SUMO介导截短型人HGF制备及抗肝纤维化作用

Preparation and anti-fibrotic activity of human truncated variant of hepatocyte growth factor with SUMO fusion in Escherichia coli

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m RNA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。

Objective To determine the effect of human truncated variant of hepatocyte growth factor （tvNK1） on transforming growth factor-β1 （TGF-βl）-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 coll. The SUMO-tvNK1 protein was purified with Ni2＋-affinity chromatograph and cleaved with a SUMO- specific protease （Ulpl） to obtain the tvNK1. The proliferation of TGF-[31-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 al （Collal） and a-smooth muscle actin （a-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 isopropylq3-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 Collal and a-SMA in TGF-β1-induced LX-2 cells. Conclusion The recombinant WNK1 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.