三疣梭子蟹C-型凝集素的原核表达和活性检测

Prokaryotic expression of C-type lectin like-domain protein gene of Portunus tritubrculatus and activity analysis of recombinant protein

关键词:  三疣梭子蟹  C-型凝集素  原核表达  活性检测

Key words:  Portunus trituberculatus  C-type lectin like-domain protein  Prokaryocyte expression  Activity analysis

摘要:

通过RT-PCR扩增三疣梭子蟹C-型凝集素（C-type lectin like-domain protein, CTLD）基因序列，将目的基因克隆入质粒pET32a，构建原核表达重组质粒pET32a-CTLD。将重组质粒转化入大肠杆菌BL21中，通过IPTG诱导CTLD的表达，对其表达产物进行SDS-PAGE和Western blot检测。用His GraviTrap Ni 亲和层析柱及超滤离心管纯化目的蛋白，并对其进行活性检验。结果表明，构建得到CTLD基因的原核表达载体，双酶切鉴定得到与预期片段相符的外源基因插入片段，经测序与目的序列完全一致；诱导表达出相对分子质量（M）39 600的目的蛋白，经SDS-PAGE和Western blot检测，融合蛋白成功表达；重组C-型凝集素对兔的红细胞没有明显的凝集作用，但对小鼠红细胞的凝集效价是22，而对所选5种细菌的凝集作用存在差异，其中对大肠杆菌具有明显的凝集作用，而对溶藻弧菌、哈维氏弧菌、鳗弧菌和金黄色葡萄球菌均无凝集现象。本研究结果为进一步研究C-型凝集素在免疫防御中的作用与地位提供依据。

Abstract:

The total RNA extracted from the hemocyte sample of Portunus trituberculatus was used to amplify the DNA sequence encoding an open reading frame for C-type lectin like-domain protein (noted CTLD) by RT-PCR. The sequence was then cloned into pMD18-T vector and was sequenced. The recombinant plasmid was digested by BamH I and Xho I. The target gene was subsequently inserted into the pET-32a(+) vector，which was also digested with the corresponding restriction endonuclease. The recombinant plasmid p32a-CTLD was transformed into Escherichia coli BL21(DE3) and then induced by isopropylthio-β-D-galactoside (IPTG) .The fusion protein was purified through His-Band resin chelating chromatography and Amicon Ultra centrifugal filter devices，and agglutination activity was assayed finally. Results showed that the CTLD recombinant plasmid was successfully obtained by double enzyme identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and western-blot analysis showed that the fusion protein was successfully expressed. The fusion protein of about M39600 was successfully induced, which was identified by SDS-PAGE and western blot analysis. For the agglutination assay, no obvious agglutination was detected on the rabbit erythrocytes, while the agglutination titer of the mice erythrocytes was 22. No obvious agglutination was observed when Vibrio alginolyticus，V. harueyi ，V. anguillarum， Staphylococcus aureus were incubated with the recombinant protein. However, mass agglutination was observed after E. coli was incubated with the recombinant protein for 1h. This study provides a good foundation for further studies on the function of C-type lectin.