人白细胞介素10基因慢病毒载体的构建及重组

The construction of recombinant lentiviral-vector with human interleukin-10 gene

目的拟构建含人白细胞介素10(hIL-10)基因的慢病毒载体(LV-hIL-10),为进一步探讨慢性疼痛的治疗研究奠定基础。方法从pCYIL-10质粒中选择适当的引物进行PCR后得到含PmeI多功能酶切位点的IL-10基因片段,将其构建到慢病毒载体pWPXL上得到重组的pWPXL-IL-10质粒,对所抽提的质粒进行酶切及测序检测正确后大量提取备用。将重组质粒pWPXL-IL-10、包膜质粒pMD2.G和包装质粒psPAX2共转染293T细胞后包装出复制缺陷的慢病毒颗粒,进行病毒滴度的测定。结果重组质粒酶切鉴定结果显示,pWPXL-IL-10质粒中有一个530bp左右的插入片断,大小与IL-l0cDNA(534bp)基本符合。测序结果显示pWPXL-IL-10质粒上插入片段的序列与基因库中hIL-10基因序列完全一致。转染后获得了高滴度(2×1010)、高纯度的病毒颗粒。结论成功构建了含人白细胞介素10基因的慢病毒载体LV-hIL-10,为慢性疼痛治疗的研究奠定了基础。

Objective To construct contains human interleukin-10 gene recombinant lentiviral-vector （LV-IL-10） and to form a basis to further explore the therapy of chronic pain, Methods hIL-10 gene fragment was isolated and amplified from pCYIL-10 plasmid by PCR, and was cloned into pWPXL-GFP. The inserted hIL-10 fragment was verified by Pme I digestion and DNA sequencing. The recombinant plasmid pWPXL-IL-10-GFP, envelope plasmid pMD2. G and packaging plasmid psPAX2 were cotransfected into 293T cells, to pack out lentivirus particle that has the ability of duplicated-deficiency, then virus titer determination was undertaken. Results The 530bp IL-10 gene fragment was amplified from pCYIL-10 plasmid by PCR, and was recombinated into pWPXL-GFP plasmid. DNA sequencing confirmed that the cloned gene segment was 100% homologous to the published hIL-10 sequence in genebank. High titer（2 × 10^10） and highly purified lentiviral particles was obtained. Conclusions The lentivirus vector LV-hIL-10 was constructed successfully, which form a basis of research of chronic pain therapy.