本文選題：分子進(jìn)化 + 病毒進(jìn)化�。� 參考：《浙江大學(xué)》2007年博士論文

【摘要】： 分子進(jìn)化研究可以幫助病毒學(xué)家了解病毒在已知限定條件下進(jìn)化時(shí)所發(fā)生的分子事件。我們計(jì)劃以甲肝病毒為模型，研究病毒在人源二倍體細(xì)胞長期連續(xù)傳代過程中的進(jìn)化熱點(diǎn)、進(jìn)化時(shí)序、進(jìn)化方向等。本文中，我們將甲肝病毒野毒H_2W在人胚肺二倍體細(xì)胞KMB_(17)連續(xù)傳代至30代，在傳代的中問代次應(yīng)用冷適應(yīng)法變異病毒，然后挑選六個(gè)子代病毒進(jìn)行基因序列分析。對H_2W及六個(gè)子代病毒的基因組RNA進(jìn)行RT-PCR擴(kuò)增，并通過PCR產(chǎn)物直接測序法測定其全長基因組序列。通過對所獲序列進(jìn)行同源性分析，總結(jié)甲肝病毒野毒在人源二倍體細(xì)胞連續(xù)傳代過程中的分子進(jìn)化信息。同時(shí)我們將所獲序列與HM175、LA等標(biāo)準(zhǔn)病毒株，以及L-A-1、MBB、HAV7等細(xì)胞適應(yīng)病毒株進(jìn)行進(jìn)化樹分析，以研究甲肝病毒在人源二倍體細(xì)胞中的進(jìn)化方向等。實(shí)驗(yàn)結(jié)果顯示第五代之前絕大多數(shù)變異已完成，第5代相比于原代野毒株，共有648個(gè)核苷酸發(fā)生變異，其中40個(gè)導(dǎo)致了氨基酸改變。從第5代到第10代僅發(fā)生一個(gè)核苷酸變異，位于5’NCR區(qū)；第10代到第14代，僅發(fā)生一個(gè)核苷酸變異，即4222位U／C突變導(dǎo)致Phe／Ser的氨基酸變異；第14代到第15代僅發(fā)生一個(gè)變異， 位于5’NCR區(qū)；從第15代到第30代，再未觀察到任何變異發(fā)生。從變異的熱點(diǎn)看，許多與病毒適應(yīng)細(xì)胞相關(guān)的熱點(diǎn)變異均發(fā)生于第5代附近，如128至136位的連續(xù)9bp缺失、176至177位的11bp插入序列，特別是3889位的C／U變異是病毒適應(yīng)細(xì)胞的標(biāo)志性變異。與毒力相關(guān)的熱點(diǎn)變異則發(fā)生第10代到第14代之間。從病毒基因變異的時(shí)序來看，大多數(shù)變異完成于第5代附近，其中包括與細(xì)胞適應(yīng)相關(guān)的關(guān)鍵變異；第5代到第15代之間僅發(fā)生極少量的變異，其中包括位于4222點(diǎn)的U／C變異，這一變異與毒力高度相關(guān)，其發(fā)生時(shí)序?yàn)榈?0到第14代之間；第15代之后的連續(xù)傳代中病毒基因組表現(xiàn)為高度穩(wěn)定。本文所獲序列與文獻(xiàn)序列進(jìn)行進(jìn)化樹分析表明，H_2W在進(jìn)化樹上趨近于GBM系列，而他的六個(gè)子代病毒在進(jìn)化樹上距離其親本較遠(yuǎn)，更加接近于L-A-1、MBB等細(xì)胞適應(yīng)株。本文所獲得的分子進(jìn)化的時(shí)序、熱點(diǎn)、基因穩(wěn)定性等資料有助于解決活疫苗病毒株育種中相關(guān)技術(shù)問題，同時(shí)對于監(jiān)測活病毒疫苗株的遺傳穩(wěn)定性等方面的研究均可提供理論支持和技術(shù)儲(chǔ)備。 第一部分 甲型肝炎病毒野毒在人源二倍體細(xì)胞連續(xù)傳代中的分子進(jìn)化研究目的 以甲肝病毒為模型，研究RNA病毒在人源二倍體細(xì)胞連續(xù)適應(yīng)傳代培養(yǎng)中的分子進(jìn)化，包括進(jìn)化熱點(diǎn)、時(shí)序、方向等。 