摘要
目的构建可在大肠杆菌中表达编码人结核杆菌(TB)热休克蛋白70(HSP70)基因的表达质粒,并经纯化后研究重组蛋白rHSP70抗肿瘤效应。方法采用基因工程技术将HSP70基因经PCR及末端修饰后装入大肠杆菌质粒pRSET-B中,构建成新的重组质粒pMT70-3。诱导表达纯化后经Western-blot、抗原肽结合试验、native-page鉴定,在有ADP、MgCl2存在时利用重组人 TB的HSP70与小鼠淋巴细胞白血病细胞系L1210的上清于37℃温育形成HSP70-肿瘤抗原肽复合物,以此作为抗原进行体内肿瘤免疫保护试验。结果 HSP70基因的表达质粒构建成功,其产物人TB rHSP70经纯化后具有生物活性。实验动物的腹水量较对照组下降,表明该复合物可引起一定的免疫保护反应(与对照组比较P<0．05)。结论人TB rHSP70-肿瘤抗原肽复合物免疫刺激后可使小鼠产生针对同种肿瘤的强烈免疫作用,与其剂量有一定的相关性。人TB HSP70不仅可起到免疫佐剂的作用,且肿瘤抗原肽复合物也起到一定的抗肿瘤作用。
Objective To construct recombinant prokaryotic plasmid DNA encoding mycobacterium tuberculosis （TB） heat shock protein 70 （HSP70） - Dnak gene obtained from pMT70, then purify its characterizes and the role of rHSP70 in tumor therapy. Methods Amplified by polymerase chain reaction （PCR） and with terminal modification, the Dnak gene was cloned into the vector with T7 promoter and was confirmed by restriction endonuclease digestion. So a new plasmid pMT70 - 3 was constructed and transformed in E. Coli. Strain BL21DE3. The engineering bacteria were induced by IPTG. Western - blot, natine - page and scanning were used to analyze the results. The human HSP70 were induced with peptides of lymphocytic leukemia cells of L1210 of mice in binding buffer which contain MgCl2 and ADP at 37℃, then mice were given rHSP70 peptide - complex by SV injection twice at weekly intervals. Results The Dnak gene could be highly expressed in E. Coli as soluble protein. The expressing efficiency was 65% of the total cell protein and the soluble protein was 90% of the expressed protein. The rHSP70 - peptide complex could improve the immune protective function which had significant difference compared with controls （P 〈 0.05）. Conclusions The prokaryotic plasmid DNA encoding Dnak gene has been constructed successfully. The human TB HSP70 can induce intensity immunifaction as immunoadjuvant and its role in the therapy for tumor is explored that rHSP70-peptide complex can produce immune protective effects against the same type tumor attacking.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2006年第10期627-629,共3页
Journal of Applied Clinical Pediatrics
基金
河南省教育厅自然科学研究项目资助(20011800002)
关键词
结核杆菌
原核质粒
热休克蛋白70
抗原肽
肿瘤
mycobacterium tuberculosis
prokaryotic plasmid
heart shock protein 70
antigen peptide
tumor
