抗AIDS新型导向毒素CVN-LP1融合基因的构建及原核表达

目的：构建重组抗病毒融合蛋白CVN-LP1原核表达载体，并进行表达和鉴定。 方法：将本室已经构建好的pET-CVN和pET-LP1，用EcoRⅠ和HindⅢ同时进行双酶切，将所得的CVN片段连入pET-LP1载体上，构建pET-CVN-LP1表达载体，转入大肠杆菌BL21(DE3)，IPTG诱导表达。用SDS-PAGE，Western-blot等方法分析鉴定表达产物。 结果：重组载体pET-CVN-LP1经PCR和双酶切鉴定，证实构建成功。将其导入大肠杆菌BL21(DE3)中表达，表达产物相对分子量为20KD左右，与理论预期值完全相符。凝胶成像分析表明最高表达量可占菌体总蛋白的16.86%。SDS-PAGE、Western-blot分析表明目的蛋白得到了很好的表达。 结论：构建了pET-CVN-LP1原核表达载体，并成功地获得表达，为进一步研究其生物学功能奠定了坚实的基础。

Constrction and prokaryotic expression of a new anti-HIV targeted toxin

Objective: To construct prokaryotic expression vector containing CVN-LP1 gene ,and identify the expression of CVN-LP1 protein. Methods: pET-CVN and pET-LP1 was restriction endonuclease digested by EcoRI and HindIII, and the CVN gene was inserted into pET-LP1 plasmid. The vector was transformed into BL21(DE3) to construct a prokaryotic expression system. The expressed product was identified by SDS-PAGE and Western blot assay. Results: The recombinant plasmid pET-CVN-LP1 was constructed, restriction endonuclease digestion and PCR identification proved that the CVN was correctly cloned into vector pET-LP1. SDS-PAGE and Western-blot analysis showed that CVN-LP1 was successfully expressed in E.coli BL21(DE3) ,The relative molecular mass (Mr) of the expression protein was 20kDa,according with the predicted Mr value. And the expression yield was about 49.58% of total bacterial protein. Conclusion: The successful expression of CVN-LP1 gene in E.coli provides a support for further study of the anti-HIV agent.