广西巴马小型猪α干扰素基因克隆及其真核表达载体的构建

目的]克隆广西巴马小型猪α干扰素基因（poIFN-α）全部编码序列并构建其哺乳动物真核表达载体,为研发重组猪干扰素类免疫佐剂及生物治疗制剂提供参考依据.方法]应用RT-PCR扩增广西巴马小型猪poIFN-α编码序列,链接至pMD 18-T载体构建重组质粒,以测序正确的重组质粒为模板,PCR扩增带有酶切位点的poIFN-α基因,然后连接真核表达载体pTARGET构建重组表达载体pTARGET-poIFN-α,并转化感受态细胞DH5α,经双酶切初步鉴定正确后送至北京诺赛生物有限公司测序,用DNASTAR软件及Primer Premer 5.0等对获得的基因序列进行分析.结果]以广西巴马小型猪总RNA为模板扩增获得的目的片段为618 bp,包含poIFN-α基因全部编码序列;将克隆获得的poIFN-α基因插入哺乳动物真核表达载体pTARGET中,可成功构建广西巴马小型猪重组表达载体pTAR-GET-poIFN-α.广西巴马小型猪poIFN-α与GenBank上已发表的猪、鼠、人IFN-α基因同源性分别为97.0％、78.2％和66.3％;其成熟肽与普通猪相比,共有17个位点发生碱基突变,其中第63、189、198、288、544、545位点为无义突变,第88、119、163、182、186、195、263、301、306、553、560位点为错义突变.结论]IFN-α成熟肽在哺乳动物中保守性较差;克隆获得的广西巴马小型猪poIFN-α基因能成功插入哺乳动物真核表达载体pTARGET中构建重组表达载体pTAR-GET-poIFN-α,为研发重组猪干扰素类生物治疗制剂、免疫佐剂、构建小型猪疾病动物模型等奠定了基础.

Cloning interferon-α gene of Guangxi Bama mini-pig and constructing its eukaryotic expression vector

Objective]All coded sequences of Bama-mini pig inteferon-ot gene were cloned and its eukaryotic ex- pression vector was constructed to provide references for restructuring swine interferon immunologic adjuvant and biothera- py preparations. Method ]A pair of specific primers for porcine interferon-alphal （polFN-α） was designed and synthesized according to gene sequence,and polFN-α gene was amplified by RT-PCR from peripherals blood lymphocytes of Bama- mini pig. Then poIFN-α CDS was cloned into pMD18-T vector and sequenced. Moreover, polFN-α gene with enzyme sites was cloned into eukaryotic expression vector pTARGET to construct recombinant expression plasmid pTARGET- poIFN-α and it was transformed into E.eoli 5α （DH5α. Recombinant poIFN-α was sent for test in SinoGenoMax Co. ,Ltd. The obtained sequence was analyzed using DNASTAR and Primer Premer 5.0. Result]The amplified polFN-oL sequence was 618 bp, containing the complete polFN-α CDS. The pTARGET-poIFN-ol recombinant was set up successfully. The results of DNA sequence analysis showed that Guangxi Bama mini-pig polFN-α CDS nucleotide sequence homologous rate was 97.0%, 78.2% and 66.3% compared with swine ,human and mouse IFN-α reported in GenBank, respectively. Com- pared with pig＇s IFN-α cDNA sequence, there were 17 loci base mutations in Guangxi Bama mini-pig, in which 63, 189, 198, 288, 544 and 545 loci were nonsense mutations, and 88, 119, 163, 182, 186, 195, 263, 301, 306, 553, 560 loci were missense mutations. Conclusion]It was concluded that 1NF-α mature peptide was not conservative enough among the mammals. 1FN-o~ cDNA sequence of Guangxi Bama-mini pig was successfully inserted in the mammalian ex- pression vector pTARGET, and the recombinant vector could be further used for swine interferon experiments in biological preparation, immunologic adiuvant, and animal model of small-size swine disease.