猪白细胞介素-12的分子克隆与序列测定

Cloning and Sequencing of Porcine IL-12

为研究白细胞介素－１２在机体免疫应答中的作用，从猪外周血提取单个核细胞（ＰＢＭＣｓ），用含ＬＰＳ的培养基培养细胞，从培养２ ｈ和１２ ｈ的活化细胞中分别提取总ＲＮＡ，用ＲＴ－ＰＣＲ方法扩增出编码猪ＩＬ－１２两个亚基的基因，其大小分别为７２０ ｂｐ（Ｐ３５）和１ ０４４ ｂｐ（Ｐ４０），将扩增产物经琼脂糖凝胶电泳后，回收纯化，用ＳａｃＩ和ＳａｌＩ双酶切后，分别将其连接至ｐＵＣ１８质粒上，进行质粒ＰＣＲ和ＳａｃＩ、ＳａｌＩ双酶切鉴定后筛选阳性克隆，并进行序列测定和分析。序列分析表明Ｐ３５基因开放阅读框含有６６９个核苷酸，序列与人、小鼠和羊同源性分别为７９．２％，３７．６％ ，８６．１％，Ｐ４０基因开放阅读框含有９７５个核苷酸，与人和小鼠、羊基因序列的同源性分别为７９．５％，５３．１％，８４．３％。结果表明克隆到了猪白细胞介素－１２ Ｐ３５和Ｐ４０两个亚基的基因，得到的Ｐ３５基因序列与国外报道完全一致，Ｐ４０基因序列与发表的序列有４个碱基差异，提示Ｐ４０基因可能存在多态性。

In order to understand its function in immune system, we cloned and sequenced two genes of porcine IL-12 subunits. The PBMCs were isola ted using lymphocyte separation medium and the total RNA was extracted from PBMC s stimulated 2 hours and 12 hours by LPS, respectively. The genes of two subunit s were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The sizes of the resulted amplicons were about 720 bp(P35) and 1044 bp(P40),which were then separated by agarosegel electrophoresis, then excised and purified the target segments from the gel. The target segments were cloned into pUC18 after digested by SacI and SalI. The products were identified by PCR and digestion with SacI and SalI. Two clones from both P35 and P40 positive recombinants were sequenced and analyzed . The results showed that the gene of P35 has only one ORF, which composed of 669 bp nucleosides. The sequence was 79.2%, 37. 6% and 86.1% identical to human, murine and ovine, respectively. The gene of P40 also has one ORF composed of 975bp nucleosides, with sequence identity of 79.5% , 53.1% and 84.3% to human, murine and ovine. The cloned gene of P35 of porcine IL-12 was the same as that of the published one from GenBank. But the gene of P 40 had 4 nucleosides which were different from the published before, suggesting that the P40 gene of porcine IL-12 may be polymorphic.