摘要
目的制备兔抗人KiSS-1多克隆抗体。方法聚合酶链反应(PCR)法扩增出KiSS-1基因,将其克隆入原核表达载体pET28中,转化大肠埃希菌BL21(DE3)。采用异丙基-D-硫代半乳糖苷(IPTG)诱导重组蛋白的表达,采用镍离子螯合琼脂糖凝胶(Ni 2+-NTA)亲和层析法纯化重组蛋白,以之为免疫原免疫家兔,制备多克隆抗体,并用酶联免疫吸附测定(ELISA)、Western blot及免疫组织化学法检测抗体。结果成功构建了KiSS-1原核表达载体pET28/KiSS-1,高效表达并纯化KiSS-1蛋白,间接ELISA检测所制备抗体的效价为1∶6 400。Western blot鉴定制备的抗体与KiSS-1蛋白能特异性结合,免疫组织化学法检测结果表明该抗体识别人卵巢癌组织中的天然KiSS-1。结论成功制备了效价高、特异性较强的兔抗人KiSS-1抗体,为进一步探讨KiSS-1在卵巢癌的发生、发展过程中的作用奠定了基础。
Objective To prepare rabbit anti-human KISS-1 polyclonal antibody. Methods Polymerase chain reaction（PCR） was employed to amplified KISS-1 gene fragments and cloned into prokaryotic expression vector pET28 and transformed into E. coli BL21 （DE3）. Isopropyl-beta-D-thiogalactopyranoside（IPTG） was used to induce the recombinant protein expression, nickel-chelating agarose geI（Ni^2＋-NTA） affinity chromatography was employed to purified the recombinant protein which served as immunogen to immunize the rabbit. The polyclonal antibody was prepared and enzyme-linked immunosorbent assay（ELISA）, Western blot and immunohistochemieal were adopted to detect the antibody. Results The recombinant KISS-1 prokaryotic expression vector pET28/ KISS-1 was successfully constructed and KISS-1 protein was expressed and purified efficiently. The prepared antibody titer was 1 ： 6 400 by indirect ELISA detection. Western blot analysis showed that the prepared antibody combined specifically with KISS- protein. Immunohistochemistry assay showed that the antibody recognized natural KISS-1 in human ovarian cancer tissue. Conclusion Rabbit anti-human KISS-1 polyclonal antibody with high titer and specificity has been prepared successfully which lay the foundation for further research on role of KiSS in the occurrence, development of ovarian cancer.
出处
《国际检验医学杂志》
CAS
2013年第21期2803-2805,共3页
International Journal of Laboratory Medicine
