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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。
Objective To prepare rabbit anti-PLCε antiserum and express its specificity by expressing phospholipase Cε (PLCε) gene in prokaryotic system. Methods The partial PLCε gene was cloned from cDNA of human bladder cancer cells by RT-PCR, and the recombinant expression vector pGEX-6P-1 / PLCε was constructed and transformed into E. coli BL21. The GST-PLCε fusion protein was induced by IPTG. And purified by chromatography system, the rabbit was immunized after ultrafiltration and the rabbit anti-PLCε antiserum was obtained. Antiserum titer was detected by ELISA and specificity was determined by Western blot. Results The 336 bp PLCε gene was amplified by RT-PCR and the recombinant plasmid PGEX-6P-1 / PLCε was successfully constructed. The sequencing results of the plasmid showed that the sequence of the target gene was identical with the PLCε gene in GenBank. The purified GST-PLCε protein was immunized rabbits to obtain antiserum; ELISA titer 1:16 000 or more; Western blot results show that the antiserum and prokaryotic expression of GST-PLCε fusion protein and human bladder cancer T24 cell line expression Of PLCε protein specific binding. CONCLUSION: PLCε protein was expressed in prokaryotic cells and rabbit anti-human PLCε antiserum was prepared for detection of related tumors, which laid the foundation for further study of PLCε protein function and mechanism.