HSP65-MUC1 VNTR_2融合蛋白的表达、纯化及其抗肿瘤作用  

Prokaryotic expression,purification of HSP65-MUC1 VNTR_2 fusion protein and primary research on its tumoricidal effect

作　　者：郑玉玲[1] 江华[1] 王希良[1] 姜永强[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《细胞与分子免疫学杂志》2007年第11期1014-1016,1024,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30672476)

摘　　要：目的:通过基因工程手段在大肠杆菌中表达HSP65-MUC1VNTR2融合蛋白、鉴定和纯化,并研究其在动物体内的肿瘤生长抑制活性。方法:通过PCR获得结核分支杆菌HSP65基因,以重叠PCR获得人MUC1基因VNTR的2个重复序列,构建原核重组表达载体HSP65-MUC1VNTR2-pET28a(+),在大肠杆菌BL21(DE3)中利用IPTG诱导表达;利用单克隆抗体(mAb)进行Westernblot鉴定,以Q柱和凝胶过滤纯化获得纯化蛋白。通过建立转染人MUC1基因的B16黑色素瘤小鼠肿瘤模型,在C57BL/6小鼠体内研究HSP65-MUC1VNTR2融合蛋白的肿瘤生长抑制活性。结果:获得的结核分支杆菌HSP65基因和人MUC1基因VNTR区的2个重复片段,经测序证明分别与GenBank中结核杆菌HSP65基因和人MUC1基因的VNTR完全相符;构建的原核表达载体HSP65-MUC1VNTR2-pET28a(+)可稳定的可溶性表达HSP65-MUC1VNTR2蛋白;经Q柱和凝胶过滤纯化,纯化的目的蛋白纯度达95%以上;利用鼠抗人MUC1mAb对表达蛋白进行验证,结果阳性;小鼠肿瘤预防免疫实验表明,实验组小鼠抑瘤率大于对照组,且具有明显性差异。结论:成功地利用原核表达系统实现了对HSP65-MUC1VNTR2蛋白的可溶性表达,并对其体内预防实验进行了初步研究,证明该融合蛋白能明显抑制表达MUC1的肿瘤生长。为进一步评价其作为肿瘤疫苗可能性的研究打下了基础。AIM： To express the HSP65-MUC1 VNTR2 in E. coli and to evaluate its activity of inhibiting tumor growth in vivo. METHODS： HSP65 and MUC1 VNTR2 were generated by PCR method and sub-cloned to pET28a（ ＋ ） to construct the recombinant expression vector HSP65-MUC1 VNTR2-pET28a（ ＋ ）. E. coli BL21 （ DE3 ） bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell line-B16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR2 was evaluated in mice C57BL/6. RESULTS： The gene HSP65 and MUCI VNTR2 confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR2-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR2 protein could be above 95% after purification by Q ionexchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR2 fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control qroup. CONCLUSION： In summary, HSP65-MUC1 VNTR2fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.

关 键 词：热休克蛋白65 MUC1 VNTR 纯化 预防免疫 

分 类 号：R392.11[医药卫生—免疫学]