本文選題：麻疹病毒 + 核苷酸變異��； 參考：《吉林大學(xué)》2011年博士論文

【摘要】：麻疹是由麻疹病毒(Measles Virus,MV)引起的一種呼吸道傳染病,是少數(shù)可以用疫苗有效控制的病毒性傳染病之一。雖然麻疹疫苗的使用使麻疹的發(fā)病率和死亡率大幅度降低,但是至今,麻疹仍是嚴(yán)重威脅兒童健康和生命安全的全球(特別是發(fā)展中國(guó)家)公共衛(wèi)生問(wèn)題。麻疹病毒為單一血清型,一直被認(rèn)為遺傳性較為穩(wěn)定,但是近年來(lái)分離的麻疹野毒株與上個(gè)世紀(jì)60年代分離的麻疹病毒比較,在基因水平上已經(jīng)有了較大的變異,麻疹疫情的回升是否與病毒的變異有關(guān)?麻疹野毒株的變異是否會(huì)造成麻疹疫苗保護(hù)能力的降低?是否需要開(kāi)發(fā)研制新型麻疹疫苗以達(dá)到最終在全世界消除麻疹的目標(biāo)?這些已經(jīng)成為世界各國(guó)學(xué)者廣泛關(guān)注的話題。 為探討麻疹野毒株的變異情況、現(xiàn)行麻疹減毒活疫苗的免疫效果以及麻疹減毒活疫苗的減毒機(jī)制等問(wèn)題,本研究開(kāi)展了下述研究工作: 1.麻疹野毒株變異情況研究: 收集2008～2009年麻疹疑似病例標(biāo)本40份(咽拭子32份,尿液8份),用Vero/SLAM細(xì)胞成功分離到4株病毒。經(jīng)典型的細(xì)胞融合病變、麻疹單克隆抗體和N基因羧基末端450個(gè)核苷酸擴(kuò)增檢測(cè),鑒定4株病毒全部為麻疹病毒。 應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(reverse transcript polymerase chain reaction,RT-PCR)對(duì)16株麻疹野毒株(本實(shí)驗(yàn)分離的4株麻疹野毒株和本實(shí)驗(yàn)室保存的2005～2007年吉林省麻疹野毒株12株)的N基因羧基(-COOH)末端450個(gè)核苷酸片段進(jìn)行擴(kuò)增,測(cè)序后使用MEGA 4.0軟件對(duì)測(cè)定結(jié)果與國(guó)際標(biāo)準(zhǔn)株Edmonston株、疫苗株滬-191株和長(zhǎng)-47株的對(duì)應(yīng)序列進(jìn)行基因親緣性分析。結(jié)果顯示:16株麻疹野毒株之間N基因羧基端450個(gè)核苷酸和150個(gè)氨基酸同源性分別為99.11%和99.33%,其組內(nèi)遺傳距離小于0.009;16株麻疹野毒株與標(biāo)準(zhǔn)株Edmonston株比較,組間遺傳距離介于0.069～0.074之間,核苷酸和氨基酸的差異率分別為6.00%～7.55%和8.00%～8.67%;16株麻疹野毒株與疫苗株滬-191株比較,遺傳距離為0.084～0.091,核苷酸和氨基酸的差異率分別為6.67%～8.22%和10.00%～10.67%;16株麻疹野毒株與疫苗株長(zhǎng)-47株比較,遺傳距離為0.071～0.076,核苷酸和氨基酸的差異率分別為6.22%～6.67%和8.67%～9.33%。 此結(jié)果說(shuō)明16株麻疹野病毒基因序列差異較小,其組內(nèi)遺傳距離上差異不大,而與標(biāo)準(zhǔn)株Edmonston株、疫苗株滬-191株和長(zhǎng)-47株相比,組間遺傳距離差異較大,核苷酸水平和氨基酸水平上都出現(xiàn)了較大的變異。 通過(guò)以上實(shí)驗(yàn)研究證明,現(xiàn)今的麻疹野毒株與標(biāo)準(zhǔn)株Edmonston株、麻疹疫苗株相比,在基因水平上發(fā)生了較大的變化,變異逐漸被累積并且有可能影響到現(xiàn)行麻疹疫苗的免疫效果。 2.麻疹疫苗減毒機(jī)制的研究 應(yīng)用病毒蝕斑實(shí)驗(yàn)的方法對(duì)所分離的麻疹野毒株進(jìn)行純化,觀察病毒蝕斑的形態(tài)特征和出斑速度,并對(duì)挑斑后的病毒克隆株進(jìn)行病毒擴(kuò)增,觀察產(chǎn)生的細(xì)胞病變特點(diǎn),測(cè)定其毒力。模擬麻疹疫苗制備的減毒技術(shù),將分離于麻疹患者標(biāo)本的麻疹野毒株和經(jīng)純化后的麻疹克隆株進(jìn)行減毒培養(yǎng),觀察減毒效果。 