[凋亡素原核表达载体的构建及凋亡素多克隆抗体的制备]
[Construction of a prokaryotic expression vector for apoptin and preparation of polyclonal antibody of apoptin].
作者信息
Wang Jian-Sheng, Zhang Ming-Xin, Duan Xiao-Yi, Mo Fei, Wang Quan-Ying, Yang Guang-Xiao
机构信息
Department of Oncological Surgery, First Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710061, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Jul;29(7):1405-7.
OBJECTIVE
To construct a prokaryotic expression vector for apoptin and prepare polyclonal antibody of apoptin.
METHODS
Apoptin gene amplified from pGEM-T/Apoptin plasmid by PCR was cloned into pET-28a (+). E.coli BL21 (DE3) was transformed by the recombinant plasmid, and apoptin protein expression induced by IPTG was analyzed by SDS-PAGE. BALB/c mice were immunized with the protein and the titer of the antibody was determined using indirect enzyme-linked immunosorbent assay (ELISA).
RESULTS
Apoptin gene was successfully cloned into pET-28a (+), and the expression of a protein with relative molecular mass of about 17 000 was identified by SDA-PAGE. After 5 immunizations of the mice with the protein, the blood antibody titer reached 1:5x10(5).
CONCLUSION
The prokaryotic expression vector for apoptin is successfully constructed and the polyclonal antibody of apoptin is obtained, which allows further functional study of apoptin.
目的
构建凋亡素原核表达载体并制备凋亡素多克隆抗体。
方法
通过PCR从pGEM-T/Apoptin质粒中扩增凋亡素基因，将其克隆到pET-28a(+)载体中。用重组质粒转化大肠杆菌BL21(DE3)，经IPTG诱导后，通过SDS-PAGE分析凋亡素蛋白的表达情况。用该蛋白免疫BALB/c小鼠，采用间接酶联免疫吸附测定（ELISA）法测定抗体效价。
结果
凋亡素基因成功克隆到pET-28a(+)载体中，SDA-PAGE鉴定出相对分子质量约为17 000的蛋白表达。用该蛋白对小鼠进行5次免疫后，血抗体效价达到1:5×10(5)。
结论
成功构建了凋亡素原核表达载体并获得了凋亡素多克隆抗体，为进一步研究凋亡素的功能奠定了基础。
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