【作者】 张辉；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2005， 硕士

【摘要】 减毒沙门氏菌作为疫苗载体可以运送表达的外源抗原至宿主免疫系统,激发机体产生特异性体液、细胞(包括CD4~+T细胞、CTL等)和粘膜免疫应答。目前,虽然减毒沙门氏菌作为疫苗载体的可行性得到确认,但迄今减毒沙门氏菌或重组沙门氏菌激发机体产生免疫应答的机理还不尽清楚,尤其是在诱导细胞免疫应答方面的机理还需进一步研究。对于特异性细胞免疫应答的检测方法有多种,其中酶联免疫斑点法(enzyme-linked immunospot assay, ELISPOT)是近年来用于检测细胞因子,评价T淋巴细胞功能和特异性的一种新方法。由于该法特异性强、敏感度高,因此已广泛用于分析和评价特异性CD8~+T细胞免疫应答的研究中。在本研究中,为了进一步研究减毒沙门氏菌运送外源抗原诱导细胞免疫应答的机制,首先建立了检测特异性细胞免疫的ELISPOT法,并运用该法分析了携带卵清白蛋白(OVA)和淋巴细胞脉络丛脑膜炎病毒核蛋白(LCMV NP)CD8~+T细胞表位的减毒鼠伤寒沙门氏菌诱导的细胞免疫应答规律。 1、检测重组沙门氏菌特异性细胞免疫应答的ELISPOT法建立 为建立检测重组沙门氏菌特异性细胞免疫的ELISPOT法,先构建了融合表达OVA257～264aa(H-2~b)、LCMVNP118～132aa(H-2~d)CD8~+T细胞表位和绿色荧光蛋白(GFP)的原核表达质粒ptG2F,通过电穿孔法转化减毒鼠伤寒沙门氏菌SL7207,获得重组菌SL7207(ptG2F)。以1×10~6cfu剂量的重组菌静脉注射免疫更多还原

【Abstract】 Attenuated Salmonella have been successfully used as vaccine vector to deliver exogenous antigen. It can drectly deliver antigen to immune system, and also stimulate specific humoral responses, cellular (CD4~+ T cell, CTL) immune responses and mucosal immune responses. Presently, the feasibility of attenuated Salmonella as vaccine vector has been identified, but the mechanism of immune responses stimulated by attenuated Salmonella or recombinant Salmonella has not yet elucidated completely, especially in cellular immune response. There are several methods for detecting specific cellular immune responses. And ELISPOT (enzyme-linked immunospot) assay has been considered as a means for detecting cytokines and become a new method in evaluating the function and specificity of T lymphocyte. Because of the strong specificity and high sensitivity, the assay has been used in analyzing and evaluating cellular immune responses specific for CD8~+ T cells already. In order to investigate the mechanism of immune responses induced by attenuated Salmonella delivering exogenous antigen, the ELISPOT assay for detecting the specific cellular immunity has been established, and analyzed the cellular immune responses induced by attenuated Salmonella typhimurium harboring CD8~+ T epitopes of Ovalbumin (OVA) and ofLymphocytic Choriomeningitis Virus nucleus protein (LCMV NP).1. Establishment of ELISPOT assay for detection of cellular immuneresponses specific for recombinant SalmonellaIn order to establish the ELISPOT assay for detection of cellular immune responses specific for recombinant Salmonella, prokaryotic expression plasmid ptG2F, carrying the oligonucleotide fragment encoding CD8~+ T cell epitopes of OVA 257-264aa (H-2~d), and LCMV NP 118-132aa (H-2~b) fused with green fluorescent protein (GFP) gene was constructed firstly and electro-transformed into the attenuated strain SL7207 of Salmonella typhimurium, the recombinant bacteria were named as SL7207(ptG2F). C57BL/6 and BALB/c mice were immunized intravenously with 1× 10~6cfu SL7207(ptG2F) in two weeks intervals. For establishment of the ELISPOT assay, splenocytes were collected from mice after second and third immunization, and then specific IFN- Y and IL-4 secreting cells were detected with OVA 257~264aa and LCMV NP 118~132aa peptides as stimulus. The results showed that the cellular immune responses specific for recombinant bacteria SL7207(ptG2F) can be detected by the ELISPOT assay. When OVA CD8~+ T cell epitope is presented by the immunized mice, the immune responses are biased toward Thl response after second immunization and then switch to the Thl and Th2 balance after third immunization. In contrast, when the LCMV NP CD8~+ T cell epitope is presented, the level of Th2 immune response is higher than that of Thl after both second and third immunization. Altogether, these results indicates that the ELISOPT assay can detect the specific cellular immune response induced by recombinant Salmonella, and it also shown that attenuated Salmonella can effectively deliver the CD8~+ T cell epitopes and induce specific cellular immune responses.2. Kinetic analysis of cellular immune responses initiated by recombinant Salmonella through oral immunizationTo analyze the dynamic change of cellular immune responses against CD8+ T cell epitope delivered by recombinant Salmonella after oral immunization. BALB/c mice were immunized orally with SL7207(ptG2F) in two weeks intervals. IFN- Y and IL-4 secreting cells were detected by ELISPOT assay in murine peyer’s patches cells after second , third , forth and fifth immunization respectively. Meanwhile, antibodies against GFP were determined in serum, fecal extract and intestinal mucosa by ELISA. The results showed that recombinant bacteria SL7207(ptG2F) can induce cellular immune responses specific for LCMV NP CD8+ T epitope, and present the different types after different times point of immunization. IFN- Y and IL-4 secreting cells specific for LCMV NP CD8+ T cell epitope could be detected after second immunization, and the frequenc更多还原