【作者】 余跃飞；

【导师】 温博贵；

【作者基本信息】 汕头大学 ， 病理学， 2004， 博士

【摘要】 恙虫病又称丛林斑疹伤寒(Scrub typhus)，是通过恙螨叮咬而传播，临床上主要以突然高热、浅表淋巴结肿大、虫咬处溃疡焦痂和皮疹为特征，其病原体为恙虫病东方体(Orientia tsutsugamushi)。尽管该病可用抗菌药如四环素、多西环素和氯霉素等进行有效治疗，但是再感染和复发经常发生。另外，还出现耐药性恙虫病东方体菌株。因此，用恙虫病疫苗对易感人群进行预防接种仍有必要。传统的恙虫病疫苗研究主要是集中在灭活和减毒的菌苗，但一直未获满意结果。因此，有必要应用新技术来开发新型恙虫病疫苗。 56kDa和47kDa蛋白是恙虫病东方体的主要外膜蛋白。研究证明，这两种外膜蛋白均存在菌体表面并具有良好的免疫原性，已成为恙虫病东方体亚单位疫苗的重要候选分子。58kDa蛋白是恙虫病东方体的热休克蛋白，属于Hsp60家族。恙虫病东方体58kDa蛋白抗原具有种特异性，提示该蛋白可能是一种潜在的保护性抗原。 本研究首先用PCR方法扩增了恙虫病东方体Karp株的47kDa、56kDa、58kDa蛋白基因，并对这些基因片段进行了克隆和序列测定。将这些基因分别插入到表达质粒pQE30中，构建了单抗原基因重组原核表达质粒(pQE30／47、pQE30／56、pQE30／58)和双抗原基因重组的原核表达质粒(pQE30／56-47、pQE30／58-47)。用重组质粒转化E.coli和IPTG诱导重组子表达目的蛋白。SDS-PAGE分析证明重组质粒转化E. coli细胞分别表达了47kDa、56kDa、58kDa重组蛋白以及56-47kDa和58-47kDa融合蛋白；蛋白免疫印迹分析证明重组蛋白均可与恙虫病东方体Karp株免疫血清发生特异反应。 为研究表达蛋白的免疫原性，用Ni-NTA亲和层析柱将重组蛋白纯化后免疫Balb／C小鼠。在第一次加强免疫后第10天，用ELISA在免疫小鼠的血清中检测到特异性抗体，表明重组蛋白能够刺激机体产生体液免疫应答。用MTT法分析免疫小鼠的脾细胞，发现这些重组蛋白均能刺激同源蛋白免疫小鼠的脾脏淋巴细胞增殖，提示这些重组蛋白能够刺激机体产生细胞免疫应答。56-kDa重组蛋白诱导机体产生的体液免疫应答的水平最高，而47-kDa重组蛋白诱导机体产生的细汕头大学医学院博七研究生学位论文胞免疫应答的水平最高。56一47kDa融合蛋白诱导的体液和细胞免疫应答水平分别与56一kDa重组蛋白和47一kDa重组蛋白所诱导的相当。 在第二次加强免疫后第14天，用10LD。，Karp株攻击免疫的小鼠，结果发现56一47kDa融合蛋白免疫组的死亡率比56一kDa重组蛋白或47一kDa重组蛋白免疫组低，与阴性对照组有显著差异。病理学检查显示56一47kDa融合蛋白免疫保护小鼠的肺、肝、肾未发现明显病理变化，而死亡小鼠的这些脏器均有严重病变。这些结果证明56一47kDa融合蛋白能更有效地诱导机体产生抗恙虫病东方体免疫应答，提示56一47kDa蛋白含有Karp株56一kDa和47一kDa外膜蛋白的抗原表位，既具有56一kDa外膜蛋白的良好体液免疫诱导性又具有47一kDa外膜蛋白的良好细胞免疫诱导性。 用Karp株56和47一kDa蛋白基因分别构建了单个基因重组的真核表达质粒pcDNA/47和pcDNA/56，并将这两个基因融合构建了双抗原基因重组的真核表达质粒pcDNA/瑟一47，用重组真核表达质粒分别转染真核细胞(COS一7细胞)。用56一和47一kDa蛋白免疫血清分别对转染的细胞作间接免疫荧光染色，结果证明56一和47一kDa蛋白基因以及它们的融合基因均在相应的转染的细胞内表达了目的蛋白。 将真核表达质粒pcDNA/47、pcDNA/56、pcDNA/56一47以肌肉多点注射的方式免疫Balb/c小鼠。在第二次加强免疫后第10天采集免疫小鼠的血清做EL工SA和收集脾细胞做淋巴细胞增殖试验，结果显示重组真核表达质粒联合同源重组蛋白免疫的小鼠比单独用重组真核表达质粒免疫小鼠产生较高水平的特异性抗体和特异性淋巴细胞增殖。Karp株的攻毒试验显示pcDNA/47或pcDNA/56免疫的小鼠死亡率高于pcDNA/56一47免疫组，而裸pcDNA/56一47的免疫效果又显著低于纳米脂质体包封的pcDNA/56一47。 本研究的结果表明56一47kDa融合蛋白具有恙虫病东方体Karp株56一kDa和47一kDa外膜蛋白的抗原表位和免疫原性，该融合蛋白能比单一外膜蛋白更有效地诱导机体产生抗恙虫病东方体的体液和细胞免疫应答。用56一47kDa融合蛋白基因构建的真核表达质粒免疫机体，也能有效地诱导机体产生抗恙虫病东方体汇!头大学医学院博l:研究生学位论文的体液和细胞免疫应答。56一47kDa融合基因与56一47kDa融合蛋白的联合免疫可能是替代恙虫病东方体全菌抗原免疫的一种较理想的预防恙虫病东方体感染的方式。更多还原

