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

作　　者：王小花 李玉婷[2] 董艳[2] 刘亚威[2] 刘海峰 WANG Xiao-hua;LI Yu-ting;DONG Yan(Medical Research Center,Mudanjiang Medical University,Mudanjiang,Heilongjiang Province 157011,China)

机构地区：[1]牡丹江医学院医药研究中心,黑龙江牡丹江157011 [2]牡丹江医学院基础医学院病原微生物与免疫实验室

出　　处：《中国公共卫生》2018年第11期1532-1536,共5页Chinese Journal of Public Health

基　　金：国家自然科学基金(81500471;81700544);黑龙江省自然科学基金(H2015080);黑龙江省卫生厅项目(2016–355)

摘　　要：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.

关 键 词：截短型人肝细胞生长因子 小分子泛素相关修饰蛋白(SUMO) 转化生长因子β1(TGF-β1) 肝星状细胞 

分 类 号：R575.2[医药卫生—消化系统]