方法 將甲肝病毒野毒H_2W在人源二倍體細(xì)胞KMg_(17)中連續(xù)傳代培養(yǎng)至30代。每隔5代選取其細(xì)胞適應(yīng)子代病毒共六個(gè)，用PCR產(chǎn)物直接測序法測定H_2W及選定的六個(gè)子代病毒的全基因序列。然后用序列分析軟件對所獲序列進(jìn)行分析，總結(jié)甲肝病毒野毒在長期的細(xì)胞連續(xù)適應(yīng)培養(yǎng)中的進(jìn)化熱點(diǎn)、進(jìn)化時(shí)序。通過對所獲基因序列與其他已知甲肝病毒株特別是細(xì)胞適應(yīng)株的進(jìn)化樹分析，研究甲肝病毒在人源二倍體細(xì)胞中的進(jìn)化方向。 結(jié)果 1．獲得了H_2W及其六個(gè)子代病毒的接近全長的基因序列，并存入GenBank，錄入號(hào)分別為：H_2W：EF406357；H_2K_5：EF406358；H_2K_(10)：EF406359；H_2K_(15)：EF406360；H_2K_(20)：EF406361；H_2K_(25)：EF406362；H_2K_(30)：EF406363 2．基于VP1／2A區(qū)168bp核苷酸片段的差異性比較，結(jié)果表明甲肝病毒野毒在適應(yīng)KMB_(17)細(xì)胞的過程中，其基因亞型由IA轉(zhuǎn)變?yōu)镮B。 3．在H_2W適應(yīng)細(xì)胞的前5代，一共有648個(gè)核苷酸發(fā)生變異，其中有40個(gè)導(dǎo)致氨基酸改變。這些變異中包含以下熱點(diǎn)變異，如第128至136位的9bp缺失、第176至177位的11bp插入、第5018到5023位的6bp缺失(導(dǎo)致二個(gè)親水氨基酸Asn、Asp缺失)、第3889位的C／U突變(導(dǎo)致Ala／Val的氨基酸變異)等是甲肝病毒野毒在適應(yīng)細(xì)胞過程中普遍發(fā)生的熱點(diǎn)變異。在第10代到第15代之間，，發(fā)生一個(gè)毒力相關(guān)的熱點(diǎn)變異，即4222位發(fā)生U／C點(diǎn)突變，該變異導(dǎo)致Phe／Ser的氨基酸突變。 4．從原代到第5代，共發(fā)生648個(gè)核苷酸變異。從第5代到第10代，僅一個(gè)核苷酸發(fā)生變異，203位缺失1bp。從第10代到第15代，僅二個(gè)核苷酸發(fā)生變異，其一位于4222位的U／C點(diǎn)突變，導(dǎo)致Phe／Ser的氨基酸突變。另一個(gè)則位于5’NCR區(qū)。第15代到第30代，無任何突變發(fā)生。絕大多數(shù)的變異完成于第5代附近，其中包括與細(xì)胞適應(yīng)相關(guān)的熱點(diǎn)變異，其后長達(dá)25代的連續(xù)傳代中僅累積發(fā)生三個(gè)變異，其中包含一個(gè)與毒力相關(guān)的變異，發(fā)生于第10代至第15代之間。 5．進(jìn)化樹分析結(jié)果顯示，H_2W在進(jìn)化樹上比較趨向于GBM系列，而他的六個(gè)子代病毒則更加趨向于L-A-1、MBB等細(xì)胞適應(yīng)株。他們在進(jìn)化樹上的距離較遠(yuǎn)。 結(jié)論 1．首次觀察到病毒在細(xì)胞中的完整進(jìn)化時(shí)序：獲得了甲肝病毒野毒在人二倍體細(xì)胞的進(jìn)化時(shí)序，細(xì)胞適應(yīng)相關(guān)基因的變異大約完成于第5代，毒力相關(guān)基因的變異介于第10代至15代之間。 2．甲肝病毒野毒適應(yīng)人二倍體細(xì)胞連續(xù)傳代的特點(diǎn)表現(xiàn)為：快速變異后的穩(wěn)定遺傳。甲肝病毒基因組在人二倍體細(xì)胞連續(xù)傳代中的穩(wěn)定性極高。