摘要
参照Digby发表的鸡γ-干扰素的核苷酸序列,设计一对引物,应用RT-PCR技术,从ConA诱导过的鸡脾淋巴细胞中特异性地扩增出大小约为500bp的基因片段。将该扩增基因进行克隆测序,结果表明其序列与Digby报道的CHIFN-γ基因序列的同源性达100%。将该基因插入原核表达载体pGEX-4T-1中构建成重组表达载体pGEX-CHIFN-γ,并将其导入大肠杆菌BL21中,诱导表达的重组蛋白经SDS-PAGE分析,分子量约为43ku,表明所克隆的CHIFN-γ基因在原核细胞中得到了表达。
The total RNA was abstracted from the concanavalin A（ Con A） -activated spleen lymphocytes. A pair of specific primers was designed by Oligo software based on the sequence of Chicken interferon-gamma submitted by Digby in GenBank （accession No. U27465）. A 500 bp DNA fragment was amplified by reverse transcriptionpolymerase chain reaction （RT-PCR） and then ligated into T-easy vector for sequencing. The result shows that the fragment contained the complete open reading frame of CHIFN-γ gene. In comparison with GenBank data, the homology of the nucleotide sequence is 100% . The sequence was inserted into pGEX-4T-1 and the recombined expressing vector was constructed, then transformed into Escherichia coli BL21. which were further induced by IPTG and cultured, and a fusion protein was obtained with about 43 000 of molecular weight in SDS-PAGE. This result showed that the cloned CHIFN-γ gene was expressed in prokaryotic cells.
出处
《中国兽药杂志》
2006年第7期12-16,共5页
Chinese Journal of Veterinary Drug
基金
国家高技术研究发展计划(863)项目(2003AA241110)
国家自然科学基金资助项目(30270992)
关键词
Γ-干扰素
鸡
克隆
表达
interferon-γ
chicken
cloning
expression
