FABP5基因重组突变体蛋白抗前列腺癌细胞活性分析

Analysis on Activity of RecombinantMutant Protein of FABP5 Gene Against Prostate Cancer Cells

为构建脂肪酸结合蛋白5(FABP5)突变体的原核表达体系,评价突变体蛋白质体外抗前列腺癌细胞的活性。利用定点突变技术,突变FABP5蛋白脂肪酸结合的3个关键位点,并构建原核表达体系,对重组蛋白质进行原核表达、分离纯化。通过细胞毒性、细胞划痕和细胞侵袭试验,评价重组FABP5突变体蛋白质对前列腺癌细胞22RV1和PC3增殖、迁移和侵袭的影响。结果显示定点突变后的DNA与表达载体pQE32连接并转入大肠杆菌(Escherichia coli)BL21(DE3),经序列测定正确,构建了重组表达工程菌,并诱导表达的重组蛋白经亲和层析纯化获得纯度较高的重组蛋白;三突变体对细胞的增殖、迁移和侵袭抑制作用比3个单突变体和3个双突变体抑制效果明显;单突变体和双突变体组内对细胞的增殖、迁移和侵袭抑制作用差异较小;而野生型重组蛋白质对两株细胞的增殖、迁移和侵袭具有促进作用。本研究从所有突变体中筛选出FABP5的三突变体重组蛋白质对前列腺癌细胞抑制作用较好,为后续开发去势抵抗性前列腺癌(CRPC)蛋白质药物提供参考。

To construct the prokaryotic expression system of fatty acid binding protein 5(FABP5)gene mutants and evaluate the anti-prostate cancer activity of the mutants in vitro,site-directed mutation technique was used to mutate FABP5 protein's three key sites of fatty acid binding,and a prokaryotic expression system was constructed to express,isolate and purify the recombinant protein.Cytotoxicity,scratch wound,and cell invasion tests were used to evaluate the effects of recombinant FABP5 mutant proteins on proliferation,migration,and invasion of 22RV1 and PC3 prostate cancer cells.The results showed that the DNA after fixed-point mutation was linked to the expression vector pQE32 and transferred into Escherichia coli BL21(DE3),the sequence was determined to be correct,the recombinant expressed engineering bacteria were constructed,and the recombinant protein induced by affinity chromatography was purified to a higher purity.The inhibition effect of the three mutants on cell proliferation,migration and invasion was more obvious than that of the three single mutants and three double mutants;There was little difference in cell proliferation,migration and invasion inhibition between single and double mutant groups.The wild-type recombinant protein promoted the proliferation,migration and invasion of the two cells.In this study,the three mutants of FABP5 protein screened out from all mutants had a good inhibitory effect on prostate cancer cells.This result provided a reference for the subsequent development of castration resistant prostate cancer(CRPC)protein drugs.