中国人白细胞介素-12cDNA基因的克隆及序列分析与比较

Cloning and Sequence Analysis of Chinese Interleukin 12 cDNA

为研究中国人IL 12的基因特征 ,采用逆转录巢式聚合酶链反应 (RT nPCR)从中国人脐带血单核细胞中分别克隆了P35、P40两亚基cDNA基因 ,包括完整的前体蛋白编码序列 ,其中P35cDNA编码 2 19个氨基酸的多肽 ,P40cDNA编码 32 8个氨基酸的多肽 ,与国外序列 (NKSF、CLMF)比较结果发现 :所克隆序列P35同NKSF相比 ,第44aa密码子由GTC(Val)→GTG(Val) ,但未改变氨基酸组成 ;同CLMF相比 ,2 44aaGAC(Asp)→GAT(Asp) ,2 47aa由ACG(Thr)→ATG(Met) ;P40亚基与CLMF的核苷酸、氨基酸组成完全相同 ,但与NKSF相比存在多处差异 ,2 39aaAAC(Asn)→AAG(Lys) ,2 91aaACC(Thr)→ACG(Thr) ;3株序列的变异全部集中在相同的几个碱基位置。说明IL 12cDNA基因尽管非常保守 ,但在核苷酸及氨基酸也存在一定的差异 ,其基因可能存在多态性 ,所克隆的IL 12基因具有一定的独特性。

In order to study the genetic characteristics of Chinese IL 12 gene, we cloned the cDNA genes of P40 and P35 subunits of IL 12 by RT nPCR from mononuclear cells of umbilical cord blood of a Chinese newborn, including the complete coding region of precursor protein, the nucleotide sequence was determined by the dideoxy mediated chain termination method. P35 cDNA sequence encodes a 219 amino acid polypeptide and P40 cDNA encodes a 328 amino acid polypeptide. Compared to the foreign sequences of NKSF, CLMF, we found that P35 sequence is different from that of NKSF, in which the 44 th amino acid codon changes to GTG(Val) from GTC(Val), but the deduced amino acid does not change. Compared to CLMF, 244 th amino acid codon GAC(Asp) changes to GAT(Asp), 247 th amino acid changes from Thr (ACG) to Met (ATG). Subunit P40 nucleotide sequence and deduced amino acids are identical with that of CLMF. However there are several differences between NKSF and P40: 239 th amino acid codon of P40 changes from AAC (Asn) to AAG (Lys), 291 th amino acid codon changes from ACC (Thr) to ACG (Thr). Sequence variations of these three are on the same nucleotide sites. The results indicate that although IL 12 gene is very conserved, its amino acid and nucleotide sequences have certain differences and the gene may be polymorphic. The IL 12 gene we cloned has some uniqueness.