【摘要】 克隆了鸡(Gallusgallus)的γ-干扰素cDNA,并用大肠杆菌(Escherichiacoli)进行了温度诱导型表达。用PCR方法扩增了鸡γ-干扰素基因组基因,将其克隆到载体pGEM-T上。对重组载体的插入片段进行酶切分析和部分序列测定:证明了基因的正确性;克隆的鸡γ-干扰素基因的编码序列中存在一个点突变(G→A),这一突变导致一个三联体密码子GAA变为AAA,编码的氨基酸由谷氨酸(Glu)变为赖氨酸(Lys)。用鸡γ-干扰素基因组基因构建了真核表达载体pCI-ChIFN。将这一真核表达载体转染兔成纤维细胞的细胞系中进行表达实验:提取转染后细胞裂解物中的RNA,再以此为模板进行RT-PCR,获得了完整的鸡γ-干扰素cDNA基因,同时证明鸡γ-干扰素基因组基因能在兔成纤维细胞系中表达。用鸡γ-干扰素cDNA构建了温度诱导型原核表达载体pBVcDNA,并将其转导到大肠杆菌DH5α中,经细菌发酵和温度诱导,表达了一个18kD的特异蛋白,其表达量占细菌总蛋白的8.75%。更多还原

【Abstract】 Interferon is a key member of cytokines and plays important roles in host immunity against virus infections.Chicken (Gallus yallus ) interferon-? gene was amplified by PCR and cloned into vector pGEM-T. The point mutation(G→A) was found by the sequence analysis in ChIFN-? cDNA gene, resulting in the change of codon (GAA→AAA), thus Glu became to Lys. Chicken interferon-? gene was inserted into pCI-neo to produce an eukaryotic expression vector, which was subsequently transfected into the rabbit fibroblast cells. Total RNA was extracted from the transfected cells and the ChIFN-? cDNA gene was amplified by RT-PCR showing chicken interferon-? gene transcribed in the rabbit fibroblast cell. ChIFN-? cDNA gene was cloned into pBV220 containing a temperature inducible promoter and to form a prokaryotic expression vector. Temperature-induced expression of ChIFN-? cDNA gene in E.coli DH5á induced a specific 18 kD protein band in polyacrylamide gel electrophoresis of bacteria proteins and the expression product was 8.75% of the bacteria total proteins.更多还原