胸腺因子D抗鸭乙型肝炎病毒的体内实验

发布时间：2018-09-05 15:11

【摘要】：目的:创建鸭乙型肝炎病毒(DHBV)DNA TaqMan 荧光定量聚合酶链反应(FQ-PCR)检测方法,调查成年福州麻鸭携带DHBV 情况并建立福州麻鸭后天感染乙型肝炎动物模型,观察胸腺因子D(TFD)在体内对DHBV 的抑制作用,并对其作用机制进行初步探讨。方法:用PUCm-T 载体和DHBV DNA PCR 纯化产物连接,转染TOP10 菌,筛选阳性菌落,提取质粒,制作标准品;在DHBV 基因组S 区设计一对引物,并在引物间设计和荧光标记一段TaqMan 探针,严格优化反应物的组成和扩增条件,建立TaqMan 荧光定量PCR 检测方法并与地高辛标记探针斑点杂交法进行比较。采用常规PCR 法检测成年福州麻鸭的DHBV 感染情况;筛选1d 龄DHBV DNA(-)雏鸭,经静脉途径接种DHBV DNA 强阳性血清,建立福州麻鸭后天感染乙型肝炎动物模型;随机分组,实验组、干扰素对照组和空白对照组分别用TFD、人干扰素-α1b(IFN-α1b)和0.9%氯化钠溶液处理4w,停药后观察1w。用TaqMan 荧光定量PCR 法检测治疗前后血清和肝脏中DHBV DNA 含量,并检测用药后血清ALT、AST水平及肝组织HE 染色病理。结果:建立的DHBV DNA TaqMan 荧光定量PCR 法灵敏度为1000 拷贝/m1;在10~5-10~9拷贝/m1 之间,含量与Ct 值具有很好的线性(Ct=-2.83611n(x)+41.45,r=-0.9983) ;批内误差为1.50%-2.98%,批间误差为2.95%-3.24%。共检查162 份成年福州麻鸭血清,DHBV 阳性89 份,阳性率为55%;感染1d 龄DHBVDNA(-)雏鸭84 只,感染阳性80 只,感染阳性率为95.24%。动物模型用药4w后,TFD 治疗组的病毒抑制率显著高于空白对照组(分别是8.73%和-5.12%,P0.05),抗病毒效果与用药剂量和用药时间相关,但中剂量和大剂量组的病毒抑制率无显著性差异。停药1w 后,TFD 治疗组血清DHBV DNA 含量无反跳现象,肝脏DHBV DNA 含量显著低于空白对照组。治疗4w 和停药1w 后血清ALT、AST及肝脏病理检查结果,TFD 治疗组与空白对照组无显著性差异。
[Abstract]:Objective: to establish a fluorescent quantitative polymerase chain reaction (FQ-PCR) method for detection of duck hepatitis B virus (DHBV) and to investigate the DHBV carried by adult Fuzhou duck and to establish an animal model of acquired hepatitis B infection in Fuzhou duck. To observe the inhibitory effect of thymus factor D (TFD) on DHBV in vivo and its mechanism. Methods: PUCm-T vector and DHBV DNA PCR purification product were used to connect, transfect TOP10 bacteria, screen positive colonies, extract plasmid, make standard product, design a pair of primers in the S region of DHBV genome, design and label a TaqMan probe between primers. The composition and amplification conditions of the reactants were optimized strictly, and the method of TaqMan fluorescence quantitative PCR detection was established and compared with that of digoxigenin labeled probe dot blot hybridization. Routine PCR method was used to detect DHBV infection in adult Fuzhou duck, 1 day old DHBV DNA (-) ducklings were screened, and then inoculated with DHBV DNA strong positive serum via intravenous route to establish the animal model of acquired hepatitis B infection of Fuzhou Ma duck, and were randomly divided into experimental group and experimental group. Interferon control group and blank control group were treated with TFD, human interferon 伪 1b (IFN- 伪 1b) and 0.9% sodium chloride solution for 4 weeks, respectively. The content of DHBV DNA in serum and liver was detected by TaqMan fluorescence quantitative PCR before and after treatment. The serum ALT,AST level and liver tissue HE staining pathology were also detected after treatment. Results: the sensitivity of the established DHBV DNA TaqMan fluorescence quantitative PCR method was 1000 copies / m ~ (-1), and the sensitivity of the DHBV DNA TaqMan fluorescence quantitative PCR method was 1000 copies / m ~ (-1), and the linear relationship between the content and the Ct value was found between 105-109 copies / m ~ (1) (Ct=-2.83611n (x) 41.45 ~ 0.9983), the intra-assay error was 1.50-2.98, and the inter-assay error was 2.95 ~ 3.2483. A total of 89 sera of 162 adult Fuzhou duck were positive for DHBV, the positive rate was 550.The infection rate was 95.24 in 84 DHBVDNA (-) ducklings of 1 day old. The virus inhibition rate of TFD group was significantly higher than that of the blank control group (8.73% and -5.12%, respectively) after 4 weeks of treatment. The antiviral effect was correlated with the dosage and time of administration, but there was no significant difference between the middle dose group and the large dose group. There was no rebound phenomenon in serum DHBV DNA content and the DHBV DNA content in liver was significantly lower in the control group than in the control group after 1 week of withdrawal. There was no significant difference in serum ALT,AST and hepatic pathology between the treatment group and the blank control group after 4 weeks of treatment and 1 week after withdrawal.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【参考文献】