人CXCL9重组腺病毒的制备及其表达产物生物活性检测

目的构建携带人CXCL9基因的重组腺病毒,分析其表达产物的生物学活性。方法采用PCR法从pBLAST2-hCXCL9质粒上扩增出hCXCL9基因,再将其克隆至pENTR11载体上,构建pENTR11-hCXCL9质粒,通过同源重组将hCXCL9基因片段重组至pAd/CMV/V5-DEST上,最后在293A细胞内进行包装、扩增后得到携带hCX-CL9基因的重组腺病毒。用包装好的病毒感染Hela细胞,分析目的基因的表达及其生物学活性。结果成功地将hCXCL9基因片段克隆至重组腺病毒载体上,并经293A细胞包装出病毒颗粒。该病毒感染Hela细胞后,可在培养上清液中检测到显著表达的hCXCL9。通过趋化活性分析发现,该表达产物对T淋巴细胞具有明显的趋化活性。结论成功构建了hCXCL9重组腺病毒,并可在体外高效表达具有生物活性的目的产物。

Construction of recombinant adenovirus vector carrying hCXCL9 and identification the biological activity of its expression product

Objective To establish a recombinant adenovirus vector carrying hCXCL9 gene and identify the biological activity of its expression product.Methods The hCXCL9 gene was amplified from the pBLAST2-hCXCL9 vector by polymerase chain reaction(PCR) technique,and then was cloned into pENTR11 vector.Subsequently,the coding sequence of hCXCL9 gene was subcloned into the backbone plasmid pAd/CMV/V5-DEST by the homologus recombination.Finally,the recombinant adenoviruses were packaged and amplified in 293A cells.The expression of hCXCL9 and its biological activity were detected after infecting Hela cells.Results The hCXCL9 gene was successfully cloned into pAd/CMV/V5-DEST plasmid and generated the recombinant adenoviruses carrying hCXCL9 gene(Ad-hCXCL9).After infecting Hela cells,the expression of hCXCL9 can be detected in the culture supernatant.The biological activity assay for chemotaxis showed that the expressed products could specifically direct the migration of T cells.Conclusion The recombinant adenovirus vector carrying hCXCL9 gene was successfully constructed,which would effectively encode of hCXCL9 with biological activity in vitro.