激光杂志
激光雜誌
격광잡지
LASER JOURNAL
2011年
5期
67-69
,共3页
李朴%史静%郭变琴%钟梁%梁勤东%涂植光
李樸%史靜%郭變琴%鐘樑%樑勤東%塗植光
리박%사정%곽변금%종량%량근동%도식광
YB-1%GST亲和层析%柱上酶切%多抗制备
YB-1%GST親和層析%柱上酶切%多抗製備
YB-1%GST친화층석%주상매절%다항제비
YB -1%prokaryotic expression%GST - amnity chromatography%column protease cligestion
目的：构建YB1-GST表达系统，建立经济高效YB-1蛋白制备方法并制备其多抗。方法：将YB-1编码序列亚克隆至表达载体pGEX-6P-1；转化表达菌并确定可溶性表达最佳条件；采用GST亲和层析与层析柱上PSP酶切融合蛋白获取无标签蛋白的YB-1，经超滤浓缩及Western blot鉴定后，进-步真空冷冻于燥，制备YB-1标准蛋白；采用大剂量YB-1长程免疫方案免疫家兔以制备其抗体。结果：成功构建了表达载体pGEX—YB1；SDS—PAGE结果显示，YB1-GST融合蛋白以可溶性表达为主；Western blot结果证实，表达产物经GST亲和层析、PSP酶切以及真空冷冻干燥后获得了纯度较高的YB-1标准蛋白，将该蛋白免疫家兔获得了较高效价与特异性的抗体。结论：建立了YB-1-GST表达系统与经济高效的YB-1蛋白纯化方法；获得了质量较高的YB-1多抗，为进-步制备YB-1单抗、深入探讨其在肿瘤细胞中的生物学功能以及YB-1定量检测方法的建立奠定了基础。
目的：構建YB1-GST錶達繫統，建立經濟高效YB-1蛋白製備方法併製備其多抗。方法：將YB-1編碼序列亞剋隆至錶達載體pGEX-6P-1；轉化錶達菌併確定可溶性錶達最佳條件；採用GST親和層析與層析柱上PSP酶切融閤蛋白穫取無標籤蛋白的YB-1，經超濾濃縮及Western blot鑒定後，進-步真空冷凍于燥，製備YB-1標準蛋白；採用大劑量YB-1長程免疫方案免疫傢兔以製備其抗體。結果：成功構建瞭錶達載體pGEX—YB1；SDS—PAGE結果顯示，YB1-GST融閤蛋白以可溶性錶達為主；Western blot結果證實，錶達產物經GST親和層析、PSP酶切以及真空冷凍榦燥後穫得瞭純度較高的YB-1標準蛋白，將該蛋白免疫傢兔穫得瞭較高效價與特異性的抗體。結論：建立瞭YB-1-GST錶達繫統與經濟高效的YB-1蛋白純化方法；穫得瞭質量較高的YB-1多抗，為進-步製備YB-1單抗、深入探討其在腫瘤細胞中的生物學功能以及YB-1定量檢測方法的建立奠定瞭基礎。
목적：구건YB1-GST표체계통，건립경제고효YB-1단백제비방법병제비기다항。방법：장YB-1편마서렬아극륭지표체재체pGEX-6P-1；전화표체균병학정가용성표체최가조건；채용GST친화층석여층석주상PSP매절융합단백획취무표첨단백적YB-1，경초려농축급Western blot감정후，진-보진공냉동우조，제비YB-1표준단백；채용대제량YB-1장정면역방안면역가토이제비기항체。결과：성공구건료표체재체pGEX—YB1；SDS—PAGE결과현시，YB1-GST융합단백이가용성표체위주；Western blot결과증실，표체산물경GST친화층석、PSP매절이급진공냉동간조후획득료순도교고적YB-1표준단백，장해단백면역가토획득료교고효개여특이성적항체。결론：건립료YB-1-GST표체계통여경제고효적YB-1단백순화방법；획득료질량교고적YB-1다항，위진-보제비YB-1단항、심입탐토기재종류세포중적생물학공능이급YB-1정량검측방법적건립전정료기출。
Aim： To construct a GST- expression system of human Y - box binding protein 1 （YB - 1）, prepare the YB - 1 standard protein and its antiserum. Methods ： The code sequence of YB - 1 was subcloned to the expression vector pGEX - 6P - 1. The recombinant vector was transformed into E. coli BL21 to express fusion protein GST- YB1. SDS- PAGE was applied to analyze the expression level and form of fusion protein. Then, YB- 1 standard protein was obtained by the means of GST - affinity chromatography and column - protease - digestion. The rabbit was immunized with YB - 1 protein to prepare the anti - YB1 polyclonal antibody. Results： The recombinant expression vector was constructed. SDS - PAGE and Western blot results showed that the GST - fusion protein was high- level expressed with soluble - form, and YB - 1 standard protein and its antisermn were obtained successfully. Conclusions： An economical, rapid method to prepare YB - 1 standard protein is established, and further obtained the high titer and affinity YB - 1 polyclonal antibody, which lay foundations for preparation of YB - 1 monoclonal antibody and development of YB - 1 quantitative analysis methods.
