改良型TAT-VP3融合蛋白安全性和对免疫系统影响的研究

发布时间：2018-04-17 04:34

本文选题：凋亡素 + TAT　；参考：《昆明医学院》2009年硕士论文

【摘要】： 目的:构建原核表达载体pGEX-6p-1/改良型TAT-VP3,并利用低温包涵体法对该融合蛋白进行纯化;探讨改良型TAT—VP3融合蛋白的安全性,及对其在免疫系统影响进行初步研究。方法:设计改良型TAT基因序列并委托生物技术公司合成,通过酶切连接的方法构建原核表达载体pGEX-6p-1/改良型TAT-VP3。经酶切及基因测序鉴定构建成功后,利用IPTG低温诱导目的蛋白表达,收集包涵体并进行纯化。再进行安全性实验及免疫药理学试验(豚鼠全身主动过敏实验;家兔热原实验;家兔肌肉刺激实验;免疫器官重量及脏器指数实验;血清溶血素测定;PHA刺激淋巴细胞转化实验;巨噬细胞功能检测;血清补体C3、C4及测定),观察其安全性,评价其对小鼠免疫系统的影响。结果:成功构建原核表达载体pGEX-6p-1/改良型TAT-VP3,并利用低温包涵体法纯化出纯度较为理想的目的蛋白;改良型TAT-VP3融合蛋白豚鼠过敏实验豚鼠未见任何不适反应;家兔热原试验合格;家兔肌肉刺激实验未见明显异常;改良型TAT-VP3融合蛋白对胸腺重量及胸腺指数影响不明显,给药组与对照组比较无显著性差异(P＞0.05);脾脏重量各组之间比较也无显著性差异(P＞0.05),但高剂量组的脾脏指数与对照组比较有显著性差异(P＜0.05);溶血素测定实验低、高剂量组的OD值(吸光度)与对照组比较无显著性差异(P＞0.05),但随剂量的增高,颜色逐渐加深,OD值出现增高趋势;PHA刺激淋巴细胞转化实验外周血T淋巴细胞转化率无明显改变,给药组及对照组之间比较无显著性差异(P＞0.05);巨噬细胞功能检测低、高剂量组巨噬细胞吞噬百分率和吞噬指数与对照组有显著性差异(P＜0.05,P＜0.01);血清补体C3、C4的测定低、高剂量组中的C3、C4有显著性差异(P＜0.05,P＜0.01)。结论:成功构建原核表达载体pGEX-6p-1/改良型TAT-VP3,并诱导表达TAT-VP3融合蛋白,对改良型TAT-VP3融合蛋白进行了纯化;改良型TAT—VP3融合蛋白豚鼠过敏实验呈阴性豚鼠过敏试验合格;家兔热原试验合格;家兔肌肉刺激实验合格;改良型TAT—VP3融合蛋白可增强小鼠体液免疫及非特异性免疫功能,增加血清中补体C3、C4的含量,对小鼠免疫系统未发现毒副作用。
[Abstract]:Aim: to construct the prokaryotic expression vector pGEX-6p-1/ modified TAT-VP3 and purify the fusion protein by low temperature inclusion body method, and to investigate the safety of the modified TAT-VP3 fusion protein and its effect on the immune system.Methods: the modified TAT gene sequence was designed and synthesized by biotechnology company. The prokaryotic expression vector pGEX-6p-1/ modified TAT-VP3 was constructed by restriction endonuclease ligation.After restriction endonuclease digestion and gene sequencing, IPTG was used to induce the expression of the target protein, and the inclusion bodies were collected and purified.Then the safety test and immunopharmacological test (guinea pig systemic active allergy test; rabbit pyrogen test; rabbit muscle stimulation test; immune organ weight and organ index test; serum hemolysin assay; PHA stimulation lymphocyte transformation test;The function of macrophages, the serum complement C _ 3 C _ 4 and the determination of C _ 3 C _ 4 were measured, the safety of C _ 3C _ 4 and its effect on the immune system of mice were evaluated.Results: the prokaryotic expression vector pGEX-6p-1/ modified TAT-VP3 was successfully constructed and purified with low temperature inclusion body method.The effect of modified TAT-VP3 fusion protein on thymus weight and thymus index was not obvious.There was no significant difference in spleen weight and spleen weight between the two groups (P > 0.05), but the spleen index in the high dose group was significantly higher than that in the control group (P < 0.05).There was no significant difference in OD value (absorbance) between the high dose group and the control group (P > 0.05), but with the increase of the dose, the OD value increased gradually and the transformation rate of T lymphocytes in peripheral blood stimulated by PHA did not change significantly.There was no significant difference between the administration group and the control group (P > 0.05), the detection of macrophage function was low, the phagocytosis percentage and phagocytosis index of macrophage in the high dose group were significantly different from those in the control group (P < 0.05 P < 0.01), the level of complement C _ 3N _ 4 in serum was lower than that in the control group.There was significant difference in C _ 3 C _ 4 in high dose group (P < 0.05) and P < 0.01 (P < 0.01).Conclusion: the prokaryotic expression vector pGEX-6p-1/ modified TAT-VP3 was successfully constructed and the modified TAT-VP3 fusion protein was purified by inducing the expression of TAT-VP3 fusion protein.The modified TAT-VP3 fusion protein could enhance the humoral immunity and non-specific immune function of mice, increase the content of complement C _ 3N _ 4 in serum, and had no toxic and side effects on the immune system of mice.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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