兔抗人KiSS-1抗体的制备与鉴定

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.

Preparation and identification of rabbit anti-human KiSS-1 antibody

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.