【摘要】： 貝氏柯克斯體(Coxiella burnetii，又稱Q熱立克次體)為Q熱病原體。該病原體為專性細(xì)胞內(nèi)寄生菌，可通過氣溶膠傳播，對(duì)人和動(dòng)物的感染性極強(qiáng)，是一種重要的生物戰(zhàn)劑／生物恐怖劑。人、畜對(duì)貝氏柯克斯體普遍易感，畜牧相關(guān)從業(yè)人員是高危人群。不管是預(yù)防貝氏柯克斯體自然感染還是防生物武器／生物恐怖均需要有效的Q熱疫苗對(duì)易感人群進(jìn)行免疫接種。 本研究將國內(nèi)分離的貝氏柯克斯體新橋株在雞胚大量繁殖，然后從感染雞胚中分離、純化貝氏柯克斯體，用甲醛滅活純化貝氏柯克斯體，制備全細(xì)胞疫苗(WCV)，同時(shí)采用氯仿-甲醇提取WCV，制備氯仿-甲醇提取殘存組分疫苗(CMR)。采用高壓氣相色譜測定了兩種疫苗的脂肪酸，發(fā)現(xiàn)CMR較WCV減少了11種脂肪酸，其中4種為不飽和脂肪酸，4種為支鏈脂肪酸，70％為C原子數(shù)≥18的較長鏈的脂肪酸。另外，兩種疫苗的支鏈脂肪酸與直鏈脂肪酸、不飽和脂肪酸與飽和脂肪酸的比值也明顯不同。通過蛋白單相及雙向電泳分析兩種疫苗蛋白，它們的蛋白電泳圖譜和主要高豐度蛋白點(diǎn)的蛋白豐度均無明顯差異。 采用CMR和WCV免疫BALB／c小鼠，，間接免疫熒光法分析免疫小鼠血清特異性抗體水平，結(jié)果顯示CMR與WCV一樣均能誘導(dǎo)高水平特異性抗體產(chǎn)生。CMR加Al(OH)3佐劑免疫小鼠，該免疫小鼠特異性抗體水平顯著高于CMR單獨(dú)免疫。用MTT法體外分析免疫小鼠脾臟淋巴細(xì)胞增殖，結(jié)果顯示CMR誘導(dǎo)小鼠脾臟淋巴細(xì)胞增殖水平顯著高于WCV，而WCV對(duì)小鼠淋巴細(xì)胞增殖有一定程度的抑制。免疫小鼠體內(nèi)、外細(xì)胞因子的測定結(jié)果顯示，CMR疫苗能誘導(dǎo)TNF-α、IFN-γ及IL-10等多種細(xì)胞因子的分泌。 用貝氏柯克斯體Ⅰ相強(qiáng)毒株(包括疫苗制備株和美國分離Nine Mile株和歐洲分離的Henzerling)攻擊免疫小鼠，用熒光定量PCR分析小鼠脾臟貝氏柯克斯體的量。結(jié)果顯示CMR與WCV一樣能有效保護(hù)小鼠抵抗貝氏柯克斯體的攻擊。CMR加Al(OH)3佐劑免疫小鼠的脾臟貝氏柯克斯體的量顯著少于CMR單獨(dú)免疫。 用大劑量WCV免疫小鼠，在接種部位出現(xiàn)皮下結(jié)節(jié)，小鼠的脾臟明顯腫大，組織病理學(xué)檢查脾臟有不同程度的網(wǎng)狀內(nèi)皮細(xì)胞增生。檢查免疫小鼠肝臟功能指標(biāo)，ALT和AST均明顯升高。而相同大劑量CMR免疫小鼠則無上述病理學(xué)變化和肝功能損傷。 WCV疫苗是目前唯一得到美國FDA批準(zhǔn)應(yīng)用的Q熱疫苗，雖然其具有良好的免疫保護(hù)效果，但其明顯的副作用使得其應(yīng)用受到嚴(yán)格限制。本研究證明采用國內(nèi)分離株制備的CMR疫苗與WCV疫苗的免疫保護(hù)效果相當(dāng)，但是CMR疫苗的副作用明顯小于WCV，CMR疫苗的低毒性是由于氯仿-甲醇提取WCV后，清除引起機(jī)體損傷的某些脂類或顯著降低某些脂類含量密切有關(guān)。CMR疫苗的良好免疫保護(hù)效果與其高效誘導(dǎo)機(jī)體的體液和細(xì)胞免疫應(yīng)答相關(guān)。 Q熱疫苗免疫小鼠能部分對(duì)抗登革病毒的感染，顯示其具有非特異免疫保護(hù)作用。
[Abstract]:Coxiella burnetii (also known as Q-febrile rickettsia) is a Q-fever pathogen. The pathogen is a specific intracellular parasitic bacteria, which can be transmitted by aerosols and is highly infectious to humans and animals. It is an important biological warfare agent/bioterroric agent. People, livestock are generally susceptible to Coxiella burnetii, and livestock-related practitioners are at high risk. Population. Effective Q-fever vaccines are needed to immunize susceptible populations, whether to prevent natural Coxiella baileyi infection or biological weapons/bioterrorism.
