CAJ | 학술논문

目的：构建人丙氨酸转氨酶（ ALT）高效重组表达体系，为ALT相关参考物质的原料制备奠定基础。方法利用生物信息学方法优化编码人ALT核苷酸序列中的密码子，合成人ALT新的基因，并将其克隆至pRSF-Duet表达载体中。重组表达质粒pRSF-Duet-ALT转化大肠埃希菌BL21，以2 mmol/L的异丙基硫代半乳糖苷（ IPTG）诱导目的蛋白的表达，并通过改变诱导剂浓度、振摇速度及诱导温度优化表达条件，以产生可溶性蛋白。可溶性蛋白经镍离子柱亲和层析及分子筛层析纯化，以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（ SDS-PAGE）及蛋白质免疫印迹（ Western blot）鉴定蛋白的性质，并对重组蛋白的活性、不同储存条件下在血清基质中的稳定性进行检测。结果经过优化的ALT核苷酸密码子在大肠埃希菌中的使用频率均在10％以上；经表达优化，在IPTG 终浓度为2 mmol/L、25℃、150 rpm振摇8 h诱导条件下，可增加可溶性蛋白的产率；纯化后的目的蛋白经电泳及免疫印迹鉴定为谷丙转氨酶，活性可达80000 U/L。重组ALT在2～8℃、25℃均可稳定2～8 d，相对偏差均在5％以内。结论通过密码子优化，成功构建了重组ALT的高效表达系统，本重组蛋白可以达到作为参考物质原料的要求。（中华检验医学杂志，2014，37：767-771）
목적：구건인병안산전안매（ ALT）고효중조표체체계，위ALT상관삼고물질적원료제비전정기출。방법이용생물신식학방법우화편마인ALT핵감산서렬중적밀마자，합성인ALT신적기인，병장기극륭지pRSF-Duet표체재체중。중조표체질립pRSF-Duet-ALT전화대장애희균BL21，이2 mmol/L적이병기류대반유당감（ IPTG）유도목적단백적표체，병통과개변유도제농도、진요속도급유도온도우화표체조건，이산생가용성단백。가용성단백경얼리자주친화층석급분자사층석순화，이십이완기류산납-취병희선알응효전영（ SDS-PAGE）급단백질면역인적（ Western blot）감정단백적성질，병대중조단백적활성、불동저존조건하재혈청기질중적은정성진행검측。결과경과우화적ALT핵감산밀마자재대장애희균중적사용빈솔균재10％이상；경표체우화，재IPTG 종농도위2 mmol/L、25℃、150 rpm진요8 h유도조건하，가증가가용성단백적산솔；순화후적목적단백경전영급면역인적감정위곡병전안매，활성가체80000 U/L。중조ALT재2～8℃、25℃균가은정2～8 d，상대편차균재5％이내。결론통과밀마자우화，성공구건료중조ALT적고효표체계통，본중조단백가이체도작위삼고물질원료적요구。（중화검험의학잡지，2014，37：767-771）
Objective To develop an efficient recombinant expression system of alanine aminotransferase ( ALT ) in order to build the foundation for the preparation of feedstock related ALT reference materials.Methods A new human ALT gene was synthesized by optimizing the codons of the nucleic acid sequence encoding human ALT using bioinformatic tools, and then it was cloned into pRSF-Duet expression vector.The recombinant plasmid pRSF-Duet-ALT was transformed into E.coli BL21 and the target protein expression was induced by 2 mmol/L isopropyl β-D-1-thiogalactopyranoside ( IPTG ) .The expression condition for soluble protein was optimized by changing the inducer concentration, shaking speed and induction temperature. The soluble protein was purified by nickel ion affinity chromatography and dextran molecular sieve chromatography, and identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis( SDS-PAGE) and Western blot analysis.The activity and stability of recombinant proteins in serum matrix under different storage conditions were detected.Results The usage frequency in E.coli of ALT codons was more than 10%after codon optimization.The expressions of soluble proteins were increased by optimizing the induced expression conditions, including a final concentration of 2 mmol/L of IPTG, and continued incubation with shaking at 150 rpm for 8 h at 25℃.The purified protein was identified as ALT by SDS-PAGE and Western blot with ALT activities of up to 80 000 U/L.Recombinant ALT could be stable for 2-8 d at 2-8 ℃or 25 ℃with a relative standard deviation of less than 5%.Conclusions An efficient recombinant expression system of ALT was developed successfully by codons optimization.The obtained recombinant protein could achieve the requirements of reference material feedstock.