重组人神经肽Y融合蛋白的原核表达、纯化及其多克隆抗体制备与鉴定

Prokaryotic Expression and Purification of Recombinant Human Neuropeptide Y Fusion Protein and Preparation and Identification of the Polyclonal Antibody

目的：构建人神经肽Y（NPY）基因的原核表达载体，诱导表达重组蛋白，制备兔抗人NPY抗血清。方法：取已构建好且经测序确认无误的再组质粒pET28a—NPY转化大肠杆菌BL21（DE3），IPTG诱导表达融合蛋白，并经SDS—PAGE检测和Western印迹鉴定，表达产物包涵体经Ni^2＋-NTA亲和层析纯化，以纯化后的融合蛋白pET28a—NPY为抗原免疫家兔，获得抗血清，Western blotting、ELISA法鉴定获得的抗血清。结果经IPTG诱导含有pET28a—NPY重组质粒的DE3菌，表达出重组人NPY融合蛋白。重组蛋白经Ni^2＋-NTA亲和层析进行纯化后，得到了较高纯度的融合蛋白，用纯化的融合蛋白免疫家兔制备了多克隆抗体，Western blotting、ELISA法证实多克隆抗体制备成功。结论：成功表达了人NPY蛋白并获得了其多克隆抗体，为进一步研究人NPY蛋白的功能奠定了基础。

OBJECTIVE： To eonstruct a prokaryotic plasmid expressing the recombinant proteins of human neuropeptide Y （NPY） and prepare the rabbit polyclonal anti - human - NPY antibodies. METHODS： The recombinant plasmid pET28a - NPY which had been constructed and sequentially confirmed was transformed into E. coil BL21 （DE3） and recombinant protein expression was induced by IPTG. SDS - PAGE and Western blot analysis were carried out to study the expression level of the recombinant proteins. The recombinant protein was purified by Ni^2＋ - NTA affinity chromatography from the inclusion body. Polyclonal antibodies were developed by immunizing rabbits with the purified recombinant protein, and their titers were analyzed by Western blotting and ELISA. RESULTS： Expression of recombinant human NPY protein was induced by IPTG in the E. Coli BL21 cell transformed with recombinant plasmid pET28a- NPY. Highly purified NPY fusion protein was obtained by Ni^2＋ -NTA affinity chromatography. Western blotting and EL1SA analysis demonstrated that highly specific anti - human NPY polyclonal antibody was obtained by imnmnizing rabbit with the purified recombinant protein. CONCLUSIONS： The recombinant protein NPY is successfully expressed and the polyclonal antibody is obtained, which lays a foundation for the further study on the function of NPY protein.