P-5m八肽多克隆抗体的制备及应用

Preparation and Application of P-5m Octapeptide Polyclonal Antibodies

目的制备P-5m八肽的多克隆抗体并进行初步鉴定和应用,为研究P-5m八肽的功能及作用机制获得重要的实验工具.方法将9-氟甲氧羰基(Fmoc-)固相合成法合成的P-5m八肽纯化后与载体蛋白-血蓝素联接;取偶联后的多肽皮下注射免疫新西兰白兔,并加强免疫得到抗血清,以C18反相色谱分离柱分离纯化;通过间接ELISA方法测定血清效价;利用Transwell实验和Western blot方法初步鉴定多克隆抗体拮抗P-5m八肽抑制肿瘤细胞转移的效果.结果纯化后的抗体经间接ELISA测定效价均大于1∶160 000,表明获得高效价的多抗;通过Transwell实验证明该抗体能够封闭P-5m对肿瘤细胞侵袭的抑制能力;通过Western blot方法证实此抗体效价较高,特异性较强;该多克隆抗体能够封闭P-5m在SW620细胞中上调E-cadherin表达的作用,并阻遏P-5m对MMP-2和MMP-9表达的下调作用,与对照组比较差异具有统计学意义(P<0.05).结论 P-5m多克隆抗体可用于Transwell和Western blot等实验,为研究P-5m八肽的功能及抗肿瘤转移作用机制提供实验依据.

Objective To prepare polyclonal antibody against P-5m octapeptide for preliminary identification and application, and to obtain important experimental tools for studying the function and mechanism of P-5m octapeptide. Method P-5m octapeptide was synthesized by 9-fluoromethoxycarbonyl （ Fmoc-） solid phase methods, purified and linked with carrier protein-keyhole limpet hemocyanin. The conjugated peptide was subcutaneously immunized with New Zealand white rabbits and booster immunized with the conjugated peptide, the anti-serum was isolated and purified by C18 reverse phase chromatography. The serum titer was determined by indirect ELISA. Transwell and western blot assays were used to detect the effect of polyclonal antibody antagonizing P-5m octapeptide on tumor metastasis. Results The titer of the purified antibody was more than 1 ： 160000 by indirect ELISA, indicated that the high titer polyclonal antibody was obtained. Transwell experiments showed that the antibody can inhibit the inhibition of P-5m on tumor cell invasion. Western blotting method showed the polyclonal antibody was highly specific to P-5m octapeptide,and it could block the expression of E-cadherin and decrease the expression of MMP-2 and MMP-9 in SW620 cells, which was statistically significant （ P＆lt;0 . 05 ） . Conclusion The prepared P-5m polyclonal antibody could be used in transwell and western blot assays,and provided the experimental basis to find the function of P-5m protein and its mechanism of anti-tumor metastasis.