山羊c-Myc原癌基因原核表达和多克隆抗体的制备  

Prokaryotic Expression of c-Myc Protoncogene and Preparation of Polyclonal Anti-c-Myc Antibody in Capra Hircus

作　　者：张昀[1] 刘平[1] 韦友传[1] 李恭贺[1] 辛桂瑜[1] 王丽霞[1] 卢晟盛[1,2] 张明[1,2] 卢克焕[1,2] 郑喜邦[1] 

机构地区：[1]广西大学动物科技学院,南宁530005 [2]广西亚热带生物资源保护与利用重点实验室,南宁530005

出　　处：《畜牧兽医学报》2012年第6期867-871,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广西自然科学基金项目(桂科自0991043);广西亚热带生物资源保护与利用重点实验室开放课题(SB09);广西大学基金项目(X081102)

摘　　要：c-Myc原癌基因与胚胎干细胞(ES细胞)和诱导的多能性干细胞(iPS细胞)生物学特性密切相关,因此制备其相应的多克隆抗体尤为必要。本研究以pMD18T-Myc质粒为模版,PCR扩增c-Myc片段,然后将其亚克隆到pSE380表达载体上,获得pSE380-Myc重组质粒。该质粒转化工程菌BL21(DE3),IPTG诱导表达His-Myc融合蛋白,随后用SDS-PAGE电泳及Western blot加以验证。在变性条件下用Ni-NTA argrose介质分离纯化His-Myc融合蛋白,并以此为抗原免疫新西兰大白兔。经过4次免疫,采集血液、分离血清,用Western blot检测抗体特异性。结果表明:(1)pSE380-Myc重组质粒在工程菌BL21(DE3)得到了高效表达;(2)得到了高纯度高含量的His-Myc融合蛋白;(3)经Wstern blot检测发现,c-Myc多克隆抗体与His-Myc融合蛋白能够特异性结合。本研究获得的特异性山羊c-Myc多克隆抗体,为进一步研究山羊ES细胞和iPS细胞的自我更新机制奠定了基础。It is necessary to prepare polyclonal antibody of c-Myc in Capra Hircus since c-Myc protoncogene plays an important role in maintaining biological characteristics of embryonic stem cells （ES cells） and inducing pluripotent stem cells （iPS cells）. The plasmid pMD18T-Myc was used as templete to amplify c-Myc fragment, which was subcloned into vector pSE380 to con- struct recombinant plasmid pSE380-Myc. The plasmid was then transformed into E. coli BL21 （DE3）, and His-Myc fusion protein was expressed with induction of IPTG, which was subse- quently verified by SDS-PAGE and Western blot assay. Purified with Ni-NTA argrose under denaturing conditon, His-Myc fusion protein was applied as antigen to immunize New Zealand White rabbits, whose blood was collected, serum （polyclonal anti-c-Myc antibody） was isolated after 4 times of immunization, and its specificity was detected with Western blot. The results showed that： （1） The recombinant plasmid pSE380-Myc was efficiently expressed in E. coli BL21（DE3）; （2） His-Myc fusion protein with higher purity was obtained; （3） Western blot analysis illustrated that the polyclonal anti-c-Myc antibody could specifically respond to His-Myc fusion protein. In conclusion, the polyclonal anti-c-Myc antibody obtained in the present study will lay a foundation for the research of self-renewing mechanism of ES cells and iPS cells in Capra Hircus.

关 键 词：山羊 C-MYC基因 蛋白纯化 多克隆抗体 

分 类 号：S827[农业科学—畜牧学] Q786[农业科学—畜牧兽医]