AFP/HSP70重组蛋白表达载体抗人肝癌细胞免疫的实验研究

THE IMMUNITY STUDY OF CONSTRUCTED EUKARYOTIC EXPRESSION VECTOR pBBS212-AFP/HSP70 ON HCC CELL LINE

目的 :构建真核表达载体 p BBS2 12 - AFP/ HSP70 ,进行肝癌基因工程疫苗主动免疫的初步研究。方法 :构建的真核表达载体 p BBS2 12 - AFP/ HSP70 ,磷酸钙共沉淀法转染 SP2 / 0细胞 ,放射免疫和双夹心 EL ISA法鉴定 AFP和 AFP/ HSP70融合蛋白的表达。以上构建真核载体进行BAL B/ C小鼠股四头肌注射 ,免疫 3次后 ,取脾制备悬液 ,经 PHA和 AFP刺激后 ,与 Hep G2细胞混合培养 ,MTT检测细胞毒反应。结果 :构建的 p BBS2 12 - AFP/ HSP70真核载体经磷酸钙共沉淀法转染 ,建立了分泌表达 AFP/ HSP70融合蛋白的 SP2 / 0细胞株。上真核载体免疫小鼠后脾细胞与肝癌细胞混合培养 ,MTT检测显示对 AFP表达较高的 Hep G2细胞呈现明确的杀伤效应 ,而空载体免疫组混合细胞培养表现较弱的效应。结论 :p BBS2 12 - AFP/ HSP70真核载体的构建及其抗人肝癌细胞的体外细胞毒实验初步显示 p BBS2 12 - AFP/ HSP70作为基因工程疫苗具有一定的意义 ,为今后深入实验研究奠定了良好的基础。

Objective:In order to study the recombinant polypeptide vaccine active immunotherapy to hepatocarcinoma,an eukaryotic expression vector for AFP gene fragment was constructed.Methods:The constructed pBBS212-AFP/HSP70 vector were transfected to SP2/0 cells by calcium phosphorate co-precipitation.BALB/C mice were immuned with thus constructed vector by i.m. Mice spleen cells irritated by PHA and AFP were co-cultured with hepatocarcinoma cell line HepG2.The cytotoxity effect was detected by MTT.Results:The SP2/0 cell line secreting AFP/HSP70 was successfully established.MTT showed a definite effect in HepG2 cells ,while a weak effect in empty vecter immuned groups.Conclusion:The successful constructing eukaryotic vector pBBS212-AFP/HSP70 and the in vitro cytotoxity test revealed a certain significance for pBBS212-AFP/HSP70 vector as a DNA vaccine. The research will lay a better basis for carrying out further studies on the active immunotherapy of recombinant polypeptide vaccine to hepatocarcinoma.