CAJ | 학술논문

透明质酸是一种长链大分子糖胺聚糖,存在于各组织器官中,是细胞外基质的重要组成部分.透明质酸不仅能够与其他胞外多糖及蛋白构成骨架,参与组织器官的结构形成,也可以与细胞表面相关蛋白受体相互作用,通过相关信号通路影响细胞的生存、增殖以及迁移.研究证实,透明质酸与肿瘤细胞的生存、迁移,肿瘤血管发生等均有密切关系,组织中透明质酸含量的变化也与某些病理状况如慢性炎症相关.因此,细胞表面或组织中的透明质酸含量的定量检测,对于疾病的诊断及预后都具有重要意义.目前透明质酸的检测方法主要依靠抗体,通过酶联免疫检测体液中的透明质酸,而缺乏在细胞或组织水平上检测透明质酸的手段.本研究将三种人源的透明质酸结合蛋白(P32,CDC37以及 RHAMM)与绿色荧光蛋白(EGFP)进行融合,在大肠杆菌中进行可溶性表达,经亲和纯化,获得了三种能够与透明质酸结合的蛋白探针.通过在溶液与细胞水平上比较与透明质酸结合的能力,从中筛选出了最适合用来进行透明质酸检测的荧光蛋白探针 RHC-EGFP.
투명질산시일충장련대분자당알취당,존재우각조직기관중,시세포외기질적중요조성부분.투명질산불부능구여기타포외다당급단백구성골가,삼여조직기관적결구형성,야가이여세포표면상관단백수체상호작용,통과상관신호통로영향세포적생존、증식이급천이.연구증실,투명질산여종류세포적생존、천이,종류혈관발생등균유밀절관계,조직중투명질산함량적변화야여모사병리상황여만성염증상관.인차,세포표면혹조직중적투명질산함량적정량검측,대우질병적진단급예후도구유중요의의.목전투명질산적검측방법주요의고항체,통과매련면역검측체액중적투명질산,이결핍재세포혹조직수평상검측투명질산적수단.본연구장삼충인원적투명질산결합단백(P32,CDC37이급 RHAMM)여록색형광단백(EGFP)진행융합,재대장간균중진행가용성표체,경친화순화,획득료삼충능구여투명질산결합적단백탐침.통과재용액여세포수평상비교여투명질산결합적능력,종중사선출료최괄합용래진행투명질산검측적형광단백탐침 RHC-EGFP.
Hyaluronic acid(HA)is a long chain glycosaminoglycan with large molecular weight,existing in all kinds of tissue and organs as an important component of extracellular matrix(ECM).HA can not only form scaffold for tissue and organs when combined with other glycosaminoglycan and proteins but also influence cell survival, proliferation and migration through distinct signal ways.Some research has provided that HA has strong relationship with the survival of carcinoma cells,angiogenesis in tumor and other pathological phenomenon.Therefore, quantification of HA level on cell surface or in tissue samples has great meaning in judging the developing level and prognosis of cancer.Up to now,the main method in HA detection is Enzyme-Linked Immunosorbent Assay(ELISA) which can only get HA level in serum but not on cell surface or in tissue samples.We constructed three fusion proteins which combined green fluorescence protein with three hyaluronic acid binding protein from human(P32, CDC37 and RHAMM),and expressed them in E .coli in a soluble form.Purified by affinity chromatography,three hyaluronic acid binding protein probe were gotten from cell lysate.Compared their hyaluronic acid binding abilities in solution and cell level,we selected the fittest protein probe RHC-EGFP for hyaluronic acid assay.