家蚕天然Hemolin蛋白原核表达

Prokaryotic expression of natural Hemolin protein from Bombyx mori

Hemolin蛋白是一种类免疫球蛋白,属于昆虫的一种重要的先天性免疫球蛋白,家蚕(Bombyx mori)Hemolin蛋白与其他昆虫的序列具有高度的同源性,为了进一步分析家蚕Hemolin蛋白的功能,以杆状病毒感染的家蚕蚕蛹为原料,通过抗体亲和层析获得高纯度的天然Hemolin蛋白。结果表明:重组表达菌株pET-32a-hemolin经终浓度为0.1 mmol/L IPTG低温诱导后可在大肠杆菌BL21中成功表达;通过镍离子金属螯合亲和层析并经200 mmol/L咪唑洗脱可以获得较纯的重组Hemolin蛋白;免疫新西兰大白兔产生特异性多克隆抗体通过蛋白A亲和层析柱纯化后可获得高纯度的Hemolin抗体,并经抗体亲和层析获得的天然Hemolin蛋白纯度高,可用于对Hemolin蛋白功能的进一步研究。文章提供的分离纯化高纯度家蚕天然Hemolin蛋白的方法能够推进天然免疫蛋白的纯化,为进一步研究家蚕天然Hemolin蛋白的晶体结构、蛋白质抑菌功能以及抗病毒活性奠定基础。

Hemolin is an immunoglobulin-like protein(immunoglobulin, Ig), which belongs to an important innate immunoglobulin of insects. Hemolin in Bombyx mori has high homology with the sequences of other insects. In order to further analyze the function of Hemolin protein in Bombyx mori, in this study, baculovirus-infected pupaes of Bombyx mori were used as raw materials, and high-purity natural Hemolin protein was obtained by antibody affinity chromatography. The results show that the recombinant expression strain pET-32 a-hemolin can be successfully expressed in E.coli BL21 after low temperature induction by IPTG with a final concentration of 0.1 mmol/L.Relatively pure recombinant Hemolin protein can be obtained through nickel ion metal chelating affinity chromatography and elution with 200 mmol/L imidazole. High-purity Hemolin antibody can be obtained after specific polyclonal antibodies from immunizing New Zealand white rabbits are purified by Protein A affinity chromatography column, and natural Hemolin protein obtained by antibody affinity chromatography has high purity, which can be used for further research on the function of Hemolin protein. The method to separate and purify natural Hemolin protein in this paper can promote purification of natural immunoprotein and lays a foundation for further studying crystal structure, bacteria inhibition function and antiviral activity of natural Hemolin protein in Bombyx mori.