肾素前体和成熟肾素的原核表达及纯化

Prokaryotic expression and purification of human renin and mature renin

目的原核表达及纯化肾素前体(renin,REN)和成熟肾素(REN-340),并检测其浓度。方法根据NCBI中检索获得的人REN基因序列(5972),经E.coli密码子优化后,人工合成REN及REN-340基因片段,克隆至载体p ET-28a(+),构建重组表达质粒6×His-REN-p ET和6×His-REN-340-p ET。转化至感受态E.coli BL21(DE3),于不同诱导温度(20、25、30、37℃)及不同终浓度(0.1、0.2、0.4、0.5 mmol/L)IPTG条件下进行诱导表达。采用最佳诱导条件诱导表达重组REN和REN-340,经Ni-NTA镍离子亲和层析后,ELISA法检测其浓度。结果经双酶切及测序鉴定,重组表达质粒6×His-REN-p ET和6×His-REN-340-p ET构建正确。重组REN和REN-340的最佳诱导温度分别为37和20℃,最佳IPTG终浓度分别为0.1和0.2 mmol/L。纯化的重组REN和RNE-340浓度分别为9148.21和15115.31 m IU/m L。结论制备的重组REN和REN-340蛋白浓度较高,且生产周期较短,有望用于制备室间质量评价(external quality assessment,EQA)的候选质控品和标准物质。

Objective To express and purify the human renin(REN)and mature renin(REN-340)in prokaryotic cells,and detect the concentration.Methods According to the human REN gene sequence(5972)registered in NCBI,REN and REN-340 gene fragments were artificially synthesized and cloned into vector pET-28a(+)after E.coli codon optimization,and the recombinant expression plasmids 6×His-REN-pET and 6×His-REN-340-pET were constructed.The recombinant plasmids were transformed into competent E.coli BL21(DE3)and induced at different induction temperatures(20,25,30 and 37 C)and different final concentrations(0.1,0.2,0.4 and 0.5 mmol/L)of IPTG.Recombinant REN and REN-340 were induced under the optimal induction conditions,and the concentrations were detected by ELISA after nickel ion affinity chromatography with Ni-NTA.Results The recombinant expression plasmids 6×His-REN-pET and 6×His-REN-340-pET were constructed correctly as identified by restriction analysis and sequencing.The optimum induction temperature of recombinant REN and REN-340 was 37 C and 20℃,and the best final concentration of IPTG was 0.1 and 0.2 mmol/L,respectively.The concentrations of purified recombinant REN and REN-340 were 9148.21 and 15115.31 mIU/mL,respectively.Conclusion The prepared recombinant REN and REN-340O proteins have high concentration with short production cycle,which are expected to be used as candidate quality control products and standard substances for external quality assessment(EQA).