中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2009年
11期
965-968
,共4页
夏孝强%崔义元%李芳华%陈米娜
夏孝彊%崔義元%李芳華%陳米娜
하효강%최의원%리방화%진미나
Orai2%多克隆抗体%过表达%内源性%敲除小鼠鉴定
Orai2%多剋隆抗體%過錶達%內源性%敲除小鼠鑒定
Orai2%다극륭항체%과표체%내원성%고제소서감정
Orai2%Polyclonal antibody%Overexpressed%Endogenous%Knockout mouse identification
目的:原核表达跨膜蛋白Orai2的GST融合蛋白,获得高敏感性、高特异性的兔抗Orai2多克隆抗体,用于Orai2条件性基因敲除小鼠鉴定及Orai2蛋白功能研究.方法:PCR扩增Orai2 CDS编码序列,亚克隆到编码GST的pGEX-6p-1原核表达载体上,将编码Orai2的pGEX-6p-1-Orai2重组质粒化学转化BL21(DE3)感受态细胞,IPTG诱导GST-Orai2融合蛋白表达,经固化的谷胱苷肽琼脂糖珠亲和纯化,透析浓缩,获得用于免疫的高纯度GST-Orai2融合蛋白.SDS-PAGE鉴定后,将纯化的融合蛋白辅以弗氏佐剂,按常规方法免疫新西兰大白兔以制备多克隆抗体.Western blot检测抗体效价和特异性,并进行Orai2条件性基因敲除小鼠和同窝野生型小鼠脑组织鉴定.结果:经亲和纯化后的GST-Orai2融合蛋白纯度较高,BCA蛋白定量检测试剂盒测定蛋白浓度约0.35 mg/ml.抗Orai2多抗抗体效价达1:10 000.能特异性识别转染真核细胞获得的过表达Orai2,以及小鼠脑组织内源性的Orai2蛋白,而不与Orai1发生交叉反应.同时,用自制的Orai2多克隆抗体检测发现,与同窝野生型小鼠相比,Orai2条件性基因敲除小鼠脑组织中Orai2蛋白表达明显降低.结论:成功表达并纯化了GST-Orai2融合蛋白,获得了高敏感度、特异性的抗Orai2蛋白的多克隆抗体,该抗Orai2多抗能特异性识别过表达的和小鼠脑组织内源性Orai2蛋白,能对Orai2条件性基因敲除小鼠和同窝野生型小鼠脑组织进行鉴定,且与Orai1没有交叉反应,为进一步研究Orai2功能奠定了基础.
目的:原覈錶達跨膜蛋白Orai2的GST融閤蛋白,穫得高敏感性、高特異性的兔抗Orai2多剋隆抗體,用于Orai2條件性基因敲除小鼠鑒定及Orai2蛋白功能研究.方法:PCR擴增Orai2 CDS編碼序列,亞剋隆到編碼GST的pGEX-6p-1原覈錶達載體上,將編碼Orai2的pGEX-6p-1-Orai2重組質粒化學轉化BL21(DE3)感受態細胞,IPTG誘導GST-Orai2融閤蛋白錶達,經固化的穀胱苷肽瓊脂糖珠親和純化,透析濃縮,穫得用于免疫的高純度GST-Orai2融閤蛋白.SDS-PAGE鑒定後,將純化的融閤蛋白輔以弗氏佐劑,按常規方法免疫新西蘭大白兔以製備多剋隆抗體.Western blot檢測抗體效價和特異性,併進行Orai2條件性基因敲除小鼠和同窩野生型小鼠腦組織鑒定.結果:經親和純化後的GST-Orai2融閤蛋白純度較高,BCA蛋白定量檢測試劑盒測定蛋白濃度約0.35 mg/ml.抗Orai2多抗抗體效價達1:10 000.能特異性識彆轉染真覈細胞穫得的過錶達Orai2,以及小鼠腦組織內源性的Orai2蛋白,而不與Orai1髮生交扠反應.同時,用自製的Orai2多剋隆抗體檢測髮現,與同窩野生型小鼠相比,Orai2條件性基因敲除小鼠腦組織中Orai2蛋白錶達明顯降低.結論:成功錶達併純化瞭GST-Orai2融閤蛋白,穫得瞭高敏感度、特異性的抗Orai2蛋白的多剋隆抗體,該抗Orai2多抗能特異性識彆過錶達的和小鼠腦組織內源性Orai2蛋白,能對Orai2條件性基因敲除小鼠和同窩野生型小鼠腦組織進行鑒定,且與Orai1沒有交扠反應,為進一步研究Orai2功能奠定瞭基礎.
