TGF-β1和TGF-βⅡ型受体酵母表达载体构建

  目的  研究截短型人肝细胞生长因子突变体（tvNK1）对转化生长因子β1（TGF-β1）诱导人肝星状细胞LX-2纤维化的影响。  方法  把经PCR和双酶切的小分子泛素相关修饰蛋白（SUMO）和tvNK1的融合基因SUMO-tvNK1插入原核表达载体pET28a，诱导表达和Ni²⁺亲和层析纯化SUMO-tvNK1；进一步经SUMO蛋白酶Ulp1水解和Ni²⁺亲和层析纯化tvNK1，MTT法观察其对TGF-β1诱导LX-2纤维化细胞增殖的影响，qPCR及蛋白印迹（WB）检测其对I型胶原蛋白（col1α1）和α – 平滑肌球蛋白（α-SMA）蛋白表达的影响。  结果  宿主菌Rosetta/pET28a-SUMO-tvNK1在37 ℃经1 mmol/L IPTG诱导5 h获得SUMO-tvNK1的可溶性表达，将超声破碎后上清液经Ni²⁺亲和层析成功获得SUMO-tvNK1，进一步经Ulp1水解和Ni²⁺亲和层析纯化tvNK1，SDS-PAGE鉴定其分子量约为22.0 kDa，MTT显示20和40 ng/mL的tvNK1能明显抑制TGF-β1活化的人肝星状细胞LX-2增殖，qPCR及WB显示20和40 ng/mL的tvNK1能显著抑制活化的LX-2细胞中Col1α1和 α-SMA在mRNA和蛋白水平表达。  结论  SUMO蛋白可用于制备tvNK1，其能抑制TGF-β1活化的LX-2细胞增殖并具有抗纤维化作用，为进一步体内外功能研究打下基础。

Anti-fibrotic effect of thymoquinone on hepatic stellate cells

  Objective  To determine the effect of human truncated variant of hepatocyte growth factor (tvNK1) on transforming growth factor-β1 (TGF-β1)-induced fibrosis in LX-2 cells.  Methods  The small ubiquitin-related modifier (SUMO) and tvNK1 genes were obtained with PCR and the fusion gene SUMO-tvNK1 with over-lap PCR. The expression vector pET28a/SUMO-tvNK1 was constructed and the soluble recombinant SUMO-tvNK1 protein was expressed in Escherichia coli. The SUMO-tvNK1 protein was purified with Ni²⁺-affinity chromatograph and cleaved with a SUMO-specific protease (Ulp1) to obtain the tvNK1. The proliferation of TGF-β1-induced LX-2 cells was determined with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The mRNA and protein expression of collagen type 1 α1 (Col1α1) and α-smooth muscle actin (α-SMA) were assayed with quantitative PCR (qPCR) and Western blot (WB), respectively.  Results  The expression vector pET28a/SUMO-tvNK1 was successfully obtained and the soluble SUMO-tvNK1 protein was induced after 5 hours of induction at 37℃ with 1 mM isopropyl-β-d-thiogalactoside (IPTG) and purified with Ni²⁺-affinity chromatograph. The molecular weight of the purified tvNK1 is 22.0 kilodalton based on the result of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of MTT assay, qPCR and WB demonstrated that at the concentration of 20 and 40 ng/ml, the recombinant tvNK1 could obviously inhibit the proliferation of TGF-β1-induced LX-2 cells and reduce the mRNA and protein expression of Col1α1 and α-SMA in TGF-β1-induced LX-2 cells.  Conclusion  The recombinant tvNK1 protein was obtained with a SUMO fusion procedure and the prepared tvNK1 can inhibit the proliferation TGF-β1-induced LX-2 cells and is of anti-fibrotic activity. These findings provide a basis for further in vitro researches.