CAJ | 학술논문

目的：构建可行的MICB ( major histocompatibility complex class Ⅰ chain-related gene B)基因原核表达系统及蛋白质纯化体系,利用表达纯化蛋白建立ELISA方法初步应用于肾移植患者中MICB抗体的检测。方法外周血RNA经RT-PCR和特异性引物扩增获得MICB基因cDNA, MICB cDNA和原核表达载体pET-28a分别双酶切后连接构建重组表达载体,转染宿主菌E. coli BL21 DE3,IPTG诱导表达。亲和层析的Ni-NTA Spin纯化蛋白质,并通过Western blot鉴定。利用纯化蛋白建立ELISA法检测肾移植患者中MICB抗体。结果构建3个MICB常见等位基因2、3外显子的pET-28a-MICB重组表达体系,经Ni-NTA Spin纯化获得重组的MICB蛋白,Western blot鉴定。利用3个不同MICB蛋白质成功构建了ELISA方法,初步检测24份肾移植患者标本MICB抗体,显示不同的重组蛋白敏感性存在差异。结论本研究建立MICB基因克隆与体外表达技术,鉴定并纯化具有生物活性的蛋白质,同时建立MICB抗体检测的ELISA方法,为进一步研究MICB与移植免疫的关系提供基础资料。
목적：구건가행적MICB ( major histocompatibility complex class Ⅰ chain-related gene B)기인원핵표체계통급단백질순화체계,이용표체순화단백건립ELISA방법초보응용우신이식환자중MICB항체적검측。방법외주혈RNA경RT-PCR화특이성인물확증획득MICB기인cDNA, MICB cDNA화원핵표체재체pET-28a분별쌍매절후련접구건중조표체재체,전염숙주균E. coli BL21 DE3,IPTG유도표체。친화층석적Ni-NTA Spin순화단백질,병통과Western blot감정。이용순화단백건립ELISA법검측신이식환자중MICB항체。결과구건3개MICB상견등위기인2、3외현자적pET-28a-MICB중조표체체계,경Ni-NTA Spin순화획득중조적MICB단백,Western blot감정。이용3개불동MICB단백질성공구건료ELISA방법,초보검측24빈신이식환자표본MICB항체,현시불동적중조단백민감성존재차이。결론본연구건립MICB기인극륭여체외표체기술,감정병순화구유생물활성적단백질,동시건립MICB항체검측적ELISA방법,위진일보연구MICB여이식면역적관계제공기출자료。
Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney trans-plantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correla-tions between MICB protein and transplantation immunity.