日本血吸虫天然分子编码基因重组蛋白疫苗rSjHGPRT的安全性评价

发布时间：2018-01-03 17:08

本文关键词：日本血吸虫天然分子编码基因重组蛋白疫苗rSjHGPRT的安全性评价　出处：《中南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的本研究旨在通过注射日本血吸虫天然分子编码基因重组蛋白疫苗rSjHGPRT分别观察家兔的皮下注射的局部刺激反应、豚鼠的全身过敏状况及小鼠的急性毒性作用，初步评价该蛋白分子疫苗的临床前安全性，为其进一步开发应用奠定工作基础。方法通过IPTG诱导，转化含SjHGPRT／pQE30的E.coliM15，大量表达rSjHGPRT；采用SDS-PAGE、电洗脱、透析法分离纯化重组蛋白；皮下注射家兔，肉眼观察局部病理改变，显微镜下观察组织病理学变化；接种豚鼠，按规定的技术原则观察和评价豚鼠的全身过敏反应；采用最大剂量法，，接种小鼠后观察其ALT、AST、Cr、BUN和血常规参数等反映急性毒性作用的指标。结果 1、采用IPTG诱导法，大量表达了rSjHGPRT，采用SDS—PAGE，电洗脱和透析等方法成功分离、纯化重组蛋白，纯化产物经细菌学检测为阴性。 2、家兔皮下注射rSjHGPRT后至72小时，局部无充血，出血，水肿等改变，组织病理学检查可见注射部位少量炎性细胞浸润，未发现水肿、坏死等组织学改变。 3、豚鼠接种rSjHGPRT的全身过敏实验显示，实验组仅一只出现弱阳性过敏反应，且很快缓解，其余豚鼠未见过敏症状。 4、小鼠急性毒性实验过程中，实验组小鼠未出现明显的行为异常，进食、饮水及活动与对照组小鼠无明显差异。小鼠体重变化未出现有统计学意义的改变，检测实验小鼠肝、肾功能及血常规亦未发现有统计学意义的结果。结论 1、成功表达、分离、纯化了重组蛋白rSjHGPRT。 2、家兔皮下注射rSjHGPRT后局部刺激反应和组织病理学改变。 3、rSjHGPRT接种豚鼠几乎无全身过敏反应，实验组中个别动物出现一过性过敏反应症状，也能很快自行缓解。 4、接种rSjHGPRT对小鼠无明显急性毒性作用。
[Abstract]:Objective to observe the local stimuli of subcutaneous injection of Schistosoma japonicum natural molecular encoding gene recombinant protein vaccine (rSjHGPRT) in rabbits. The systemic allergy of guinea pigs and the acute toxicity of mice were evaluated preliminarily to evaluate the preclinical safety of the protein molecular vaccine in order to lay a foundation for its further development and application. Methods E. coli M15 containing SjHGPRT/pQE30 was transformed into E. coli M15 by IPTG, and rSjHGPRT was expressed in large quantities. The recombinant protein was purified by SDS-PAGE, electroelution and dialysis. Local pathological changes were observed by subcutaneous injection of rabbits and histopathological changes were observed under microscope. After inoculating guinea pigs, the systemic allergic reactions of guinea pigs were observed and evaluated according to the prescribed technical principles. The maximum dose method was used to observe the parameters of alt ASTX Cr bun and blood routine to reflect the acute toxicity of mice after inoculation. Results 1. RSjHGPRT was expressed by IPTG induction method. The recombinant protein was purified by SDS-PAGE, electroelution and dialysis. The purified product was negative by bacteriological examination. 2. There were no changes of hyperemia, hemorrhage, edema and so on after subcutaneous injection of rSjHGPRT to 72 hours. Histopathological examination showed a small amount of inflammatory cell infiltration at the injection site, and no edema was found. Necrosis and other histological changes. 3. The systemic allergy test of guinea pigs inoculated with rSjHGPRT showed that only one of the experimental groups had a weak positive allergic reaction, and the other guinea pigs had no allergic symptoms. 4. In the course of acute toxicity test, the mice in the experimental group had no obvious behavioral abnormality, no significant difference in food intake, drinking water and activity, but no significant changes in body weight in the experimental group compared with those in the control group. No significant results were found in liver, kidney function and blood routine examination. Conclusion 1. The recombinant protein rSjHGPRT was successfully expressed, isolated and purified. 2. Local stimulation and histopathological changes after subcutaneous injection of rSjHGPRT in rabbits. 3The guinea pigs inoculated with rSjHGPRT had almost no systemic allergic reaction, and the symptoms of transient allergic reaction appeared in some animals in the experimental group. 4. Inoculation of rSjHGPRT had no obvious acute toxicity in mice.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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