進(jìn)化方向上趨近于其他細(xì)胞適應(yīng)株如MBB、L-A-1。 3．分子進(jìn)化可用于甲肝病毒的毒力基因定位研究、毒力基因變異條件的研究以及甲肝病毒基因組遺傳穩(wěn)定性研究。 4．甲肝病毒是一個(gè)研究病毒分子進(jìn)化的極有效的模型：可在多種細(xì)胞系培養(yǎng)(猴腎、肝瘤細(xì)胞、非肝二倍體細(xì)胞等等)；基因組較小，可方便地進(jìn)行全基因組序列測定。 第二部分 甲型肝炎減毒活疫苗病毒株H_2K的培育 研究目的 將甲肝病毒野毒直接適應(yīng)于人源二倍體細(xì)胞以選育一株毒力弱化且免疫原性良好的甲肝減毒活疫苗病毒株。 方法 將甲肝病毒野毒株H_2W適應(yīng)到人源二倍細(xì)胞KMB_(17)，并連續(xù)傳代培養(yǎng)至30代，在傳代培養(yǎng)的中間代次(第15至20代)使用低溫適應(yīng)法變異毒力基因。以分子進(jìn)化的相關(guān)研究方法研究毒力基因的定位、毒力基因的變異條件、毒力基因的變異時(shí)序以及基因組的穩(wěn)定性特別是變異后的毒力基因的穩(wěn)定性。根據(jù)毒力基因的變異時(shí)序選擇合適代次的細(xì)胞適應(yīng)株進(jìn)行猴體動(dòng)物實(shí)驗(yàn)，評價(jià)減毒株的毒力及免疫原性。 結(jié)果 1．H_2W在細(xì)胞傳代過程的第5代開始，其繁殖滴度穩(wěn)步增長至較高水平，其后不再增長。 2．通過序列比較研究，發(fā)現(xiàn)4222位點(diǎn)是甲肝病毒的主要毒力決定位點(diǎn)，該位點(diǎn)的變異發(fā)生于第10至第14代，其后在長達(dá)15代(第16至30代)的連續(xù)傳代中該變異一直穩(wěn)定遺傳。 3．低溫適應(yīng)不是甲肝病毒毒力基因變異的主要原因，這一點(diǎn)與流感病毒、呼吸道合胞病毒等不同。在特定細(xì)胞進(jìn)行長達(dá)10代以上的適應(yīng)傳代可能是甲肝病毒減毒的主要原因，其中細(xì)胞系的來源尤為重要。 4．甲肝病毒野毒在適應(yīng)細(xì)胞生長后，其后的連續(xù)25代(第5代到第30代)培養(yǎng)中僅積累三個(gè)變異，基因組表現(xiàn)出極高的穩(wěn)定性。 5．猴體毒力試驗(yàn)結(jié)果表明實(shí)驗(yàn)組均抗體陽轉(zhuǎn)且維持較高抗體水平，ALT轉(zhuǎn)氨酶水平無異常，肝組織未見充血、水腫、變性和壞死等病理改變。H_2K_(25)是一株較理想的活疫苗毒種候選株。 結(jié)論 1．HAV野毒在KMB17細(xì)胞連續(xù)傳代后可獲減毒，動(dòng)物試驗(yàn)證實(shí)免疫原性佳、毒力弱化。 2．經(jīng)KMB17細(xì)胞適應(yīng)后的基因組高度穩(wěn)定，毒力基因變異后穩(wěn)定遺傳。 3．成功培育了一株減毒活疫苗毒種候選株，其具備以下特征：繁殖滴度高、毒力弱化、免疫原性佳、基因組高度穩(wěn)定。
[Abstract]:The molecular evolution of hepatitis A virus in human diploid cells was studied by RT - PCR .
In the 10th to 14th passages , only one nucleotide mutation occurred , that is , 4222 U / C mutation resulted in the amino acid variation of Phe / Ser ;
Only one variation occurred in the 14th to 15th generations ,









