【摘要】：目的:創(chuàng)建鴨乙型肝炎病毒(DHBV)DNA TaqMan 熒光定量聚合酶鏈反應(yīng)(FQ-PCR)檢測(cè)方法,調(diào)查成年福州麻鴨攜帶DHBV 情況并建立福州麻鴨后天感染乙型肝炎動(dòng)物模型,觀察胸腺因子D(TFD)在體內(nèi)對(duì)DHBV 的抑制作用,并對(duì)其作用機(jī)制進(jìn)行初步探討。 方法:用PUCm-T 載體和DHBV DNA PCR 純化產(chǎn)物連接,轉(zhuǎn)染TOP10 菌,篩選陽(yáng)性菌落,提取質(zhì)粒,制作標(biāo)準(zhǔn)品;在DHBV 基因組S 區(qū)設(shè)計(jì)一對(duì)引物,并在引物間設(shè)計(jì)和熒光標(biāo)記一段TaqMan 探針,嚴(yán)格優(yōu)化反應(yīng)物的組成和擴(kuò)增條件,建立TaqMan 熒光定量PCR 檢測(cè)方法并與地高辛標(biāo)記探針斑點(diǎn)雜交法進(jìn)行比較。采用常規(guī)PCR 法檢測(cè)成年福州麻鴨的DHBV 感染情況;篩選1d 齡DHBV DNA(-)雛鴨,經(jīng)靜脈途徑接種DHBV DNA 強(qiáng)陽(yáng)性血清,建立福州麻鴨后天感染乙型肝炎動(dòng)物模型;隨機(jī)分組,實(shí)驗(yàn)組、干擾素對(duì)照組和空白對(duì)照組分別用TFD、人干擾素-α1b(IFN-α1b)和0.9%氯化鈉溶液處理4w,停藥后觀察1w。用TaqMan 熒光定量PCR 法檢測(cè)治療前后血清和肝臟中DHBV DNA 含量,并檢測(cè)用藥后血清ALT、AST水平及肝組織HE 染色病理。 結(jié)果:建立的DHBV DNA TaqMan 熒光定量PCR 法靈敏度為1000 拷貝/m1;在10~5-10~9拷貝/m1 之間,含量與Ct 值具有很好的線性(Ct=-2.83611n(x)+41.45,r=-0.9983) ;批內(nèi)誤差為1.50%-2.98%,批間誤差為2.95%-3.24%。共檢查162 份成年福州麻鴨血清,DHBV 陽(yáng)性89 份,陽(yáng)性率為55%;感染1d 齡DHBVDNA(-)雛鴨84 只,感染陽(yáng)性80 只,感染陽(yáng)性率為95.24%。動(dòng)物模型用藥4w后,TFD 治療組的病毒抑制率顯著高于空白對(duì)照組(分別是8.73%和-5.12%,P0.05),抗病毒效果與用藥劑量和用藥時(shí)間相關(guān),但中劑量和大劑量組的病毒抑制率無(wú)顯著性差異。停藥1w 后,TFD 治療組血清DHBV DNA 含量無(wú)反跳現(xiàn)象,肝臟DHBV DNA 含量顯著低于空白對(duì)照組。治療4w 和停藥1w 后血清ALT、AST及肝臟病理檢查結(jié)果,TFD 治療組與空白對(duì)照組無(wú)顯著性差異。
[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 偽 1b (IFN- 偽 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

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