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By reverse transcription-polymerase chain reaction (RT-PCR), a cDNA encoding chicken (interferon-gamma) (chIFN-γ) was cloned from Luoyang local chicken embryo splenocytes induced by (Newcastle) disease virus Ⅰstrain. The result showed that the optimal time at which the mRNA of IFN-γ gene was extracted from spleen cells of chicken embryo is 48 h after the embryo was inoculated by 1∶50 and 1∶100 diluted Newcastle disease virus. Sequence analysis showed that the chIFN-γ cDNA is 492 bp and encodes a protein including... By reverse transcription-polymerase chain reaction (RT-PCR), a cDNA encoding chicken (interferon-gamma) (chIFN-γ) was cloned from Luoyang local chicken embryo splenocytes induced by (Newcastle) disease virus Ⅰstrain. The result showed that the optimal time at which the mRNA of IFN-γ gene was extracted from spleen cells of chicken embryo is 48 h after the embryo was inoculated by 1∶50 and 1∶100 diluted Newcastle disease virus. Sequence analysis showed that the chIFN-γ cDNA is 492 bp and encodes a protein including 164 amino acids. Compared with the reported nucleotide acid sequence of IFN-γ gene and the corresponding amino acid sequence of IFN-γ, the nucleotide acid sequence of the IFN-γ gene cloned by us and the amino acid sequence deduced from the cloned IFN-γ gene shared 99.7% and 100% homology, respectively. It was concluded from the above-mentioned results that the encoding region of IFN-γ gene is highly conservative. 应用反转录 聚合酶链式反应 (RT PCR)技术 ,从鸡新城疫I系疫苗接种的洛阳土种鸡胚脾提取的RNA中扩增到干扰素 γ(IFN γ)基因cDNA。结果表明 ,以 1∶5 0或 1∶10 0稀释度接种后第 4 8h提取的mRNA扩增效果最佳。序列测定显示 ,所克隆的洛阳土种鸡IFN γ基因cDNA与所报道的鸡IFN γ基因cDNA同源性为 99.7% ,相应的氨基酸序列同源性为 10 0 % ,说明鸡IFN γ基因编码区高度保守。 A cDNA encoding Chicken interleukin-2(IL-2)was amplified by reverse transcription-polymerase chain reaction(RT-PCR)from Luoyang local Chicken embryo spleenocytes . Sequences analysis showed that the target gene was Chicken IL-2 and no termination codon.The IL-2 gene was inserted into the bacterial plasmid PET-28a, resulting in the construction of PET-28a-IL2 prokaryotic expression plasmids. The recombiant plasmid PET-28a-IL2 containing IL-2 gene were identified by restriction enzymes analysis and PCR method... A cDNA encoding Chicken interleukin-2(IL-2)was amplified by reverse transcription-polymerase chain reaction(RT-PCR)from Luoyang local Chicken embryo spleenocytes . Sequences analysis showed that the target gene was Chicken IL-2 and no termination codon.The IL-2 gene was inserted into the bacterial plasmid PET-28a, resulting in the construction of PET-28a-IL2 prokaryotic expression plasmids. The recombiant plasmid PET-28a-IL2 containing IL-2 gene were identified by restriction enzymes analysis and PCR method and sequenced to confirm its rightness. The recombinant fusion protein was highly expression in E. coli BL21(DE_(3))four hours later induced by IPTG and the molecular weight is about 14 ku. These provide a basis for the study on the biological property of chicken IL-2. 应用RT-PCR技术从新城疫I系疫苗诱导的洛阳土种鸡胚脾淋巴细胞中扩增到全长0.43kb的鸡IL-2基因cDNA,将IL-2cDNA克隆到PMD-T载体,测序结果表明,该基因为不含终止密码子的目的基因。将该目的基因克隆到原核表达载体PET-28a,获得的重组质粒PET-28a-IL2经酶切、PCR及序列测定,表明IL-2基因插入的位点、大小与读码框均正确。PET-28a-IL2在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物经SDS-PAGE电泳分析,在14ku处出现一条约占总蛋白21%的蛋白带,表明所克隆的IL-2基因在大肠杆菌中得到良好的表达,为进一步研究IL-2的生物学特性奠定了基础。
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