【Abstract】 Scrub typhus is a zoonosis caused by Orientia tsutusgamushi(Ot) and transmitted by trombiculid mites. The typical symptoms of scrub typhus include fever, headache, rash, eschar, and lymphadenopathy. Although the disease can be effectively treated with the antibiotic doxycycline, reinfection and relapses occur frequently. In addition, there is a possibility that antibiotic-resistant strains emerge. As a result of such potential problems, the development of scrub typhus vaccines should be pursued. To date, considerable efforts have been invested in developing a vaccine for scrub typhus, but no vaccines for humans are available at present. It is very urgent to develop novel vaccine against scrub typhus.Both 47- and 56-kDa proteins are considered as the major surface proteins of Ot, which has a potential importance in development of subunit vaccine against scrub typhus. The antigenic uniqueness of the 58-kDa heat shock protein of Ot indicates that this protein may be a potentially protective antigen.The genes encoding 47-kDa, 56-kDa, and 58-kDa proteins were amplified from the genomes of Ot. Karp strain by PCR, and the gene fragments were cloned and sequenced. The gene fragments encoding 47-kDa, 56-kDa, and 58-kDa proteins were linked to the prokaryotic expression vector pQE30, constructing recombinant plasmids pQE30/47, pQESO/56, and pQE30/58. In addition, the recombinant plasmids pQE30/56-47 harboring 47-and 56-kDa protein gene and pQE30/58-47 harboring 47- and 58-kDa protein gene were constructed. The transformants harboring the recombinant plasmids were induced to expresse the recombinant proteins with IPTG The expression products 47-kDa, 56-kDa, and 58-kDa proteins as well as 56-47kDa and 58-47kDa fusion protein were detected by SDS-PAGE and the proteins were verified by immunoblot analysis with antiserum to Ot Karp.To study the immunogenicity of the recombinant proteins, the recombinant proteins purified by affinity chromatograph were used to immunize Balb/c mice. After a booster injection, the IgGs to the recombinant proteins of Ot. Karp were detected in the sera from the immunized mice by ELISA. The antigen-specific spleen cell proliferative responses weredemonstrated in the immunized mice by MTT method. The results suggest that the recombinant proteins are the protective antigens capable to stimulate mice to produce specific humoral and cellular immunoresponses. The animals immunized with 56-47kDa fusion protein exhibited profound humoral and cellular immunoresponses, including higher levels of specific IgG antibodies, higher levels of gamma interferon (IFN-y) and interleukin-2 (IL-2), and stronger proliferation of splenic cells in response to homogenous antigen as compared with mice immunized with 47-kDa, 56-kDa, 58-kDa recombinant protein, or 58-47kDa fusion protein.The mice were challenged with 10 LD5o Ot Karp 14 days after second boost immunization. The mortality of mice immunized with 56-47kDa fusion protein was lower than that of the mice immunized with another recombinant protein after challenge of Ot Karp. The level of protection afforded by 56-47kDa fusion protein was 50%, higher than other groups and significantly higher than the negative controls (P <0.05). The mild pathological changes in lungs, livers, and kidneys were observed in the survival mice immunized with 56-47kDa fusion protein. However, the severe pathological changes in these organs were recognized in the unimmunized mice. These results suggest that the mice immunized with 56-47kDa fusion protein exhibited a more increased resistance to Ot Karp as compared with another recombinant protein-immunized mice. These results strongly suggest that 56-47kDa fusion protein contains antigenic epitopes of the 56- and 47-kDa outer membrane protein of Ot Karp and it is a more suitable candidate for replacing whole-cell antigen of Ot Karp to induce protective immunity against scrub typhus.The genes encoding 47- and 56-kDa outer membrane protein were used to construct the recombinant eukaryotic expression plasmids pcDNA/47, pcDNA/56, and pcDN更多还原