本文選題：嗜肝DNA病毒 + 鴨乙型肝炎病毒��； 參考：《華中科技大學(xué)》2006年博士論文

【摘要】： 目的 1.克隆湖北麻鴨乙型肝炎病毒(DHBV)一新毒株的全基因組并進(jìn)行序列分析,為建立湖北麻鴨乙型肝炎動(dòng)物模型提供必要的前提條件。 2.構(gòu)建DHBV感染性克隆,通過體外細(xì)胞轉(zhuǎn)染獲得單一來源、基因序列清楚的體內(nèi)實(shí)驗(yàn)的感染毒種。 3.建立標(biāo)準(zhǔn)化的湖北麻鴨乙型肝炎病毒動(dòng)物模型,并初步觀察了拉米呋啶(3TC)體內(nèi)抗鴨乙型肝炎病毒(DHBV)的作用。 方法 1.通過對(duì)湖北麻鴨自然攜帶DHBV的調(diào)查篩選出DHBV陽性血清,用聚合酶鏈反應(yīng)擴(kuò)增一分離株的DHBV全基因,并進(jìn)行克隆與序列測(cè)定分析。 2.以該毒株為模板,從質(zhì)粒pMD-DHBV和DHBV陽性血清中獲取三部分基因片段,總長(zhǎng)為4.4kb,分別克隆至載體PCR2.1的Sac I和Not I酶切位點(diǎn),構(gòu)建含1.5倍鴨乙型肝炎病毒全基因的重組質(zhì)粒PRC2.1-DHBV1.5,并酶切鑒定其插入方向。 3.將構(gòu)建的重組質(zhì)粒經(jīng)脂質(zhì)體LipofectineTM2000導(dǎo)入雞肝癌細(xì)胞系LMH細(xì)胞系中轉(zhuǎn)染表達(dá),Southern blot檢測(cè)DHBV復(fù)制水平,電鏡觀察DHBV完整病毒顆粒。 4.將收集的轉(zhuǎn)染細(xì)胞培養(yǎng)上清液純化后作為體內(nèi)實(shí)驗(yàn)感染的標(biāo)準(zhǔn)毒種,通過腹腔注射接種2日齡雛鴨,連續(xù)觀察50天,用實(shí)時(shí)熒光定量PCR(Real-Time PCR)檢測(cè)該模型動(dòng)物血清DHBV DNA出現(xiàn)和持續(xù)時(shí)間。 5.藥物組鴨則于轉(zhuǎn)染上清液感染接種3天后給予拉米呋啶(3TC),藥量為25mg/kg/d,開始連續(xù)5天空腹灌胃給藥,后一周3次,再持續(xù)3周,停藥后觀察2周,檢測(cè)該期間感染鴨血清中病毒含量的變化情況,肝臟組織復(fù)制中間體的存在及肝臟病理學(xué)檢查。 結(jié)果 1.湖北麻鴨自然攜帶DHBV率為10%;分離株(HB-DHBV)的基因組全長(zhǎng)3024bp,有編碼P,S和C蛋白的三個(gè)開放閱讀框;與GenBank中17株DHBV基因組比較,核苷酸同源性介于89.3%~963.5%之間;S蛋白、C蛋白和P蛋白的功能區(qū)序列均高度保守;而P蛋白特異性氨基酸分析表明,該分離株更接近于中國(guó)基因型的分離株。 2.經(jīng)酶切鑒定獲得正向插入的1.5倍鴨乙型肝炎病毒全基因重組質(zhì)粒PCR2.1-DHBV1.5。 3. 1.5倍加長(zhǎng)DHBV基因組質(zhì)粒能夠在LMH細(xì)胞內(nèi)存在各種病毒復(fù)制中間體,由此也證明所構(gòu)建的重組體可以用于體內(nèi)基因免疫。 4.2日齡雛鴨通過腹腔接種后即可容易感染DHBV,實(shí)驗(yàn)感染率達(dá)到87.5%(14/16);雛鴨在接種后第7天出現(xiàn)病毒血癥,血清病毒含量于第11天達(dá)到高峰值,后逐步降低,但在觀察期內(nèi)仍維持較高水平。 