【作者】 陈涛；

【导师】 曹祥荣； 李震；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2002， 硕士

【摘要】 干扰素(IFN)是一类重要的细胞因子，具种属特异性，在同种细胞上具有广谱的抗病毒、抗细胞增殖、免疫调节等多种生物活性。其中IFN-α／β是机体的第一抵御病毒体系。猪是一种重要的经济动物，利用重组DNA技术生产有广泛抗病毒效应的基因工程猪α型干扰素(PoIFN-α)无疑有重要意义。 本研究从猪肝细胞中提取基因组DNA，运用常规PCR方法扩增得到一个内部含终止子的假基因，登录Genebank号为AF350425。比较此假基因序列和PoIFNα1基因序列的差别，结合AS-PCR方法及SOE技术获得了猪α型干扰素基因的成熟肽编码区。所得序列记为PoIFN-α1a，与PoIFN-α1基因成熟肽编码区相比有5个碱基差别，导致3个氨基酸不同。 首先以非融合蛋白形式进行表达。将PoIFN-α1a插入到原核表达载体pGRW，转化大肠杆菌DH5α，得到的重组菌株经诱导有以包涵体形式表达的目的蛋白条带出现，表达量约10％。提取包涵体，变、复性后，以WISH／VSV系统检测其抗病毒活性约为3200IU／mg。而采用巨引物PCR法介导的定点突变将PoIFN-α1a第86位的Cys突变为Tyr，同时将N端首位氨基酸(Cys)的密码子TGT同义突变为大肠杆菌偏爱的密码子TGC，突变后基因记作PoIFN-α1b。将其与融合蛋白表达载体pGEX-4T-3连接，转化大肠杆菌BL-21，诱导后表达产物占菌体总蛋白的20％。将以包涵体形式表达的目的蛋白经变、复性处理，并以FPLC进一步纯化，得到产物在WISH／VSV系统上的抗病毒活性为5200IU／mg。更多还原

【Abstract】 Interferons (IFNs), a family of cytokines, have many kinds of biological activities, such as interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. IFN- a / P is the first system of body to defend virus attacking. Pig production is important for food industry and the development of recombant porcine interferon- a ( rPoIFN- a ) is strongly desirable for prevention of pig virus diseases.A pseudogene with a termination codon TAG in the middle was isolated from porcine liver by PCR method. The sequence had been send to Genebank and the accession number was AF350425. Then a PoIFN-a gene was produced by AS-PCR and SOE using four special primers based on the differences between the sequence of the pseudogene and PoIFN- Q 1. hi the sequence of the PoIFN- a gene five nucleotides were different from the sequence of PoIFN-a i, which leaded to three amino acids difference.To obtain a recombinant mature PoIFN- a , plasmid pGRW-PoIFN- a 1 a was constructed. The expression of the unfused protein in the strain E. coli DH5 a was 10%, and the antiviral activity of the protein was 3200IU/mg. Then a site-directed mutation of Cys(86) to Tyr was made by megaprimer PCR ; meanwhile TGT, the first codon of PoIFN- a , was changed to TGC , which was a bias codon to E. coli. In order to increase the expression level of PoIFN- a , the mutated sequence was inserted into the fused protein expression plasmid pGEX-4T-3. The expressed fused protein, which was found in insoluble form, counted for 20% of the total cell proteins. After denaruration, renaturation and purification, a higher antiviral activity was measured (5200IU/mg).更多还原