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Studies on Safety and Immune System Influence of the Modified Fusion Protein Type of TAT-VP3

Author: ZhangWenYi
Tutor: WangJianSong；ZuoYiGang；YangDeLin
School: Kunming Medical College
Course: Surgery
Keywords: Apoptin TAT Fusion protein Security Immune Pharmacology
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 45
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Abstract


Objective: To construct prokaryotic expression vector pGEX-6p-1 / modified TAT-VP3, and use of low-temperature inclusion bodies of the fusion protein was purified; explore the safety of modified TAT-VP3 fusion protein, and its impact in the immune system a preliminary study. Methods: The design of improved the TAT gene sequences and commissioned by the biotechnology company synthesis, digested connected, prokaryotic expression vector pGEX-6p-1 / modified TAT-VP3. Successfully constructed by restriction enzyme digestion and gene sequencing, IPTG low-temperature-induced expression of the target protein, to collect the inclusion bodies and purified. Further safety studies and immune pharmacology test (guinea pig Active systemic allergy test; rabbit pyrogen experiment; rabbit muscle stimulation experiments; immune organ weight and organ index experiments; serum hemolysin determination; PHA stimulated lymphocyte transformation test; macrophage function testing; Serum Complement C3, C4, and determination), observed its security, evaluate its impact on the immune system in mice. Results: successfully constructed prokaryotic expression vector pGEX-6p-1 / modified TAT-VP3, and the use of low-temperature inclusion body purification purity ideal target protein; modified TAT-VP3 fusion protein guinea pig allergic guinea pigs no discomfort ; rabbit fever original test qualified; rabbit muscle stimulation experiments no obvious abnormalities; improved TAT-VP3 fusion protein no significant effect on the thymus weight and thymus index, administration and control groups showed no significant difference (P> 0.05 ); between spleen weight of each group comparison also no significant difference (P> 0.05), but the high-dose group, the spleen index and control group comparison there a significant difference (P <0.05); hemolytic factors determination experimental low-and high-dose group OD (absorbance) compared with the control group, no significant difference (P> 0.05), but increased with the dose, the color gradually deepened, OD values ??tended to increase; PHA stimulated lymphocyte transformation test peripheral T lymphocyte transformation rate no change significantly between the administration and control groups showed no significant difference (P> 0.05); macrophage function detects low and high dose groups macrophage phagocytic percentage and phagocytic index with the control group there is a significant difference (P < 0.05, P <0.01); Serum Complement C3, C4 Determination lower in the high dose group C3, C4 significant difference (P <0.05, P <0.01). Conclusion: successfully construct the prokaryotic expression vector pGEX-6p-1 / improved the TAT-VP3, and induction of expression of TAT-VP3 fusion protein, modified of TAT-VP3 fusion proteins were purified; improved of TAT-VP3 fusion protein guinea pig allergic experimental negative guinea pig allergy tests qualified; rabbit pyrogen test qualified; rabbit muscle stimulation experiments qualified; improved TAT-VP3 fusion protein can enhance humoral immunity in mice and non-specific immune function, increased serum complement C3 and C4 content found no toxic effects on the immune system.

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CLC: > Medicine, health > Basic Medical > Medical Immunology
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