5.在3TC治療期間,實(shí)驗(yàn)動(dòng)物體內(nèi)病毒血癥處于較低水平,病毒復(fù)制受到明顯抑制,被感染肝細(xì)胞的數(shù)量也明顯減少,且無明顯的毒性作用;在停藥3天后,病毒血癥即出現(xiàn)反跳現(xiàn)象,此后又有上升趨勢(shì)。 結(jié)論 1.湖北麻鴨是研究實(shí)驗(yàn)性DHBV的良好鴨種;HB-DHBV屬于DHBV中國(guó)基因型中的一個(gè)亞型;ε區(qū)二級(jí)結(jié)構(gòu)的不同造成DHBV各基因型間的差別。 2.經(jīng)體外重組,獲得攜帶1.5倍加長(zhǎng)DHBV基因組片段的重組質(zhì)粒PCR2.1-DHBV1.5,并可在雞肝癌細(xì)胞LMH中介導(dǎo)高水平病毒復(fù)制;獲得含可自我復(fù)制病毒顆粒的轉(zhuǎn)染細(xì)胞上清液作為動(dòng)物體內(nèi)感染接種物。 3.通過接種轉(zhuǎn)染細(xì)胞上清液成功建立湖北麻鴨乙型肝炎病毒動(dòng)物模型,藥物評(píng)估實(shí)驗(yàn)證明其良好的穩(wěn)定性和實(shí)用性;該模型也為研究DHBV生物學(xué)特性提供了有力的手段。 本研究的創(chuàng)新點(diǎn) 1.在國(guó)際上首次克隆出湖北麻鴨DHBV分離株的完整基因組,其基因序列已收錄于GenBank數(shù)據(jù)庫(kù)中,序列號(hào)為DQ276978。 2.在國(guó)內(nèi)首次成功構(gòu)建1.5倍DHBV DNA感染性克隆,通過轉(zhuǎn)染LMH細(xì)胞系證實(shí)其具有高水平病毒復(fù)制能力。 3.在國(guó)內(nèi)首次應(yīng)用轉(zhuǎn)染細(xì)胞上清液作為動(dòng)物體內(nèi)感染接種物,將DHBV感染性克隆注射到雛鴨體內(nèi),成功建立DHBV慢性感染鴨模型。 本研究的意義 1.本實(shí)驗(yàn)通過調(diào)查湖北地區(qū)麻鴨自然攜帶DHBV的情況,進(jìn)而對(duì)分離出的一株DHBV新毒株進(jìn)行全基因組克隆和序列分析,不僅進(jìn)一步充實(shí)對(duì)現(xiàn)有該病毒的基礎(chǔ)理論研究,也為建立湖北麻鴨乙型肝炎動(dòng)物模型提供必要的前提條件。 2.以該毒株為模板,運(yùn)用三片法構(gòu)建了1.5倍鴨乙型肝炎病毒全基因重組質(zhì)粒,然后將克隆質(zhì)粒轉(zhuǎn)染LMH細(xì)胞系,收集含單一來源、基因序列清楚并可自我復(fù)制病毒顆粒的細(xì)胞上清液作為動(dòng)物體內(nèi)感染接種物。相對(duì)于陽性血清庫(kù)的建立,該方法更便于實(shí)驗(yàn)室操作,特別排除了病毒株準(zhǔn)種的干擾,且由于該重組質(zhì)粒的基因序列存在可操作性(缺失、替換),故該模型適合研究不同種系DHBV生物學(xué)和組織學(xué)特性,也可研究體內(nèi)特異性的病毒突變株的生長(zhǎng)動(dòng)態(tài)情況以及用于研究嗜肝病毒與細(xì)胞的相互作用及篩選抗病毒藥物。
[Abstract]:Purpose






