Construct ion of recombinant adenovirus vector pDC316-hIL-24 and the detection of viral titer
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摘要:
目的构建重组腺病毒载体pDC316-hIL-24，并测定病毒滴度。 方法从HEK293细胞中提取mRNA，设计特异性引物，通过RT-PCR法扩增hIL-24基因全编码区序列。将测序正确的片段用BglⅡ和HindⅢ双酶切定向插入到pDC316-EGFP穿梭质粒中。构建重组穿梭质粒pDC316-EGFP-hIL-24，在脂质体介导下与腺病毒辅助大质粒pBHGlox（delta）E1，3Cre共转染293细胞，包装产生复制缺陷型重组腺病毒pDC316-hIL-24，经HEK293细胞扩增，TCID50法测定重组腺病毒滴度。 结果成功构建出表达hIL-24基因的重组腺病毒载体（pDC316-hIL-24），获得了高滴度表达hIL-24基因的重组腺病毒。 结论重组腺病毒表达载体（pDC315-hIL-24）的成功构建及重组腺病毒的获得有利于进一步开展肿瘤的转基因治疗研究。 -
关键词:
- hIL-24 /
- 重组腺病毒载体 /
- 病毒滴度 /
- pDC316-hIL-24
Abstract:ObjectiveTo onstruct recombinant adenovirus vector pDC316-hIL-24 and detect the viral titer. MethodsTotal mRNA was ex tracted from HEK293 cells, and specific primer pairs were designed in order to clone human interleukin-24 (hIL-24) gene cDNA by RT-PCR method. hIL-24 gene was directional constructed into shuttle plasmidr (pDC316-EGFP) after digestion by restrictive endoenzyme BglⅡ, and HindⅢ. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-EGFP and large adenovirus helper plasmid pBHGlox (delta) E1, 3Cre in mediation of liposome. The constructed recombinant adenovirus vector was named pDC316-hIL-24. Recombinant adenovirus was amplificated and purificated in HEK293 ce lls. ResultsRecombinant adenovirus vector pDC316-hIL-24 was constructed successfully and high titer recombinant adenovirus was obtained. ConclusionThe construction of recombinant adenovirus vector pDC316-hIL-24 and the obtain of recombinant adenovirus will lay down the basis for developing gene therapy of cancer. -
Key words:
- hIL-24 /
- recombinant adenovirus vector /
- viral titer /
- pDC316-hIL-24
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图 1 RT-PCR产物电泳图
Figure 1. Identification the hIL-24 gene by PCR, 1: hIL-24; 2: DNA MARKER
图 3 pDC316-EGFP-hIL-24质粒经BglⅡ及HindⅢ双酶切
Figure 3. Identification the pDC316-EGFP-hIL-24 by BglⅡ and HindⅢ enzyme. 1: pDC316 -EGFP-hIL-24, 2: DNA Marker.
图 4 出现CPE反应的293细胞
Figure 4. 293 Cells with a CPE response (Original magnification: ×10).
图 5 pDC316 hIL-24的表达的绿色荧光蛋白
Figure 5. Identification of gfp (O riginal magnification: ×100).
图 6 Ad.mda-7.egfp的PCR鉴定
Figure 6. identification of Ad.mda-7.egfp by PCR M: DNAMarker 2000; 1: PCR positive.
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