鸭乙型肝炎病毒湖北株的鉴定分析及其体内实验研究模型的建立

发布时间：2018-04-19 14:48

本文选题：嗜肝DNA病毒 + 鸭乙型肝炎病毒　；参考：《华中科技大学》2006年博士论文

【摘要】： 目的 1.克隆湖北麻鸭乙型肝炎病毒(DHBV)一新毒株的全基因组并进行序列分析,为建立湖北麻鸭乙型肝炎动物模型提供必要的前提条件。 2.构建DHBV感染性克隆,通过体外细胞转染获得单一来源、基因序列清楚的体内实验的感染毒种。 3.建立标准化的湖北麻鸭乙型肝炎病毒动物模型,并初步观察了拉米呋啶(3TC)体内抗鸭乙型肝炎病毒(DHBV)的作用。方法 1.通过对湖北麻鸭自然携带DHBV的调查筛选出DHBV阳性血清,用聚合酶链反应扩增一分离株的DHBV全基因,并进行克隆与序列测定分析。 2.以该毒株为模板,从质粒pMD-DHBV和DHBV阳性血清中获取三部分基因片段,总长为4.4kb,分别克隆至载体PCR2.1的Sac I和Not I酶切位点,构建含1.5倍鸭乙型肝炎病毒全基因的重组质粒PRC2.1-DHBV1.5,并酶切鉴定其插入方向。 3.将构建的重组质粒经脂质体LipofectineTM2000导入鸡肝癌细胞系LMH细胞系中转染表达,Southern blot检测DHBV复制水平,电镜观察DHBV完整病毒颗粒。 4.将收集的转染细胞培养上清液纯化后作为体内实验感染的标准毒种,通过腹腔注射接种2日龄雏鸭,连续观察50天,用实时荧光定量PCR(Real-Time PCR)检测该模型动物血清DHBV DNA出现和持续时间。 5.药物组鸭则于转染上清液感染接种3天后给予拉米呋啶(3TC),药量为25mg/kg/d,开始连续5天空腹灌胃给药,后一周3次,再持续3周,停药后观察2周,检测该期间感染鸭血清中病毒含量的变化情况,肝脏组织复制中间体的存在及肝脏病理学检查。结果 1.湖北麻鸭自然携带DHBV率为10%;分离株(HB-DHBV)的基因组全长3024bp,有编码P,S和C蛋白的三个开放阅读框;与GenBank中17株DHBV基因组比较,核苷酸同源性介于89.3%~963.5%之间;S蛋白、C蛋白和P蛋白的功能区序列均高度保守;而P蛋白特异性氨基酸分析表明,该分离株更接近于中国基因型的分离株。 2.经酶切鉴定获得正向插入的1.5倍鸭乙型肝炎病毒全基因重组质粒PCR2.1-DHBV1.5。 3. 1.5倍加长DHBV基因组质粒能够在LMH细胞内存在各种病毒复制中间体,由此也证明所构建的重组体可以用于体内基因免疫。 4.2日龄雏鸭通过腹腔接种后即可容易感染DHBV,实验感染率达到87.5%(14/16);雏鸭在接种后第7天出现病毒血症,血清病毒含量于第11天达到高峰值,后逐步降低,但在观察期内仍维持较高水平。 5.在3TC治疗期间,实验动物体内病毒血症处于较低水平,病毒复制受到明显抑制,被感染肝细胞的数量也明显减少,且无明显的毒性作用;在停药3天后,病毒血症即出现反跳现象,此后又有上升趋势。结论 1.湖北麻鸭是研究实验性DHBV的良好鸭种;HB-DHBV属于DHBV中国基因型中的一个亚型;ε区二级结构的不同造成DHBV各基因型间的差别。 2.经体外重组,获得携带1.5倍加长DHBV基因组片段的重组质粒PCR2.1-DHBV1.5,并可在鸡肝癌细胞LMH中介导高水平病毒复制;获得含可自我复制病毒颗粒的转染细胞上清液作为动物体内感染接种物。 3.通过接种转染细胞上清液成功建立湖北麻鸭乙型肝炎病毒动物模型,药物评估实验证明其良好的稳定性和实用性;该模型也为研究DHBV生物学特性提供了有力的手段。本研究的创新点 1.在国际上首次克隆出湖北麻鸭DHBV分离株的完整基因组,其基因序列已收录于GenBank数据库中,序列号为DQ276978。 2.在国内首次成功构建1.5倍DHBV DNA感染性克隆,通过转染LMH细胞系证实其具有高水平病毒复制能力。 3.在国内首次应用转染细胞上清液作为动物体内感染接种物,将DHBV感染性克隆注射到雏鸭体内,成功建立DHBV慢性感染鸭模型。本研究的意义 1.本实验通过调查湖北地区麻鸭自然携带DHBV的情况,进而对分离出的一株DHBV新毒株进行全基因组克隆和序列分析,不仅进一步充实对现有该病毒的基础理论研究,也为建立湖北麻鸭乙型肝炎动物模型提供必要的前提条件。 2.以该毒株为模板,运用三片法构建了1.5倍鸭乙型肝炎病毒全基因重组质粒,然后将克隆质粒转染LMH细胞系,收集含单一来源、基因序列清楚并可自我复制病毒颗粒的细胞上清液作为动物体内感染接种物。相对于阳性血清库的建立,该方法更便于实验室操作,特别排除了病毒株准种的干扰,且由于该重组质粒的基因序列存在可操作性(缺失、替换),故该模型适合研究不同种系DHBV生物学和组织学特性,也可研究体内特异性的病毒突变株的生长动态情况以及用于研究嗜肝病毒与细胞的相互作用及筛选抗病毒药物。
[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 .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R373

【参考文献】