本文選題：凋亡素 + TAT�。� 參考：《昆明醫(yī)學(xué)院》2009年碩士論文

【摘要】： 目的:構(gòu)建原核表達(dá)載體pGEX-6p-1/改良型TAT-VP3,并利用低溫包涵體法對(duì)該融合蛋白進(jìn)行純化;探討改良型TAT—VP3融合蛋白的安全性,及對(duì)其在免疫系統(tǒng)影響進(jìn)行初步研究。 方法:設(shè)計(jì)改良型TAT基因序列并委托生物技術(shù)公司合成,通過酶切連接的方法構(gòu)建原核表達(dá)載體pGEX-6p-1/改良型TAT-VP3。經(jīng)酶切及基因測序鑒定構(gòu)建成功后,利用IPTG低溫誘導(dǎo)目的蛋白表達(dá),收集包涵體并進(jìn)行純化。再進(jìn)行安全性實(shí)驗(yàn)及免疫藥理學(xué)試驗(yàn)(豚鼠全身主動(dòng)過敏實(shí)驗(yàn);家兔熱原實(shí)驗(yàn);家兔肌肉刺激實(shí)驗(yàn);免疫器官重量及臟器指數(shù)實(shí)驗(yàn);血清溶血素測定;PHA刺激淋巴細(xì)胞轉(zhuǎn)化實(shí)驗(yàn);巨噬細(xì)胞功能檢測;血清補(bǔ)體C3、C4及測定),觀察其安全性,評(píng)價(jià)其對(duì)小鼠免疫系統(tǒng)的影響。 結(jié)果:成功構(gòu)建原核表達(dá)載體pGEX-6p-1/改良型TAT-VP3,并利用低溫包涵體法純化出純度較為理想的目的蛋白;改良型TAT-VP3融合蛋白豚鼠過敏實(shí)驗(yàn)豚鼠未見任何不適反應(yīng);家兔熱原試驗(yàn)合格;家兔肌肉刺激實(shí)驗(yàn)未見明顯異常;改良型TAT-VP3融合蛋白對(duì)胸腺重量及胸腺指數(shù)影響不明顯,給藥組與對(duì)照組比較無顯著性差異(P＞0.05);脾臟重量各組之間比較也無顯著性差異(P＞0.05),但高劑量組的脾臟指數(shù)與對(duì)照組比較有顯著性差異(P＜0.05);溶血素測定實(shí)驗(yàn)低、高劑量組的OD值(吸光度)與對(duì)照組比較無顯著性差異(P＞0.05),但隨劑量的增高,顏色逐漸加深,OD值出現(xiàn)增高趨勢(shì);PHA刺激淋巴細(xì)胞轉(zhuǎn)化實(shí)驗(yàn)外周血T淋巴細(xì)胞轉(zhuǎn)化率無明顯改變,給藥組及對(duì)照組之間比較無顯著性差異(P＞0.05);巨噬細(xì)胞功能檢測低、高劑量組巨噬細(xì)胞吞噬百分率和吞噬指數(shù)與對(duì)照組有顯著性差異(P＜0.05,P＜0.01);血清補(bǔ)體C3、C4的測定低、高劑量組中的C3、C4有顯著性差異(P＜0.05,P＜0.01)。 結(jié)論:成功構(gòu)建原核表達(dá)載體pGEX-6p-1/改良型TAT-VP3,并誘導(dǎo)表達(dá)TAT-VP3融合蛋白,對(duì)改良型TAT-VP3融合蛋白進(jìn)行了純化;改良型TAT—VP3融合蛋白豚鼠過敏實(shí)驗(yàn)呈陰性豚鼠過敏試驗(yàn)合格;家兔熱原試驗(yàn)合格;家兔肌肉刺激實(shí)驗(yàn)合格;改良型TAT—VP3融合蛋白可增強(qiáng)小鼠體液免疫及非特異性免疫功能,增加血清中補(bǔ)體C3、C4的含量,對(duì)小鼠免疫系統(tǒng)未發(fā)現(xiàn)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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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本文編號(hào)：1762112