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小鼠Ii基因克隆、表达及其抗体制备
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摘要:
为了获取小鼠Ii编码基因,利用原核表达系统进行诱导表达,提取纯化目的蛋白并制备其相应抗体,应用RT-PCR获取小鼠Ii编码基因;插入pGEX-4T-1表达载体,SDS-PAGE分析蛋白表达;纯化融合蛋白,免疫家兔,制备抗血清.结果扩增出了与预期大小相同的700 bp的特异性基因条带,其与GenBank登录的鼠Ii基因同源性高达99.2%~99.8%;SDS-PAGE检测到大小约49.5 ku的特异性蛋白条带;应用琼脂双向免疫扩散、间接ELISA、Western-blot、间接免疫荧光法等进行抗体活性鉴定,证明获得了特异性强、具有良好生物活性的抗体.成功克隆了小鼠Ii编码基因,有效进行了诱导表达,并制备了具有良好免疫学活性的特异性抗Ii抗体.
关键词:  恒定链  基因克隆  表达  抗体
DOI:
基金项目:国家自然科学基金?
Cloning and expression of mouse Ii gene and preparation of its antibody
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Abstract:
For cloning and sequence analysis on expression of mice Ii gene and preparation of its antibody,Ii gene was cloned from RNA by RT-PCR with a pair of designed primers;After sequence and homology analysis,this fragment was inserted into pGEX-4T-1 plasmid,and transformed into the strain BL21.Then the bacteria were induced and cultured,expressed protein was observed on SDS-PAGE;Fusion protein was extracted and purificated,which was immuned the rabbits,antiserum was collected.As a results,the length of this clon...
Key words:  invariant chain  gene cloning  expression  antibody  

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