1 . The whole genome of a new strain of hepatitis B virus ( DHBV ) in Hubei province was cloned and sequenced .






2 . constructing DHBV infectious clones , obtaining single source through in vitro cell transfection , and infecting virus seeds in vivo experiments with clear gene sequence .






3 . Establish a standardized animal model of duck hepatitis B virus in Hubei , and observe the effect of lamivudine ( 3TC ) on duck hepatitis B virus ( DHBV ) in vivo .






method






1 . The DHBV positive serum was screened by the investigation of DHBV in Hubei . The whole gene of DHBV was amplified by polymerase chain reaction and cloned and sequenced .






2 . The recombinant plasmid PRC2.1 - DHBV1.5 containing 1.5 times the whole gene of duck hepatitis B virus was constructed from the plasmid pMD - DHBV and DHBV positive serum by using the strain as the template , and the insertion direction of the recombinant plasmid PRC2.1 - DHBV1.5 containing 1.5 times the whole gene of duck hepatitis B virus was constructed .






3 . The recombinant plasmid constructed was transfected into LMH cell line of chicken liver cancer cell line by LipofectineTM2000 . Southern blot was used to detect DHBV replication level .






4 . After purification of the supernatant of transfected cell culture supernatant , the serum DHBV DNA appeared and lasted for 50 days with real - time fluorescence quantitative PCR ( Real - Time PCR ) .






5 . The drug group was given lafutidine ( 3TC ) after 3 days after infection with the supernatant , the dosage was 25 mg / kg / day , 5 days after oral gavage , 3 weeks in the last week , 3 weeks after drug withdrawal , 2 weeks after drug withdrawal , the changes of virus content in the infected duck serum during the period , the presence of the liver tissue replication intermediate and the liver pathology examination were detected .






Results






1 . In Hubei , the rate of DHBV was 10 % . The total length of the isolate ( HB - DHBV ) was 3024bp . There were three open reading frames encoding P , S and C proteins . The nucleotide homology was 89.3 % ~ 963.5 % , compared with the 17 DHBV genome in GenBank . The specific amino acid analysis of P protein showed that the isolates were closer to the isolates of Chinese genotype .






2 . The full - gene recombinant plasmid PCR2.1 - DHBV1.5 of the forward inserted 1.5 - fold duck hepatitis B virus was obtained by enzyme digestion .






3.1 . 5 Addition of the long DHBV genome plasmid enables the presence of various viral replication intermediates in LMH cells , thereby also demonstrating that the constructed recombinant can be used in vivo gene immunization .






The infection rate of DHBV was 87.5 % ( 14 / 16 ) . After inoculation , the infection rate of DHBV was 87.5 % ( 14 / 16 ) . The virus level reached the peak at the 7th day after inoculation , and the level of serum virus decreased gradually , but remained high in the observation period .






5 . During the 3TC treatment , the vixemia of the experimental animals was low , the replication of the virus was obviously inhibited , the number of infected liver cells was also significantly reduced , and no obvious toxic effect was observed . After 3 days of drug withdrawal , the virus was anti - hop , and then there was an upward trend .






Conclusion






1 . Hubei measles duck is a good duck species for experimental DHBV ; HB - DHBV is one of the subtypes of DHBV Chinese genotype ; the difference of the two secondary structures in epsilon region leads to the difference between the genotypes of DHBV .






2 . The recombinant plasmid PCR2.1 - DHBV1.5 carrying 1.5 times of DHBV genome segment was obtained by in vitro recombination , and high - level virus replication can be mediated in chicken liver cancer cell LMH ; and the transfected cell supernatant containing self - replicating virus particles is obtained as an infected inoculum in an animal .






3 . The animal model of duck hepatitis B virus in Hubei was successfully established by inoculating supernatant of transfected cells , and its stability and practicability were proved . The model provided a powerful means to study the biological characteristics of DHBV .






Innovative points in this study






1 . The complete genome of DHBV isolates from Hubei was first cloned at the international level . The gene sequence was recorded in GenBank database , and the serial number was DQ276978 .






2 . First , 1.5 - fold DHBV DNA infectious clones were successfully constructed in China , and the LMH cell line was transfected to demonstrate its high level of virus replication .






3 . For the first time in the country , the supernatant of transfected cells was used as the inoculum in vivo , and the infected clones of DHBV were injected into ducklings , and the model of DHBV chronic infection was successfully established .






Significance of the study






1 . By investigating the nature of DHBV carrying DHBV in Hubei region , the whole genome cloning and sequence analysis of a new strain of DHBV were carried out , which not only enriched the basic theory of the virus , but also provided the necessary precondition for the establishment of the model of duck hepatitis B in Hubei .






2 . Using this strain as a template , we constructed 1.5 times duck hepatitis B virus whole gene recombinant plasmid by using three - chip method , then transfected LMH cell line by using three - chip method . The cell supernatant containing single source , gene sequence and self - replicating virus particles was collected as infection inoculum in animal .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373

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