摘要
目的通过构建原核表达系统,在大肠埃希菌中表达重组人肝再生增强因子(hALR)蛋白,为各种急慢性肝损伤及肝衰竭的治疗提供新的治疗方法奠定基础。方法利用正常人肝组织提取总RNA,经一步法逆转录聚合酶链反应(RTPCR)获取hALRcDNA全序列,构建原核表达载体hALRpET28a(+)转化大肠埃希菌(BL21),诱导表达重组hALR蛋白,以组氨酸为标签进行蛋白纯化。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)和WesternBlot鉴定重组蛋白。结果经双酶切和DNA测序证实hALRcDNA正确插入表达载体pET28a(+),成功表达并纯化得相对分子质量为23000的重组蛋白,低温诱导表达使可溶蛋白明显增加,与预期结果相符。结论成功表达和纯化获重组hALR蛋白。
Objective As to find a new way in the therapy of various acute or chronic hepatic injury and hepatic failure, the purified recombinant human augmenter of liver regeneration （hALR） was obtained from Escherichia coli BL21 （DE3）by establishing a prokaryotic expression system using prokaryotic expression plasmid pET28a（＋）. Methods The prokaryotic expression system was constructed by prokaryotic expression plasmid pET28a（＋ ） and a full length hALR cDNA obtained from human liver tissue by one-step RT-PCR. The recombinant hALR protein was expressed in Escherichia coli BL21 under IPTG induction （to a final concentration of 0. 4 retool/L）, The recombinant protein was determined by SDS-PAGE and Western-blot. Change expression condition to increase the expression of protein and purify the objective protein by His Bind Kits, Detect the purified protein by SDS-PAGE and Western-blot. Results The full length cDNA encoding hALR and histidine label was obtained and detected by agarose gel electrophoresis of about 420 bp, The correct insertion site in hALR- pET28a（＋） recombinant plasmid was confirmed by restriction endonuclease digestion and the correct DNA sequence of the insertion fragment was determined by DNA sequencing, The molecular weight of the recombinant hALR protein including histidine label was 23 000 detected by SDS-PAGE and Western blot. The protein strape in SDS-PAGE was a single line of about 23 000 after His Tag Bind Kits purification. Lower temperature can increase soluble protein expression, 23℃ was the best induction temperature as to obtain more soluble protein, Conclusions Full length hALR cDNA was obtained from human liver tissue and it was inserted correctly into the prokaryotic expression plasmid pET28a （＋）. The recombinant hALR protein was obtained and purified successfully and it could be developed into a new anti-hepatic damage product,
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2005年第3期157-160,共4页
Chinese Journal of Infectious Diseases
基金
国家863计划(2003AA205150)
国家自然科学基金(30170255)
浙江省"新世纪151人才工程"基金
浙江省卫生厅医学科研人才基金(2001.QN01
2004B064)
浙江省中医管理局青年人才基金(No:2002C037)
浙江省自然科学基金(Y204156)
