精胺/精眯N1-乙酰基转移酶重组蛋白的原核表达及其抗体制备

目的 探讨精胺/精眯N¹乙酰基转移酶（SSAT）的原核表达及抗SSAT多克隆抗体的制备。方法 RT-PCR法从人肺癌细胞总RNA中克隆SSAT cDNA后，构建SSAT原核表达质粒pET15b/SSAT并转化BL21(DE3)大肠杆菌细胞。SSAT重组蛋白经IPTG诱导表达后，利用PAGE凝胶纯化和Ni-NTA亲和层析纯化。用凝胶纯化的SSAT重组蛋白免疫家兔以制备抗SSAT多克隆抗体，所得抗体的特异性及效价用Western blot和ELISA技术检测。结果 SSAT重组蛋白可在大肠杆菌中诱导性高表达，用此蛋白免疫动物所得的SSAT抗体的效价以及特异性均较高。结论 建立了SSAT原核表达系统并成功制备出SSAT多克隆抗体。

Prokaryotic Expression and Antibody Preparation of Human Spermine/Spermidine N1acetyltransferase

ObjectiveTo obtain prokaryotic expression of human spermidine/spermine N¹ acetyltransferase（SSAT）and prepare SSAT antibody. MethodsHuman SSAT cDNA was amplified by RT-PCR from total RNA of human lung cancer line A549.The SSAT cDNA was then cloned into prokaryotic expression plasmid pET15b and transformed into BL21（DE3).The expression of SSAT in host cells was induced by IPTG and recombinant SSAT protein was purified by polyacrylamide gel electrophoresis and Ni-NTA resin.The antibody acquired from rabbits immunized by purified SSAT protein were detected by Western blot and ELISA. ResultsHuman SSAT was successfully expressed in E.coli induced by IPTG and antibody of SSAT was prepared from purified recombinant SSAT. ConclusionA method for prokaryotic expression and antibody preparation of the human SSAT was successfully established.