斜纹夜蛾GNBPⅠcDNA的克隆及其原核表达
cDNA Cloning and Prokaryotic Expression of GNBP I from Spodoptera litura
采用同源克隆结合RACE技术首次克隆了斜纹夜蛾(Spodoptera litura)GNBPⅠ(SlGNBPⅠ)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SlGNBPⅠ时空表达模式进行检测，结果表明，SlGNBPⅠ在4龄表达量最高，而且SlGNBPⅠ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，SlGNBPⅠ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SlGNBPⅠORF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-SlGNBPⅠ,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SlGNBPⅠ在昆虫识别免疫细菌中的功能奠定了蛋白基础。
he 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 of the further research on the fuction of SlGNBPⅠin the recognition of pathogens of the immune response.
郑志华、金丰良、孙强、黄婉君、李林妙、高钢
分子生物学昆虫学生物工程学
斜纹夜蛾革兰氏阴性菌结合蛋白Ⅰ克隆原核表达
Spodoptera lituraGNBPⅠcDNA cloningprokaryotic expression
郑志华,金丰良,孙强,黄婉君,李林妙,高钢.斜纹夜蛾GNBPⅠcDNA的克隆及其原核表达[EB/OL].(2012-04-19)[2025-08-18].http://www.paper.edu.cn/releasepaper/content/201204-273.点此复制
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