목적:원핵표체과막단백Orai2적GST융합단백,획득고민감성、고특이성적토항Orai2다극륭항체,용우Orai2조건성기인고제소서감정급Orai2단백공능연구.방법:PCR확증Orai2 CDS편마서렬,아극륭도편마GST적pGEX-6p-1원핵표체재체상,장편마Orai2적pGEX-6p-1-Orai2중조질립화학전화BL21(DE3)감수태세포,IPTG유도GST-Orai2융합단백표체,경고화적곡광감태경지당주친화순화,투석농축,획득용우면역적고순도GST-Orai2융합단백.SDS-PAGE감정후,장순화적융합단백보이불씨좌제,안상규방법면역신서란대백토이제비다극륭항체.Western blot검측항체효개화특이성,병진행Orai2조건성기인고제소서화동와야생형소서뇌조직감정.결과:경친화순화후적GST-Orai2융합단백순도교고,BCA단백정량검측시제합측정단백농도약0.35 mg/ml.항Orai2다항항체효개체1:10 000.능특이성식별전염진핵세포획득적과표체Orai2,이급소서뇌조직내원성적Orai2단백,이불여Orai1발생교차반응.동시,용자제적Orai2다극륭항체검측발현,여동와야생형소서상비,Orai2조건성기인고제소서뇌조직중Orai2단백표체명현강저.결론:성공표체병순화료GST-Orai2융합단백,획득료고민감도、특이성적항Orai2단백적다극륭항체,해항Orai2다항능특이성식별과표체적화소서뇌조직내원성Orai2단백,능대Orai2조건성기인고제소서화동와야생형소서뇌조직진행감정,차여Orai1몰유교차반응,위진일보연구Orai2공능전정료기출.
Objective:To prepare GST-Orai2 fusion protein and to prepare polyclonal antibody against Orai2 by immunizing rabbits.To further investigate the function of Orai2,a transmembrane protein,this antibody was used to identify the Orai2 conditional gene knockout mice.Methods:ORF of Orai2 was amplified by PCR and subcloned into pGEX-6p-1 vector.After transforming BL21 (DE3) competent cells,we succeeded in inducing the expression of GST-Orai2 fusion protein using IPTG.Then the GST-Orai2 protein was purified by immobilized Glutathione affinity chromatography and identified by SDS-PAGE.A New England rabbit was immunized with the prepared fusion protein in Freund's adjuvant to prepare specific antibody.Finally,the prepared antibody was identified by Western blot by checking its titer and specificity.Furthermore,we took use of the prepared Orai2 antibody to identify Orai2 conditional gene knockout mice,with comparing to the wildtype ones in the same cage.Results:The purity of purified GST-Orai2 reached to 90% and the concentration was 0.35 mg/ml by BCA kit.We could detect Orai2 protein even in dilution of 1:10,000.Also,the prepared polyclonal antibody agianst Orai2 could detect both overexpressed and endogenous Orai2 protein in mouse-brain,without crossing reaction with Orai1.As well,we found that the Orai2 protein expression was of obvious reduction in Orai2 conditional gene knockout mice,compared with the wildtype ones in the same cage.Conclusion:We successfully obtain the purified GST-Orai2 fusion protein and prepare specific and highly sensitive polyclonal antibody against Orai2.The antibody can be used to detect overexpressed and endogenous Orai2 protein inmouse-brain specifically,and to identify Orai2 conditional gene knockout mice,without any crossing reaction with Orai1.Our work contributes a lot to the future investigation of functions of Orai2.
