摘要
目的·构建外膜脂蛋白Lpp(Braun's lipoprotein)基因缺失的大肠埃希菌菌株,在该菌株中进行大肠埃希菌胞外生产N-糖基化重组蛋白研究。方法·利用Red同源重组系统,敲除大肠埃希菌CLM37基因组上的外膜脂蛋白Lpp基因;通过绘制生长曲线,研究Lpp基因缺失后对大肠埃希菌生长状态的影响。将受体蛋白表达载体pIG6-rFn3-Gly和空肠弯曲杆菌来源的N-糖基化基因簇载体pACYCpgl共转化大肠埃希菌CLM37?Lpp,研究大肠埃希菌胞外生产N-糖基化重组蛋白情况。结果·获得了Lpp基因缺失的大肠埃希菌菌株CLM37?Lpp,并在该菌株中成功实现了胞外生产N-糖基化重组蛋白rFn3-Gly;相比于菌株CLM37,大肠埃希菌CLM37?Lpp胞外生产重组蛋白rFn3-Gly的总量约提升了4倍,糖基化效率约提高了6倍。结论·成功构建大肠埃希菌菌株CLM37?Lpp并实现了胞外生产N-糖基化重组蛋白r Fn3-Gly,提高了糖蛋白产量及糖基化效率。
Objective · To construct the E.coli CLM37 strain with Lpp gene deletion and to study the production of N-glycosylated recombinant proteins in this E.coli strain. Methods · Firstly, Red homologous recombination system was used to knock out the Lpp gene from the genome of E.coli CLM37. And then, the growth curve was detected to study the effects of deleted Lpp gene on the growth states of E.coli strain. Finally, the vector pIG6-rFn3-Gly which expresses receptor protein and the vector pACYCpgl, which carries N-glycosylation gene cluster that derives from Campylobacter jejuni, were co-transformed into E.coli CLM37？Lpp to investigate the extracellular production of N-glycosylated recombinant proteins. Results · The E. coli CLM37？Lpp with Lpp gene deletion was obtained, and the extracellular production of N-glycosylated rFn3-Gly was successfully achieved in this strain. Compared with E. coli CLM37, the total amount of rFn3-Gly produced via extracellular production of E. coli CLM37？Lpp increased about 4 times, and the glycosylation effciency increased about 6 times. Conclusion · N-glycosylated rFn3-Gly was successfully produced via extracellular production in E. coli CLM37？Lpp, and the production of interest glycoprotein and the glycosylation effciency were improved.
作者
阮瑶
王力凡
郭龙华
付鑫
丁宁
胡学军
RUAN Yao;WANG Li-fan;GUO Long-hua;FU Xin;DING Ning;HU Xue-jun(Medical Research Centre,Dalian University,Dalian 116622,China)
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2018年第11期1306-1311,共6页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(31370937)~~
