氨基端PLCε基因的克隆、原核表达及抗血清的制备  

Preparation of rabbit polyclonal antisera against prokaryotically expressed human PLCε with GST-tag

作　　者：宋学东[1] 王胤[1] 杜红飞[1] 范砚茹[1] 梁勤东[1] 吴小侯 罗春丽[1] 

机构地区：[1]重庆医科大学检验医学院重庆医科大学临床检验诊断学教育部重点实验室,400016 [2]附属第一医院泌尿外科

出　　处：《免疫学杂志》2014年第1期33-36,共4页Immunological Journal

基　　金：PLCε对膀胱肿瘤细胞增殖调控的研究(A类);重庆市教委2008年科学技术研究项目(KJ080306)

摘　　要：目的通过在原核系统中表达人磷脂酶Cε(phospholipase Cε,PLCε)基因,制备兔抗PLCε的抗血清,并验证其特异性。方法应用RT-PCR从人膀胱癌细胞的cDNA中克隆出部分PLCε基因,构建重组表达载体PGEX-6P-1/PLCε,并转化大肠杆菌BL21,以IPTG诱导表达GST-PLCε融合蛋白,通过GST亲和层析系统纯化重组蛋白,超滤后免疫家兔,获得家兔抗PLCε的抗血清。采用ELISA检测抗血清效价,Western blot测定其特异性。结果 RT-PCR扩增出336 bp的PLCε基因,并成功构建重组质粒PGEX-6P-1/PLCε,质粒测序结果显示,目的基因序列与GenBank中PLCε基因序列完全一致。将纯化后的GST-PLCε蛋白免疫家兔,获得抗血清;ELISA效价1∶16 000以上;Western blot结果显示,该抗血清与原核表达的GST-PLCε融合蛋白和人膀胱癌T24细胞株表达的PLCε蛋白特异性结合。结论在原核细胞中表达了PLCε蛋白,制备了家兔抗人PLCε的抗血清,可用于相关肿瘤检测,为进一步研究PLCε蛋白功能和作用机制奠定了基础。This study designed to express and purify human PLCε in prokaryotic expression system and to prepare the PLCε-speeifie rabbit antiserum. Part of PLCε gene was obtained by RT-PCR from the eDNA of human bladder cancer cells, and recombined into PGEX-6P-1 vector to construct a recombinant expression vector PGEX- 6P-1/PLC~; the recombinant plasmid was transformed into E.coli BL21（DE3） and then induced with IPTG for expression; the fused GST-PLCε protein was purified by affinity GST chromatography system, and used to immunize rabbit for preparing PLCε-specific rabbit antiserum. Then the titer and specificity of rabbit antiserum were evaluated by ELISA and Western blot. Results demonstrated that the PLCε gene was partly amplified and the prokaryotic expression vector of PGEX-6P-1/PLCε was successfully constructed. The sequencing results of plasmid showed that the sequences of purpose gene were in complete accord with that of PLC epsilon gene in GenBank; the ELISA revealed that the titer of the rabbit antiserum was 1：16 000 approximately, while Western blot analysis showed that the antiserum could specifically bind with the purified GST-PLCε fused protein and the PLCε protein extracted from human bladder cancer cells. In conclusion, this study expressed GST-PLCε protein and prepared PLCε-specific rabbit antiserum successfully, which could be used in tumor detection, and also set a basis for the function and mechanism study of PLCε protein in the future.

关 键 词：人磷脂酶Cε 原核表达 抗血清制备 

分 类 号：R392.11[医药卫生—免疫学] Q786[医药卫生—基础医学]