生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
1期
76-79
,共4页
重组SCIRR39抗原表位%原核表达%多克隆抗体%脊髓损伤修复
重組SCIRR39抗原錶位%原覈錶達%多剋隆抗體%脊髓損傷脩複
중조SCIRR39항원표위%원핵표체%다극륭항체%척수손상수복
recombinant spinal cord injury and regeneration related protein 39 C-terminal epitope%prokaryotic expression%polyclonal antibody%injury and regeneration
目的:在大肠杆菌中表达大鼠脊髓损伤与修复蛋白39(SCIRR39)的C端抗原表位,并制备其多克隆抗体.方法:从大鼠脊髓全横断损伤脊髓cDNA中扩增1386 bp的Scirr39基因编码框,亚克隆该基因编码蛋白C端359~461位氨基酸残基的DNA片段,插入表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达情况,切胶纯化目的蛋白;利用多克隆抗体制备技术,制备重组SCIRR39蛋白的多克隆抗体;用ELISA方法检测抗体效价, Western印迹检测抗体的特异性.结果:SCIRR39蛋白C端抗原表位与GST的融合蛋白在大肠杆菌中以可溶形式高表达,相对分子质量为37.9×103;获得抗SCIRR39蛋白C端抗原表位的兔抗血清,其效价达到1∶104;Western印迹显示多克隆抗体能特异识别重组SCIRR39蛋白的C端抗原表位.结论:在原核系统中表达纯化了重组SCIRR39抗原表位蛋白,制备的重组蛋白多克隆抗体将用于检测SCIRR39在脊髓损伤过程中的表达变化.
目的:在大腸桿菌中錶達大鼠脊髓損傷與脩複蛋白39(SCIRR39)的C耑抗原錶位,併製備其多剋隆抗體.方法:從大鼠脊髓全橫斷損傷脊髓cDNA中擴增1386 bp的Scirr39基因編碼框,亞剋隆該基因編碼蛋白C耑359~461位氨基痠殘基的DNA片段,插入錶達載體,轉化大腸桿菌BL21(DE3),IPTG誘導錶達,SDS-PAGE分析錶達情況,切膠純化目的蛋白;利用多剋隆抗體製備技術,製備重組SCIRR39蛋白的多剋隆抗體;用ELISA方法檢測抗體效價, Western印跡檢測抗體的特異性.結果:SCIRR39蛋白C耑抗原錶位與GST的融閤蛋白在大腸桿菌中以可溶形式高錶達,相對分子質量為37.9×103;穫得抗SCIRR39蛋白C耑抗原錶位的兔抗血清,其效價達到1∶104;Western印跡顯示多剋隆抗體能特異識彆重組SCIRR39蛋白的C耑抗原錶位.結論:在原覈繫統中錶達純化瞭重組SCIRR39抗原錶位蛋白,製備的重組蛋白多剋隆抗體將用于檢測SCIRR39在脊髓損傷過程中的錶達變化.
목적:재대장간균중표체대서척수손상여수복단백39(SCIRR39)적C단항원표위,병제비기다극륭항체.방법:종대서척수전횡단손상척수cDNA중확증1386 bp적Scirr39기인편마광,아극륭해기인편마단백C단359~461위안기산잔기적DNA편단,삽입표체재체,전화대장간균BL21(DE3),IPTG유도표체,SDS-PAGE분석표체정황,절효순화목적단백;이용다극륭항체제비기술,제비중조SCIRR39단백적다극륭항체;용ELISA방법검측항체효개, Western인적검측항체적특이성.결과:SCIRR39단백C단항원표위여GST적융합단백재대장간균중이가용형식고표체,상대분자질량위37.9×103;획득항SCIRR39단백C단항원표위적토항혈청,기효개체도1∶104;Western인적현시다극륭항체능특이식별중조SCIRR39단백적C단항원표위.결론:재원핵계통중표체순화료중조SCIRR39항원표위단백,제비적중조단백다극륭항체장용우검측SCIRR39재척수손상과정중적표체변화.
Objective: To express the recombinant rat spinal cord injury and regeneration related protein 39 (SCIRR39) C-terminal epitope in E.coli BL21(DE3), and prepare polyclonal antibody against it. Methods: The gene coding SCIRR39 with the length of 1386 bp was amplified from rat spinal cord cDNA by PCR, then site 1077 to 1383 bp was amplified from it and was inserted into pGEX-4T-2 to construct the GST-SCIRR39-C ex?pression plasmid. Then it was transformed into E.coli BL21(DE3) and induced by IPTG. The recombinant GST-SCIRR39-C was visualized by SDS-PAGE and was purified from the gel, and the GST tag was removed. Poly?clonal antibody against Scirr39-C antigen was prepared by polyclonal antibody preparation techniques and detected by ELISA and Western blot. Results: The recombinant GST-SCIRR39-C was expressed at high level and partly soluble, and its molecular was 37.9 kD. The rabbit serum could specifically recognize the recombinant SCIRR39-C, and the titer reached 1∶104. Conclusion: The recombinant SCIRR39-C has been expressed in E.coli BL21(DE3) and was purified to prepare the polyclonal antibody against it. The polyclonal antibody will be used to explore the molecular mechanism of CNS injury and repair.