Located in the 5 ' NCR area ;
From the 15th to 30th generations , no variation has been observed . From the point of view of variation , many of the hotspot variations associated with the virus - adapted cells occur in the vicinity of the 5th generation , such as the deletion of 9 bp in the 128 - 136 position , the deletion of the 11bp insertion sequence in the 176 - 177 position , especially the 3889 - bit C / U mutation , which occurs between the 10th and 14th generations . Most of the variation is near the 5th generation , including the key variation related to cell adaptation .
Only a small amount of variation occurs between the 5th generation and the 15th generation , including U / C variation at 4222 points , which is highly correlated with the virulence height , with a timing of between 10th and 14th generations ;
The results show that H _ 2W approaches the series in the evolution tree , and the six progeny viruses are distant from their parents in the evolution tree , which is closer to the cells of L - A - 1 and MBB . The timing , hotspot and gene stability of the molecular evolution obtained in this paper can help to solve the related technical problems in the breeding of live vaccine virus strains , and provide theoretical support and technical reserve for the research of monitoring the genetic stability of live virus vaccine strains .









the first portion









Molecular evolution of hepatitis A virus wild virus in the continuous generation of human diploid cells









Using hepatitis A virus as a model , the molecular evolution of RNA viruses in human diploid cells was studied , including evolutionary hot spots , timing , direction and so on .









method









Human diploid cells KMg _ ( 17 ) were passaged continuously for 30 generations . The sequences of all genes were determined by direct sequencing of PCR products . The evolution of hepatitis A virus in human diploid cells was analyzed by means of sequencing analysis .









Results









1 . The gene sequences near full length of H _ 2W and its six progeny viruses were obtained and stored in GenBank . The entry numbers are : H _ 2W : EF406357 ;
H _ 2K _ 5 : EF406358
H _ 2K _ ( 10 ) : EF406359 ;
H _ 2K _ ( 15 ) : EF406360 ;
H _ 2K _ ( 20 ) : EF406361 ;
H _ 2K _ ( 25 ) : EF406362 ;
H _ 2K _ ( 30 ) : EF406363









2 . Based on the difference of the nucleotide fragments in VP1 / 2A region , the results showed that the gene subtype of hepatitis A virus was changed from IA to IB during the adaptation of KMB _ ( 17 ) cells .









3 . There were 648 nucleotides in the first 5 generations of H _ 2W - adapted cells . Among them , there were 40 mutations in amino acids , such as the deletion of 9 bp in the 128 - 136 position , the deletion of 6bp at the 176 - 177 position , the C / U mutation at position 5018 to 5023 ( resulting in the amino acid variation of Ala / Val ) , etc . , which resulted in a virus - related hotspot variation between the 10th and 15th generations , that is , 4222 - bit U / C - point mutation , which resulted in the amino acid mutation of Phe / Ser .









4 . A total of 648 nucleotide variations occurred from the first to the 5th generation . From the 5th to the 10th generation , only one nucleotide had been mutated , and the 203 - bit deletion was 1bp . The mutation occurred in only two nucleotides from the 10th to 15th generations . One of the mutations was located in the vicinity of the 5th generation , including the hotspot variation associated with the cell adaptation .









