抗新生血管蛋白KBP多克隆抗体的制备及鉴定  

Preparation and purification of rabbit anti-KBP polyclonal antibody

作　　者：陈易斐[1] 黄莉钧[1] 李磊[1] 戴智育[1] 程锐[1] 杨霞 高国全[3] 

机构地区：[1]中山大学中山医学院生物化学教研室,广东广州510080 [2]海洋微生物功能分子广东省高校重点实验室,广东广州510080 [3]教育部热带病重点实验室,广东广州510080

出　　处：《解剖学研究》2011年第2期105-109,共5页Anatomy Research

基　　金：科技部新药创制重大专项(2009ZX09103-642);国家自然科学基金(30872980;30971208;30973449;81001014);(教育部)高等学校博士点基金(20100171110049);广东省自然科学基金重点项目(10251008901000009);教育部高校基本科研业务费中山大学青年教师培育项目(10YKPY28)

摘　　要：目的激肽释放酶结合蛋白(Kallikrein-binding protein,KBP)具有抑制血管新生的高活性,但目前尚无KBP的特异性抗体,限制其进一步研究。本研究拟制备高灵敏度和高特异性的KBP抗体并对其进行初步评估。方法构建含大鼠源性KBP序列的pET-28a重组载体的大肠杆菌BL21(DE3),在此基础上,扩增表达菌,经异丙基B.D一硫代半乳糖苷(IPTG)诱导表达,Ni-NTA色谱柱亲和层析纯化获得KBP重组蛋白。利用SDS-PAGE和Western blot方法对KBP进行鉴定。以纯化产物为抗原免疫新西兰大白兔,获得兔抗KBP血清,亲和纯化获得多克隆抗体。应用Elisa和Western-blot方法检测纯化抗体的效价和特异性。结果 KBP融合蛋白得到高表达,且纯度大于85%。用该融合蛋白免疫大白兔后得到的抗KBP抗体,效价达1∶512 000,并特异性识别大鼠和人组织中天然的KBP蛋白。结论以纯化的KBP重组蛋白为抗原,获得KBP多克隆抗体,具有较好的效价和特异性,为KBP功能和作用机制的深入研究提供了必要的工具。Objective KBP,short from kallikrein binding protein,is a endogenous protein with high antiangiogenic potential.The further study about KBP is restricted because there is still not specific anti-KBP antibody.This study is designed to obtain highly sensitive and specific anti-KBP polyclonal antibody and carry on the primary evaluation of it.Methods The expression vector pET28a-KBP had been reconstructed and transformed into BL21（DE3） by our lab.Recombinant KBP protein was induced to express by IPTG and purified by Ni-NTA His Bind resin.Purified KBP was identified by SDS-PAGE and western blot analysis and used to immunize the New Zealand rabbits.The anti-KBP polyclonal antibody is prepared by affinity purification from the rabbits＇ serum after immunization.The titer of the antibody was detected by ELISA and its specificity was analyzed by Western blot.Results The KBP recombinant protein was highly expressed in E.coil and purity of purified protein is more than 85%.The titer of rabbit anti-KBP polyclonal antibody obtained after immunization and purification is about 1∶512,000.And the anti-KBP antibody indentifies native KBP of rat and human tissues specifically.Conclusion Highly special polyclonal antibody for KBP was successfully generated,which is essential for further study of the function and mechanisms of KBP.

关 键 词：KBP重组蛋白 多克隆抗体制备 抗体评价 

分 类 号：R392[医药卫生—免疫学]