實(shí)驗(yàn)結(jié)果顯示,同一株麻疹野毒株所產(chǎn)生的蝕斑,其斑塊的大小、形狀、中心和邊緣特征以及出斑的速度均存在明顯的差異,經(jīng)過(guò)挑斑后得到的各個(gè)克隆株,其毒力從6.17 lgTCID_(50)/0.1ml～7.40 lgTCID_(50)/0.1ml之間不等,挑斑擴(kuò)增后出現(xiàn)細(xì)胞病變的時(shí)間和病變的特點(diǎn)也有較大的差別。通過(guò)減毒實(shí)驗(yàn)對(duì)2株野毒株和來(lái)源于同一麻疹野毒株的6株克隆株進(jìn)行減毒培養(yǎng),結(jié)果顯示,麻疹野毒株的毒力有所下降,而克隆株的毒力未見(jiàn)明顯變化。麻疹病毒的毒力下降只發(fā)生在野毒株的減毒過(guò)程,克隆株的毒力l并無(wú)變化,提示野毒株與克隆株之間存在差異。 野毒株毒力的降低主要發(fā)生在病毒由原代人羊膜細(xì)胞(HAM)向原代雞胚細(xì)胞(CEC)的適應(yīng)過(guò)程中,而病毒由原代人胚腎細(xì)胞(HK)適應(yīng)HAM的過(guò)程和病毒在CEC上連續(xù)傳代的過(guò)程均未見(jiàn)毒力的明顯變化。結(jié)果提示,麻疹野毒株可能是一個(gè)混合體,其中包含了大量的強(qiáng)毒株和少量的弱毒株,CEC對(duì)病毒起到篩選作用,將能適應(yīng)CEC的弱毒株保留下來(lái),強(qiáng)毒株由于不能很好的適應(yīng)CEC而被淘汰,弱毒株在CEC上的傳代起到了將弱毒株大量擴(kuò)增的目的。 3.疫苗接種與麻疹流行病學(xué)研究 本研究收集了1988年至今我國(guó)麻疹的流行病學(xué)資料,采用描述性流行病學(xué)方法分析了1990～2007年我國(guó)麻疹疫苗接種率與全國(guó)發(fā)病率變化的情況和2003～2009年我國(guó)31個(gè)省的發(fā)病率差異情況,并且分析了2004～2010年全國(guó)麻疹的發(fā)病季節(jié)分布特點(diǎn)及吉林省強(qiáng)化免疫后麻疹的發(fā)病情況。 結(jié)果顯示,雖然我國(guó)麻疹疫苗的接種率一直維持較高的水平,但麻疹的發(fā)病率與死亡率在我國(guó)法定傳染病中仍居于前列,每隔2～3年就會(huì)出現(xiàn)一次小規(guī)模的暴發(fā)流行,并且各個(gè)省市自治區(qū)的麻疹發(fā)病率相差懸殊;麻疹患病出現(xiàn)高峰的時(shí)間明顯推遲,麻疹發(fā)病的季節(jié)性已經(jīng)發(fā)生了較大的變化;加強(qiáng)免疫可以在短期內(nèi)收到較好的預(yù)防效果,但麻疹的流行周期并沒(méi)有被強(qiáng)化免疫所打破,強(qiáng)化免疫的長(zhǎng)期效果有待進(jìn)一步觀察和研究。 本研究提示,有較好的計(jì)劃免疫基礎(chǔ)并不能有效的避免麻疹的暴發(fā),為達(dá)到消除麻疹的目標(biāo),除繼續(xù)加強(qiáng)麻疹野病毒的血清學(xué)和分子流行病學(xué)監(jiān)測(cè),扎實(shí)開(kāi)展兒童免疫規(guī)劃基礎(chǔ)工作外,還需對(duì)我國(guó)現(xiàn)行的麻疹免疫策略及免疫程序進(jìn)行調(diào)整,適時(shí)研究開(kāi)發(fā)更有效的新型麻疹疫苗提高疫苗的免疫效力。
[Abstract]:measles is a respiratory infectious disease caused by measles virus ( MV ) . It is one of the few viral infectious diseases that can be effectively controlled by vaccine . Although measles vaccine has been used to reduce the morbidity and mortality of measles , measles virus is a single serotype and has been considered to be genetically stable .


