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目的 :用重组Bac TRⅡ杆状病毒表达系统大量表达融合蛋白TGF βRⅡ /Fc,并对其进行纯化及鉴定 ;验证该融合蛋白是否能够阻断细胞因子TGF β1的生物学作用。 方法 :采用病毒空斑形成试验测定病毒滴度 ,并对其进行扩增。采用Pro teinG柱和快速蛋白质液相层析法 (FPLC)纯化目的蛋白。采用SDS PAGE分析纯化后的目的蛋白 ,并将蛋白电泳条带进行灰度扫描 ,计算目的蛋白的含量。采用Westernblot和夹心ELISA法 ,鉴定目的蛋白是否表达。采用MTT比色法 ,验证融合蛋白TGF βRⅡ /Fc能否阻断TGF β1对小鼠肺成纤维细胞 (L92 9)生长的作用。采用Westernblot技术 ,验证融合蛋白能否阻断TGF β1对L92 9细胞纤维黏连蛋白 (FN)生成的促进作用。结果 :本实验室构建并保存的重组Bac TRⅡ杆状病毒原液内病毒的滴度为 2× 10 12 pfu/L。蛋白电泳后灰度扫描结果表明 ,目的蛋白约占总蛋白的 10 %。夹心ELISA法和Westernblot分析证明目的蛋白已表达。融合蛋白TGF βRⅡ /Fc能够阻断TGF β1对L92 9细胞生长的抑制作用 ,以及对该细胞FN生成的促进作用。结论 :本室构建的重组Bac TRⅡ杆状病毒表达系统 ,能够表达融合蛋白TGF βRⅡ /Fc ,表达水平为 9mg/L。纯化得到的目的蛋白具有一定的生物学活性 ,可阻断TGF β1对L92 9细胞生长的?
OBJECTIVE: To express and express the fusion protein TGFβRⅡ / Fc in large quantities by recombinant Bac TR Ⅱ baculovirus expression system, and to verify whether the fusion protein can block the biological effect of TGFβ1. Methods: The viral titer was determined by virus plaque formation test and amplified. The target protein is purified using a Pro teinG column and rapid protein liquid chromatography (FPLC). The purified protein was analyzed by SDS PAGE, and the protein electrophoresis band was scanned by grayscale to calculate the content of the target protein. Western blot and sandwich ELISA were used to identify whether the target protein was expressed. MTT colorimetric method was used to verify whether the TGFβRⅡ / Fc fusion protein could block the effect of TGFβ1 on the growth of mouse lung fibroblasts (L92 9). Westernblot technique was used to verify whether the fusion protein could block the promoting effect of TGFβ1 on fibronectin (FN) production in L92 9 cells. Results: The titer of virus in the recombinant Bac TR Ⅱ baculovirus stock solution constructed and preserved in our laboratory was 2 × 10 12 pfu / L. Grayscale scanning results showed that the target protein accounted for about 10% of the total protein. Sandwich ELISA and Western blot analysis confirmed that the target protein was expressed. The fusion protein TGFβRⅡ / Fc can block the inhibitory effect of TGFβ1 on the growth of L92 9 cells and the promotion of FN production. CONCLUSION: The recombinant Bac TR II baculovirus expression system constructed in this study can express the fusion protein TGF βRⅡ / Fc at a level of 9 mg / L. Purification of the target protein has a certain biological activity, can block TGF β1 on L92 9 cell growth?