【作者】 薛猛；

【导师】 李厚达； 薛整风；

【作者基本信息】 扬州大学 ， 动物学， 2003， 硕士

【摘要】 核酸免疫是近年来发展起来的一种新的免疫方法，它是指将编码外源蛋白的基因表达载体直接导入机体以激发机体产生特异性免疫应答。由于核酸疫苗具有减毒和灭活疫苗等传统疫苗不可比拟的优越性，正备受关注。经过近年来的大量研究，已基本上对核酸疫苗临床应用的许多要素作了全面的评估，主要有疫苗的免疫学机理、安全性、临床应用效率等。本文从核酸疫苗对新生儿的免疫保护，以及对病人的免疫治疗这两方面入手，以乙肝表面抗原(HBsAg)核酸疫苗为例，探讨了核酸疫苗诱发产生母源抗体及打破免疫耐受的能力。本论文的工作由两部分组成： 1、HBsAg核酸疫苗诱导小鼠产生母源抗体 以编码人类乙型肝炎病毒表面抗原(ayw亚型)的质粒pcDNA-HBs作为疫苗，肌肉注射6周龄的BALB／c雌鼠，三星期后加强免疫一次，再过两星期后使其配种怀孕，怀孕第19天剖腹产取出胎儿并分离孕鼠及胎儿的血清。经ELISA检测证明了核酸疫苗能够激发孕鼠产生特异性的抗体并能传递给胎儿，且母体与胎儿的抗体效价相同，从而证明了核酸疫苗与传统疫苗一样能诱导产生母源抗体，这一结果为进一步利用核酸疫苗阻断乙肝及其他传染性疾病的母婴垂直传播，以及保护新生儿免受病原的感染，提供了原始资料。 2、HBsAg核酸疫苗消除HBsAg转基因小鼠的免疫耐受 为建立对HBsAg耐受的小鼠模型，利用显微注射转基因技术，构建了CMV启动子控制的HBsAg(ayw亚型)转基因小鼠。经PCR扩增，筛选出阳性小鼠。经免疫组化分析表明，在阳性小鼠的肝脏及脾脏中可检测到HBsAg的表达，而在其他组织则未检测到HBsAg的表达，说明外源基因在小鼠体内的表达具有一定的组织特异性。通过ELISA分析，在阳性小鼠的血清中未检测到ant卜HBsAg抗体，表明小鼠处于对HBsAg的免疫耐受状态。随后以peDNA-HBs质粒，肌肉注射法分三次免疫转基因小鼠，每两星期一次。对免疫后小鼠的血清进行EuSA分析，发现小鼠体内产生了特异性的anti一HBsAg抗体，这一结果提示核酸疫苗可打破机体对特定抗原的免疫耐受状态，从而为慢性乙型肝炎的治疗性疫苗的研究开辟了新的途径。 综上，本研究的结果为更好地开发利用核酸疫苗的潜在价值，提供了初步的参考资料。更多还原

【Abstract】 DNA immunization is a new form of vaccination, which means direct delivery into animals of DNA encoding antigenic proteins to elicit specific immune response. DNA vaccine is acknowledged as a promising new generation of vaccine for its multiple advantages over traditional vaccine such as attenuated or inactivated vaccine. Many factors affecting clinical application of DNA vaccine, such as the immunological mechanism, safety, efficiency, have been already evaluated in recent numerous researches. To explore the immunoprotection effect to neonates and the immunotherapy potential of DNA vaccine, we studied the ability of this vaccine in inducing maternalantibodies and breaking immunotolerance, with the hepatitis B surface antigen (HBsAg) DNA vaccine as an example. Our works consist of two parts:1. HBsAg DNA vaccine induces maternal antibodies in miceThe plasmid pcDNA-HBs encoding surface antigen of human hepatitis B virus (ayw subtype) was used as DNA vaccine to intramusclarly inoculate 6-week-old female BALB/c mice. A second immunization was given after two weeks, and the mice were crossed after another three weeks. Fetuses were taken out by caesarean birth at the nineteenth pregnant day, and the serum isolated from the pregnant mice and the fetuses was tested by ELISA. It was found that DNA vaccine stimulates the female mice to produce maternal antibodies that can transfer to fetuses and that the ELISA titer of serum from mother and fetus is identical. This result suggests that like traditional vaccines, DNA vaccine can induce maternal antibodies as well. Our experiment is the fundamental investigation in further utilizing DNA vaccine in the protection of neonates and in the interruption of maternal-infant vertical transmission of hepatitis B and other infectious diseases.2. HBsAg DNA vaccine breaks imunotolerance in HBsAg transgenic mice.To construct a murine model that is immunotolerant to HBsAg, we developed the HBsAg (ayw subtype) transgenic (Tg) mice with CMV promoter by microinjection. The Tg mice were identified by PCR. Expression of the HBsAg gene, determined by immunohistochemistry, was found to occur in the liver and the spleen but not other organs of the Tg mice, which suggests that the expression of foreign gene is tissue specific. Result of ELISA that anti-HBsAg antibodies can’t be detected in the serum of Tg mice suggests that the mice are imunological tolerant to HBsAg. Subsequently, the Tg mice were intramusclarly injected with the plasmid pcDNA-HBs for three times at biweekly intervals. Anti-HBsAg antibodies can be detected in serum of the immunized mice by ELISA, which demonstrates that DNA vaccine can overcome the imunotolerance to specific antigen in mice. Thus, a new approach was proposed in the study of therapeutic vaccine of chronic hepatitis B.In summary, these tentative experiments provide referential data to further exploit the potential value of DNA vaccine.更多还原