斜纹夜蛾GNBPⅠcDNA的克隆及其原核表达.docVIP

  斜纹夜蛾 GNBPcDNA 的克隆及其原核 表达# 李林妙，高钢，孙强，黄婉君，郑志华，金丰良** 5 10 15 20 25 30 35 （华南农业大学资源环境学院，广州 510642） 摘要：采用同源克隆结合 RACE 技术首次克隆了斜纹夜蛾(Spodoptera litura)GNBP(SlGNBP Ⅰ)基因的 cDNA 序列（GenBank 登录号：JF313110），其开放阅读框（ORF）序列为 1302 bp, 编码 433 个氨基酸，分子量为 48.57 kDa,等电点为 6.20。Real-time PCR 对 SlGNBP时空 表达模式进行检测，结果表明，SlGNBP在 4 龄表达量最高，而且 SlGNBP在斜纹夜蛾的 表皮，脂肪体、中肠和血细胞中均有表达，但在脂肪体中特异性高表达。通过微生物诱导后， Real-time PCR 检测表明，SlGNBP能被微生物诱导。为了进一步研究重组蛋白的功能，本 实验将 SlGNBPORF 序列克隆到原核表达载体 pET-32a(+)上，成功构建了原核表达载体 pET32-SlGNBP,经 IPTG 诱导下，在宿主菌 BL21（DE3）中高效表达了分子量为 65.6 kDa 的可溶性融合蛋白，经 Ni 亲和层析一步纯化获得融合蛋白，并免疫新西兰大白兔，ELISA 检测效价滴度为 1:12800,Western blot 检测表明抗血清能与融合蛋白呈阳性反应，表明所 表达的蛋白具有免疫原性。这为今后研究 SlGNBP在昆虫识别免疫细菌中的功能奠定了蛋 白基础。 关键词：斜纹夜蛾；革兰氏阴性菌结合蛋白；克隆；原核表达 cDNA Cloning and Prokaryotic Expression of GNBP I from Spodoptera litura LI Linmiao, GAO Gang, SUN Qiang, HUANG Wangjun, ZHENG Zhihua, JIN Fengliang (College of Natural Resources and Environments, South China Agricultural University, GuangZhou 510642) Abstract: The cDNA encoding Gram-negative bacterial binding protein Ⅰ (GNBPⅠ) was isolated from Spodoptera litura (SlGNBPⅠ,GenBank Accession No.JF313110) by homology cloning and rapid amplification of cDNA ends (RACE), the open reading frame (ORF) of SlGNBPⅠwas 1302 bp encoding 433 amino acid residues with the predicted molecular weight of 48.57 kDa and pI of 6.20 respectivey. The result of Real-time PCR showed that SlGNBPⅠwas expressed in the epidermis, fat body, midgut and hemocyte, but with the highest expression in fat body. The SlGNBPⅠwas ligated into pET-32a(+) vector, and then transformed into Escherichia coli BL21 (DE3).About 65.6 kDa of the recombinant fusion protein was expressed after induction with IPTG. The fusion protein was purified by nickel-column and antiserum was prepared successfully. The analysis of ELISA showed that the titer antiserum was 1:12800. Western blot showed that the fusion protein and antiserum was positive reaction, which indicating the SlGNBPⅠhad immunogenicity.These results woulg be the basis