兔抗破骨细胞蛋白质酪氨酸磷酸酶多克隆抗体的制备和应用

Preparation and application of rabbit anti-osteoclastic protein tyrosine phosphatase polyclonal antibody

目的:采用已构建的pET28a-ΔPTP-oc重组质粒纯化重组蛋白,制备兔抗破骨细胞蛋白质酪氨酸磷酸酶(PTP-oc)多克隆抗体并鉴定其性能。方法:将构建的pET28a-ΔPTP-oc重组质粒转化至BL21(DE3)中,应用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达。表达产物经过Q柱阴阳离子交换和Ni柱亲和层析获得纯化的PTP-oc重组蛋白,将纯化的重组蛋白作为抗原免疫家兔,获得PTP-oc多克隆抗体。采用间接ELISA法检测抗体效价,Western blotting法检测抗体的特异性。结果:成功纯化出PTP-oc重组蛋白。间接ELISA法检测,采用纯化诱导后的重组蛋白免疫家兔获得了兔抗PTP-oc多克隆抗体,多克隆抗体效价达1∶32 000以上,Western blotting法检测,所得多克隆抗体有较高的特异性。结论:成功纯化了PTP-oc重组蛋白,制备出PTP-oc多克隆抗体。

Objective：To purify the recombinant protein by using the constructed pET28 a-ΔPTP-oc recombinant expression plasmid,and to prepare the rabbit polyclonal antibody against osteoclastic protein tyrosine phosphatase（PTP-oc）and to identify its properties.Methods：The constructed pET28 a-ΔPTP-oc recombinant plasmid was transformed into E.coli BL21（DE3）,and the isopropyl-β-D-thiogalactoside（IPTG）was used to induce the expression of recombinant protein.The recombinant protein was purified by the method of Q fast flow agarose and Ni-NTA agarose,and the purified recombinant protein was used as an antigen to immunize the rabbit to obtain PTP-oc polyclonal antibody.The titer and specificity of the antibody were identified by indirect ELISA and Western blotting methods.Results：The recombinant PTP-oc protein was successfully purified.The indirect ELISA results showed that after immunizing the rabbit with the recombinant PTP-oc protein,the polyclonal antibody with the titer of 1∶3 2000 was gotten.The Western blotting results showed that the polyclonal antibody had high specificity.Conclusion：The PTP-oc recombinant protein is successfully purified,and the polyclonal antibody of PTP-oc is prepared successfully.