸刮片中人乳頭瘤病毒的基因分型[J];中華實驗和臨床病毒學雜志;1999年01期
6 喬友林,章文華,李凌,潘秦鏡,楊玲,吳令英,戍壽德,李愛玲,張詢,任生達,Belinson J;子宮頸癌篩查方法的橫斷面比較研究[J];中國醫(yī)學科學院學報;2002年01期
本文編號：2178894
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