生物工程學(xué)報(bào) 25 August 2009, 25(8):1121～1129
柔嫩艾美耳球蟲HSP基因的克隆,、表達(dá)及鑒定
顏彥, 韓紅玉, 黃兵, 趙其平, 董輝, 姜連連, 李玉劍, 樊玉娟, 姚倩
中國(guó)農(nóng)業(yè)科學(xué)院上海獸醫(yī)研究所 農(nóng)業(yè)部動(dòng)物寄生蟲學(xué)重點(diǎn)開放實(shí)驗(yàn)室, 上海 200241
摘 要: 為研究柔嫩艾美耳球蟲熱激蛋白(Heat shock proteins, HSPs)的生物學(xué)特性, 應(yīng)用RACE和RT-PCR技術(shù), 從柔嫩艾美耳球蟲子孢子中首次克隆獲得了EtHSP的全長(zhǎng)cDNA(GenBank Accession No. FJ911605),。EtHSP包含一個(gè)1455 bp的開放閱讀框, 編碼484個(gè)氨基酸, 預(yù)測(cè)表達(dá)蛋白的分子量大小為53.5 kD,。應(yīng)用Real-time PCR對(duì)柔嫩艾美耳球蟲不同發(fā)育階段(未孢子化卵囊、孢子化卵囊、子孢子和裂殖子)表達(dá)量進(jìn)行分析, 發(fā)現(xiàn)該基因在子孢子階段的表達(dá)明顯高于其他階段。同時(shí), 構(gòu)建了原核表達(dá)重組質(zhì)粒pET28a(+)-EtHSP, 轉(zhuǎn)化到大腸桿菌BL21(DE3)中, 經(jīng)IPTG誘導(dǎo)表達(dá)后, 對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE及Western blotting分析。結(jié)果顯示, 重組質(zhì)粒pET28a(+)-EtHSP在大腸桿菌中以包涵體形式表達(dá), 經(jīng)1 mmol/ L IPTG誘導(dǎo)6 h后的表達(dá)量最高, 該蛋白可被抗柔嫩艾美耳球蟲的多克隆抗血清識(shí)別, 表明該蛋白具有較好的反應(yīng)原性,。本研究結(jié)果為進(jìn)一步研究該基因的生物學(xué)功能奠定了基礎(chǔ),。
關(guān)鍵詞: 柔嫩艾美球蟲, 熱激蛋白, 發(fā)育階段, 免疫原性
Cloning, expression and characterization of HSP gene from Eimeria tenella
Yan Yan, Hongyu Han, Bing Huang, Qiping Zhao, Hui Dong, Lianlian Jiang, Yujian Li, Yujuan Fan,and Qian Yao
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
Abstract: In order to study the functions of the HSPs (Heat shock proteins) of Eimeria tenella, we cloned a novel gene (which designated EtHSP) coding HSP of Eimeria tenella by RT-PCR and RACE (Rapid-amplification of cDNA ends). The full-length cDNA sequence of EtHSP was 1802 bp, containing a 1455 bp ORF (Open reading frame) (GenBank Accession No. FJ911605) encoding a deduced protein of 484 amino acids. Real-time PCR revealed that the mRNA level of EtHSP was much higher in sporozoites of E. tenella than other developmental stages (unsporulated oocysts, sporulated oocysts and merozoites). We constructed the recombinant plasmids pET28a(+)-EtHSP , then transformed it into E. coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed in included bodies, with peak expression 6 h after induction by IPTG. Western blotting revealed that the protein was specifically recognized by polyclonal antibodies against E. tenella, showing that the fusion protein was native antigen.
Keywords: Eimeria tenella, HSPs, development stages, immunogenicity
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