In order to explore the variation of measles wild strain , the effect of measles attenuated live vaccine and the anti - toxic mechanism of measles attenuated live vaccine , the following research work has been carried out :


1 . Study on variation of measles wild strain :


Forty samples of suspected measles cases from 2008 to 2009 were collected ( 32 parts of pharyngeal swab and 8 parts of urine ) . Four strains of virus were isolated successfully by Vero / SLAM cells .


The results showed that 450 nucleotides and 150 amino acid sequences were 99.11 % and 99.33 % , respectively , and the genetic distance was between 0.069 and 0.074 , and the difference of nucleotide and amino acids was 6.67 % ~ 8.22 % and 10.00 % ~ 10.67 % , respectively .


The results showed that there were no significant differences in the genetic distance between the 16 measles virus gene sequences , but there was a large variation in the genetic distance between the two groups , compared with that of the standard strain , which was similar to that of the standard strain , which showed a large variation in the genetic distance , the nucleotide level and the amino acid level .


Through the above experimental studies , it has been proved that the measles wild strain has a large change in gene level compared with the standard strain , and the measles vaccine strain is gradually accumulated and has the potential to affect the immune effect of the current measles vaccine .


2 . Research on the Anti - Virus Mechanism of measles Vaccine


Using the method of virus plaque assay , the isolated measles wild strain was purified to observe the morphological characteristics and spot speed of the virus plaque , and the virus clone strain was amplified by virus . The characteristics of the produced cell lesion were observed and the virulence of the virus was observed . The attenuated technique was used to simulate the attenuated measles vaccine prepared by measles vaccine .


The results showed that there were significant differences in the size , shape , center and edge of the plaque , and the rate of plaque formation . The results showed that the virulence of the strain decreased and the virulence of the cloned strain was not changed . The virulence of the cloned strain was not changed . The difference between wild strain and clone was suggested .


The results showed that the attenuated strain could be a mixture containing a large number of virulent strains and a small number of attenuated strains .


3 . Epidemiological study on vaccination and measles


The epidemiological data of measles in China from 1988 to present were collected . The incidence of measles vaccination in China from 1990 to 2007 and the incidence of measles in 31 provinces in China from 2003 to 2009 were analyzed by descriptive epidemiological methods , and the seasonal distribution characteristics of measles in 2004 - 2010 and the incidence of measles in Jilin Province were analyzed .


The results showed that , although the rate of measles vaccination in China has been maintained at a high level , the incidence and mortality rate of measles is still at the forefront of the notifiable infectious diseases in our country , and the incidence of measles in every province and city has been greatly delayed . The time of measles occurrence is obviously delayed , and the epidemic period of measles has not been broken , and the long - term effect of enhanced immunity needs to be further observed and studied .


In order to achieve the goal of eliminating measles , we should adjust the current measles immune strategy and immune procedure in our country , and study and develop more effective new measles vaccine to improve the immune efficacy of the vaccine .
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R186

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本文編號(hào)：1948504