In this study, a new bridge strain of Coxoplasma baileyi isolated in China was propagated in chicken embryos, then isolated from infected chicken embryos, purified Coxoplasma baileyi, inactivated by formaldehyde, purified Coxoplasma baileyi, prepared whole cell vaccine (WCV), and extracted WCV with chloroform-methanol to prepare residual component vaccine (CMR). The fatty acids of the two vaccines were determined by phase chromatography, and 11 kinds of fatty acids were found to be reduced by CMR compared with WCV. Among them, 4 were unsaturated fatty acids, 4 were branched chain fatty acids, and 70% were longer chain fatty acids with C atom number (>18). In addition, the ratio of branched chain fatty acids to straight chain fatty acids, unsaturated fatty acids to saturated fatty acids of the two vaccines was not significant. Similarly, there was no significant difference between the two vaccine proteins in protein electrophoresis profiles and protein abundance of major high abundance protein spots.
CMR and WCV were used to immunize BALB/c mice. Indirect immunofluorescence assay was used to analyze the level of serum specific antibodies in immunized mice. The results showed that CMR could induce high level of specific antibodies as well as WCV. The level of specific antibodies in immunized mice immunized with CMR plus Al (OH) 3 adjuvant was significantly higher than that in CMR alone. The results showed that the proliferation level of splenic lymphocytes induced by CMR was significantly higher than that induced by WCV, while WCV inhibited the proliferation of lymphocytes to a certain extent.
Immune mice were attacked with phase I virulent Coxiella baileyi strains (including vaccine preparation strains, Nine Mile strains isolated from the United States and Henzerling strains isolated from Europe). The amount of Coxiella baileyi in the spleen of mice was analyzed by fluorescence quantitative PCR. The results showed that CMR could protect mice against Coxiella baileyi as well as WCV. CMR plus Al (OH) 3 adjuvant was immune. The amount of spleen Kirk's body in spleen of epidemic mice was significantly less than that of CMR alone.
Immunized mice with high dose of WCV showed subcutaneous nodules at the site of inoculation, spleen enlargement and reticular endothelial cell hyperplasia in different degrees. The liver function indexes, ALT and AST of immunized mice were significantly increased, while the same dose of CMR immunized mice showed no pathological changes and liver function damage. Injury.
WCV vaccine is currently the only Q-fever vaccine approved by FDA in the United States. Although it has a good immune protection effect, its obvious side effects restrict its application. This study proves that the immune protection effect of CMR vaccine prepared by domestic isolates is similar to that of WCV vaccine, but the side effects of CMR vaccine are obviously less than that of WCV vaccine. The low toxicity of WCV, CMR vaccine is due to the removal of some lipids causing injury or the significant reduction of some lipids content after chloroform-methanol extraction of WCV. The good immune protection effect of CMR vaccine is related to its highly effective induction of humoral and cellular immune responses.
The mice immunized with Q-fever vaccine could partly resist the infection of dengue virus, which showed that it had non-specific immune protection.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R376

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫長儉;陳梅玲;溫博海;劉海洪;楊曉;牛東升;;兩種Q熱疫苗的脂肪酸分析[J];中國人獸共患病學(xué)報(bào);2006年11期

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