摘要
提取经新城疫病毒诱导培养的奶牛外周血淋巴细胞总RNA,应用RT-PCR方法扩增出奶牛α干扰素成熟蛋白基因,然后将特异性片段连接到pMD18-T载体,测序结果表明,扩增片段为牛α干扰素成熟蛋白序列,与Genebank上发表的α1干扰素序列同源性为98%。然后将特异性片段连接到含分泌信号肽序列的Pichiapastoris表达载体pPICZαA上,将重组质粒经SacI酶切线性化后电转化导入毕赤酵母菌株X-33。转化子经PCR分析鉴定后利用甘油增菌和甲醇诱导,实现了BoIFN-α在毕赤酵母系统中的分泌表达。SDS-PAGE结果显示表达产物分子量约为23kDa,比其推导结果(20kDa)略大,推测可能是因为表达产物发生了一定程度的糖基化。细胞病变抑制法结果表明,重组牛IFN-α具有较高的干扰素活性,约为4.1×105U/ml,经微量蛋白测量仪测得,其表达量约为80μg/ml,比活为5.1×106U/mg。
Total RNA was isolated from bovine peripheral blood lymphocytes, which were stimulated with New Castle Disease Virus. Then the bovine IFN-α cDNA was amplified by reverse transcription polymerase chain reaction. The amplified fragment was cloned into vector pMD18-T and then sequenced. The result indicated that the cloned gene was a mature bovine BoIFN-α gene, which had the identities of 98% with the BoIFN-α1 gene published in the Genebank. The cloned gene fragment was inserted into Pichia pastoris expression vector pPICZαA. The recombinant plasmid was linearized with Sac Ⅰ and then transformed into X-33 strain by electroporation. Whether the BoIFN-α gene was integrated into the alcohol oxidase promoter （ AOX1 ） locus on yeast chromosome was verified by amplification with AOX1 primers. The SDS-PAGE result showed that cloned recombinant protein was expressed in the supernatant with molecular weight of approximated 23kDa. The expressed protein was larger, which may be due to the difference of glycosylation. The concentration of the secreted BoIFN-α was about 801μg/ml, and its antiviral activity is about 5.1 ×10^6U/mg.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第9期64-68,共5页
China Biotechnology
