摘要
目的 :克隆中国人IL 12的 p40和p35亚基cDNA ,构建含hIL 12双顺反子的腺病毒载体。 方法 :采用RT PCR从上海地区人骨髓有核细胞中克隆IL 12的双亚基cDNA ,并插入质粒 pCI中进行全基因序列分析。利用质粒 p△ElsplA和IRES序列构建同时表达p35和 p40亚基的腺病毒载体。酶切鉴定构建物中基因插入的正确性。结果 :上海地区人骨髓有核细胞中IL 12的 p40和p35cDNA基因同已报道的北京地区人DC细胞中IL 12的 p40和国外报道的NKSF ,CLMF的 p40和p35序列各有异同 ,构建的IL 12双亚基同时表达的腺病毒载体酶切鉴定正确。 结论 :人IL 12的 p40和 p35基因可能存在多态性 ,同时表达IL 12双亚基的腺病毒载体构建成功 ,对开展人IL 12基因治疗肿瘤具有重要意义。
Objective: To clone IL 12 p40 and p35 cDNA from Chinese and to construct bicistronic adenovirus vector containing human IL 12 p40 and p35 cDNA. Methods: Both p40 and p35 cDNA of human interleukin 12 were amplified by means of reverse transcription and polymerase chain reaction (RT PCR) from total RNA of human bone marrow mixed nucleated cells. The amplified cDNA fragments were inserted into pCI plasmid repectively. The total nucleotide sequences of both p40 and p35 cDNA were sequenced . Bicistronic adenovirus vectors (pAd p40 p35) which encode both human IL 12 subunits (p35 and p40) simultaneously were constructed by using p△ElsplA and internal ribosome entry site (IRES). The structure of pAd p 40 p35 is identified by means of restriction enzyme analysis . Results: IL 12 p40 and p35 cDNA sequences cloned from Shanghai bone marrow nucleated cells have similarities and differences when compared with Beijing p40, CLMF p40, CLMF p35, NKSF p40, NKSF p35. Restriction enzyme analysis shows that the struction of IL 12 bicistronic adenovirus vector was right. Conclusion: Our research data indicate that polymorphism might exist in the human p40 and p35 gene. The IL 12 bicistronic adenovirus vector might be applied to tumor immuno gene therapy.
出处
《海军医学杂志》
2001年第2期97-100,共4页
Journal of Navy Medicine
