癌症是全世界死亡的主要原因之一。 使用嵌合抗原受體 T 細胞 (CAR-T) 策略的免疫療法是癌症輔助治療中重要且強大的方法。我們的目標是製定策略來增強 T 細胞生長和這些 T 細胞攜帶的 CAR-T 基因的表達。首先，我們設計了一種重組蛋白，利用馬來腹蛇蛇毒蛋白 Rhodostomin (Rho) 和 鋸鱗蝰蛇毒蛋白Echistatin (Ech) 來改善慢病毒向 T 細胞的轉導。Rho和 Ech 蛋白是有效的整合素抑製劑，實驗室先前的研究發現它們分別對整合素α4β1和α5β1結合具有很強的親和力。這種相互作用可以增強病毒和細胞之間的接觸，從而促進病毒進入。我們使用畢赤酵母表達系統表達並純化了兩種蛋白質：Rho/Ech 細胞結合域（與整合素 α5β1和α4β1 相互作用）和纖連蛋白肝素結合域 (Fn3(12-14))（與病毒相互作用）。 迄今為止，我已成功表達 Ech-Fn3(12-14) 並將其純化，產量約為 2 mg/L。細胞粘附試驗表明我的蛋白質的 Ech 結構域表現出其生物活性。為了確定基因轉導的最佳濃度，我們比較了不同濃度的聚凝胺和我的蛋白對轉導效率的影響。結果顯示，6μg聚凝胺的轉導效率最高，而纖連蛋白片段則表現出劑量依賴性。此外，與聚凝胺和對照組相比，兩種轉導增強劑的組合增加了 1.2 至 3.5 倍。在生長培養基中添加這兩種轉導增強劑將顯著增強CAR-T慢病毒轉導至T細胞的效率。此外，我們在畢赤酵母中表達並純化了血管內皮生長因子（VEGF），產量為2.0 mg/L。這項研究建立的方法將大大提高各種免疫細胞和相關療法的有效性。

Cancer is one of the leading causes of death worldwide. Immunotherapies using chimeric antigen receptor-T cell (CAR-T) strategies are important and powerful methods in cancer adjuvant therapies. We aim to develop strategies to enhance T cell growth and expression of the CAR-T genes carried by these T cells. First, we designed a strategy to improve lentivirus transduction to T cells by utilizing snake proteins Rhodostomin (Rho) and Echistatin (Ech). The Rho and Ech proteins are potent integrin inhibitors with strong affinities for binding to cancer cells. This interaction may enhance the contact between the virus and the cell, thereby facilitating viral entry. We expressed and purified two proteins using the Pichia pastoris expression system: the Rho/Ech cell-binding domains, which interact with integrins α5β1 and α4β1, and the fibronectin heparin-binding domain (Fn3(12-14)), which interacts with the virus. To date, I have successfully expressed the Ech-Fn3(12-14) and purified it to homogeneity with a yield of ~2 mg/L. Cell adhesion assay showed that the Ech domain of my protein exhibits its bioactivities. To determine the optimal concentration for gene transduction, we compared the effect of different concentrations of polybrene and my protein on the transduction efficiency. The results showed the highest transduction efficiency with 6 μg of polybrene, while the fibronectin fragments exhibited a dose-dependent manner. Moreover, the combination of two transduction enhancers had a 1.2-to-3.5-fold increase compared to polybrene and the control group. The addition of these two transduction enhancers to the growth medium will significantly enhance the transduction efficiencies of the CAR-T lentivirus into the T cell. In addition, we expressed and purified the vascular endothelial growth factor (VEGF) in Pichia pastoris with a yield of 2.0 mg/L. Methods established in this study will greatly improve the effectiveness of various immune cells and related therapies.

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