可溶性TAT PTD-Ngb融合蛋白的原核表达、鉴定与纯化及其对皮质神经元的跨膜转导

Prokaryotic Expression, Identification, and Purification of Soluble TAT PTD-Ngb Fusion Protein and Its Transmembrane Ability in Cortical Neurons

【目的】构建含蛋白转导域(protein transduction domain,PTD)与脑红蛋白(neuroglobin,Ngb)融合基因的表达质粒,原核表达可溶性TATPTD-Ngb,并鉴定、纯化,检测TATPTD-Ngb对皮质神经元的跨膜转导功能。【方法】提取SD大鼠脑组织总RNA,通过RT-PCR法构建编码TATPTD-Ngb的融合基因,克隆入pET28b原核表达载体,转化入大肠杆菌BL21(DE3)pLysS,诱导表达,表达产物进行SDS-PAGE、Western-blot分析及可溶性鉴定,利用所表达的目的蛋白上的6个组蛋白用Ni-NTA琼脂进行亲和层析纯化,将不同浓度纯化的融合蛋白与原代培养皮质神经元共孵育2h,用Western-blot分析融合蛋白的跨膜转导。【结果】成功构建了含有TATPTD-Ngb的原核表达载体,插入片段500bp,成功表达并纯化了可溶性TATPTD-Ngb融合蛋白,相对分子质量约为20k,Westernblot鉴定显示表达蛋白具有抗原性,并具有转导入原代培养皮质神经元的功能,随给予蛋白浓度的增高进入神经元内的融合蛋白量增高。【结论】可溶性融合蛋白TATPTD-Ngb可转导入皮质神经元,对进一步研究Ngb的功能及蛋白转导技术的应用奠定了基础。

[Objective ] To clone, express, purify the soluble Ngb fusion protein containing protein transduction domain of HIV-1 trans-activator （TAT PTD-Ngb） and to verify its transmembrane ability in vitro. [Methods] The sequence coding TAT PTD-Ngb fusion gene was amplified by PCR from rat brain RNA after being anti-transcripted to cDNA and cloned into the expression plasmid pET28b. After sequence analysis, the recombinants were transducted into the E.coli. BL21（DE3）plysS, which was induced with IPTG （0.4 mmol/L） to express the TAT PTD- Ngb fusion proteins. Ni-NTA resin was used to purify the products, which were verified by means of SDS-PAGE and Western blot analysis subsequently. The primary cultured cortical neurons had been incubated in neurobasal medium with TAT PTD-Ngb at different final concentrations for 2 hours, and test the internalization of fusion protein into cells by Western blot analysis in order to examine the ability of TAT PTD-Ngb to transfer into cortical neurons. [Results] The TAT PTD-Ngb expression vector were successfully constructed, containing the insert of 500 bp. The fusion protein TAT PTD-Ngb was about 20 k, which could react immunologically with anti-his-tag monoclonal antibody. Western blot analysis displayed that TAT PTD-Ngb intemalizod into neurons and the amount of fusion protein also increased with the final concentration of TAT PTD-Ngb in the culture medium. [Conclusion ] The recombinants TAT PTD-Ngb which expressed by E.coli BL21 could be transferred into cortical neurons. Our resultswill facilitate further functional study of Ngb and clinical application of protein transduction technology.