5 . The results of evolutionary tree analysis showed that H _ 2W tended to be more popular in evolutionary trees , while his six progeny were more likely to adapt to cells such as L - A - 1 , MBB , etc . They were far away from the evolutionary tree .









Conclusion









1 . The complete evolution timing of the virus in cells was observed for the first time : the evolution timing of the hepatitis A virus wild virus in human diploid cells was obtained , and the variation of the cell adaptation related gene was approximately completed in the 5th generation , and the variation of the virulence - related genes was between the 10th and 15th generations .









2 . The characteristics of the continuous passaging of the human diploid cell in the hepatitis A virus are as follows : stable inheritance after rapid variation . The stability of the hepatitis A virus genome in the continuous passage of human diploid cells is extremely high . The evolution direction approaches other cell - adapted strains such as MBB , L - A - 1 .









3 . Molecular evolution can be used for the study of virulence gene localization of hepatitis A virus , the research on mutation conditions of virulence genes and the study on genetic stability of hepatitis A virus genome .









4 . Hepatitis A virus is a very effective model to study the evolution of virus molecules : it can be cultured in a variety of cell lines ( monkey kidney , liver tumor cell , non - hepatic diploid cell , etc . ) ;
The genome is small , and the whole genome sequence determination can be carried out conveniently .









the second part









Cultivation of attenuated hepatitis A virus strain H _ 2K









Purpose of study









the hepatitis A virus wild virus is directly applicable to human diploid cells to breed a hepatitis A virus attenuated live vaccine virus strain which is attenuated and has good immunogenicity .









method









The strain H _ 2W of hepatitis A virus strain was adapted to human diploid cell KMB _ ( 17 ) and cultured continuously for 30 generations . The virulence gene was studied by means of molecular evolution .









Results









1.H _ 2W increased steadily to a high level in the 5th passage of the cell passage process , and then no longer increased .









2 . Through a sequence comparison study , it was found that 4222 sites were the main virulence determinants of hepatitis A virus , the variation of which occurred in the 10th to 14th passages , followed by stable inheritance in successive passages up to 15 passages ( 16th to 30th generation ) .









3 . Low temperature adaptation is not the main cause of hepatitis A virus virulence gene mutation , which is different from influenza virus , respiratory cell virus and so on . It may be the main reason for virus shedding of hepatitis A virus in the specific cell for more than 10 generations , in which the source of cell line is very important .









4 . After adaptation to cell growth , hepatitis A virus wild virus accumulates only three variants in the subsequent 25 - generation ( 5th generation to 30th generation ) culture , and the genome exhibits extremely high stability .









5 . The results of toxicity test of monkey body showed that in the experimental group , the antibody was positive and maintained higher antibody level , ALT transaminase level was not abnormal , the liver tissue did not see congestion , edema , degeneration and necrosis . H _ 2K _ ( 25 ) was the ideal candidate for live vaccine virus .









Conclusion









1 . After continuous passage of KMB17 cells , HAV wild virus can be attenuated , and animal experiments prove that the immunogenicity is good and the virulence is weakened .









2 . After KMB17 cell adaptation , the genome was highly stable and the virulence gene was stable after mutation .









3 . A candidate strain of attenuated live vaccine is successfully bred , which has the following characteristics : high reproductive titer , weakened virulence , good immunogenicity and stable genome height .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373

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相關(guān)期刊論文 前2條

1 黃小琴,楊凈思,周德久,白惠珠,曹逸云,董德祥;H2株甲肝病毒經(jīng)KMB17細(xì)胞培養(yǎng)的毒力及核苷酸序列[J];中國生物制品學(xué)雜志;2000年03期

2 錢汶,陳悅青,洪艷,唐彩華,周康鳳,莊f 成,陳勇;RT-PCR-ELISA方法檢測甲肝減毒活疫苗病毒滴度的初步研究[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;2004年03期

本文編號(hào)：1911904