人IL-12的克隆、表达及生物活性的鉴定

Cloning,sequence analysis and biological characterization of human recombinant IL 12

目的：克隆中国人ＩＬ１２ｐ４０基因，构建ＩＬ１２的真核基因表达载体。方法：采用ＲＴＰＣＲ从北京地区人脐血树突状细胞（ＤＣ）中克隆ＩＬ１２ｐ４０ｃＤＮＡ基因，并进行序列分析。利用ｐｃＤＮＡ３１和ｐＬＸＰＸＳＮ构建ＩＬ１２表达载体，转染人肝癌细胞后对其进行生物学和免疫学分析。结果：北京地区人ＤＣ中ＩＬ１２ｐ４０ｃＤＮＡ基因２１０位密码子有独特的结构，为ＧＣＣ（Ａｌａ）。并且２３９和２９１位密码子与已报道的ＮＫＳＦ和ＣＬＭＦｐ４０序列各有异同，在与国外提供的ｐ３５基因共同转染细胞后能产生功能性的ＩＬ１２。用其构建的ＩＬ１２双亚基共表达逆转录病毒载体在转染肝癌细胞株并经选择后，上清液中有较高的具有刺激淋巴母细胞增殖活性的ＩＬ１２。Ｗｅｓｔｅｒｎｂｌｏｔ分析结果显示，在７０ｋＤ处有特异性的蛋白条带出现。结论：支持人ＩＬ１２ｐ４０基因可能存在着多态性的假设；

Objective:Cloning IL 12p40 from Chinese and constructing human recombinant IL 12 expression vector.Methods:By using RT PCR to clone IL 12 p40cDNA from the mRNA extracted from cord blood dendritic cells(DC) of Beijing newborns' and then sequenced.Expression vectors encoding the IL 12 subunits p35,p40 and IL 12 bicistronic expression vector were constructed by using pCDNA3.1 and pLXPXSN,and then transfected human heptocellular carcinoma(HCC) cell line by lipofection.Results:IL 12 cloned from DC of Beijing has unique structure in codon 210,being GCC(Ala).The 239 and 291 codons are the same as CLMF p40 but different from NKSF p40.Transfection with expression vectors encoding either the p40 or the p35 of hIL 12 to HCC cell line showed that only in the case of co transfection with both vectors in equivalent amount,the supernatant of the transfected cells showed biological activity of IL 12.Subsequently,the bicistronic retroviral vector encoding both p40 and p35 of hIL 12 was transfected the replicating HCC cell line.Following selection,the supernatant of the transduced cell colonies showed prominent biological activity in stimulating the proliferation of lymphoblasts.Western blot showed the appearance of the characteristic 70 kD band.Conclusion:The above data support the hypothesis that the p40 gene of human IL 12 may be polymorphic.