鸡α干扰素基因克隆、原核表达及抗病毒活性研究
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摘要
家禽传染性疾病是养禽业最人的威胁，给养殖业造成严重经济损失。干扰素(IFN)是一类重要的细胞因子，具种属特异性，具有广谱的抗病毒、抗细胞增殖、免疫调节等多种生物活性。因此研发具有广谱抗病毒作用的鸡干扰素具有重大的意义。
参照GenBank上登陆的鸡α干扰素基因序列设计一对引物，应用PCR技术直接从河南大面积饲养的罗曼鸡、海兰鸡及地方品种固始鸡肝组织基因组DNA中扩增鸡α干扰素基因。将扩增得到的特异性片段克隆入pGEM-T Easy载体中，转化感受态细胞JM109，运用蓝白斑筛选、质粒PCR鉴定及酶切鉴定筛选阳性可疑菌株，并将筛选的阳性菌株送往大连宝生物测序。测序结果表明，得到了上述3个品种鸡α干扰素基因，大小均为582bp，除去93个信号肽，共编码162个氨基酸。用DNAstar序列分析软件对其进行同源性分析，核苷酸同源性均在99.3％以上，氨基酸同源性均在97.9％以上。
在此基础上，重新设计一对引物，从pGEM-T Easy载体中扩增鸡α干扰素成熟肽蛋白基因用Sph Ⅰ和Sal Ⅰ双酶切后，再将其插入同样经Sph Ⅰ和Sal Ⅰ双酶切的原核表达载体pQE30中，构建了鸡α干扰素原核表达载体pQEIFN-α。转化感受态细胞JM109，经PCR、酶切鉴定并进行序列测定，结果表明，将ChIFN-α成熟肽蛋白基因正确地插入到原核表达载体pQE30的目的位点，重组质粒pQEIFN-α转化受体菌M15后用IPTG诱导，实现了鸡α干扰素在大肠杆菌M15中的表达，表达产物经SDS-PAGE电泳后在约20.0 Ku处出现了特异性条带，用1.0mmoL／LIPTG于37℃诱导8h表达产物最佳，用鼠抗鸡IFN-α单克隆抗体作一抗，辣根过氧化物酶标记的羊抗鼠抗体作二抗，Western blotting检测出现了特异性的条带。表达产物以包涵体形式存在，包涵体经变性、复性处理后，用细胞病变抑制法测定活性，结果表明重组鸡α干扰素在Vero细胞上抗VSV的病毒活性为1.16×10~3U／mg。
与人和哺乳动物比较，鸡IFN-α的研究相对来说比较滞后。本研究利用体外表达技术成功研制重组IFN-α蛋白，为研制抗鸡IFN-α的多克隆抗体和单克隆抗体以及ChIFN-α蛋白的生物学特性的研究奠定基础。
Domestic infective diseases which cause the tremendous economic losses to poultry industry, provoke the serious threat of poultry industry. 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.Therefore the research on the chicken interferon has the important meaning.
One pair of primers were designed according to the sequences of chicken Interferon-alpha published in GenBank. Chicken interferon-alpha gene were amplified from genome DNA extracted from liver tissue of luoman chicken, hainan chicken and gushi chicken, respectively,and were linked to pGEM-T Easy vector, then transformed to competent cells of JM109. By blue-white spot screening , idenfication of plasmid PCR and enzyme digestion,positive strain was sent to Takara for sequencing. And sequencing results showed that chicken Interferon-alpha gene of luoman chicken, hainan chicken and gushi chicken were obtained.The amplified fragment of ChIFN-α was 582bp in length, which includes one open-reading frame, encoding 162 amino acid residues..The results of homology analysis with other chicken interferon-alpha gene published previously in GenBank by DNAStar analysis reveal there was identity of 99.3% at nucleotide homology and that of 97.9% at amino acid homology.
On this basis,another pair of primers were designed. Maturity protein gene of IFN-α was amplified from pGEM- ChlFN-α and was digested by SphI and SalI,then was inserted into pQE30 of the prokaryotic expression vector which was digested by SphI and SalI, the recombinant expressed plasmid pQEIFN-α was constructured ,then transformed to competent cells of JM109. pQEIFN-α was identificated by PCR, enzyme digestion and DNA sequencing conclusively. The result indicated that the gene of encoding ChIFN-α maturity protein was inserted correctly to the target site of
参照GenBank上登陆的鸡α干扰素基因序列设计一对引物，应用PCR技术直接从河南大面积饲养的罗曼鸡、海兰鸡及地方品种固始鸡肝组织基因组DNA中扩增鸡α干扰素基因。将扩增得到的特异性片段克隆入pGEM-T Easy载体中，转化感受态细胞JM109，运用蓝白斑筛选、质粒PCR鉴定及酶切鉴定筛选阳性可疑菌株，并将筛选的阳性菌株送往大连宝生物测序。测序结果表明，得到了上述3个品种鸡α干扰素基因，大小均为582bp，除去93个信号肽，共编码162个氨基酸。用DNAstar序列分析软件对其进行同源性分析，核苷酸同源性均在99.3％以上，氨基酸同源性均在97.9％以上。
在此基础上，重新设计一对引物，从pGEM-T Easy载体中扩增鸡α干扰素成熟肽蛋白基因用Sph Ⅰ和Sal Ⅰ双酶切后，再将其插入同样经Sph Ⅰ和Sal Ⅰ双酶切的原核表达载体pQE30中，构建了鸡α干扰素原核表达载体pQEIFN-α。转化感受态细胞JM109，经PCR、酶切鉴定并进行序列测定，结果表明，将ChIFN-α成熟肽蛋白基因正确地插入到原核表达载体pQE30的目的位点，重组质粒pQEIFN-α转化受体菌M15后用IPTG诱导，实现了鸡α干扰素在大肠杆菌M15中的表达，表达产物经SDS-PAGE电泳后在约20.0 Ku处出现了特异性条带，用1.0mmoL／LIPTG于37℃诱导8h表达产物最佳，用鼠抗鸡IFN-α单克隆抗体作一抗，辣根过氧化物酶标记的羊抗鼠抗体作二抗，Western blotting检测出现了特异性的条带。表达产物以包涵体形式存在，包涵体经变性、复性处理后，用细胞病变抑制法测定活性，结果表明重组鸡α干扰素在Vero细胞上抗VSV的病毒活性为1.16×10~3U／mg。
与人和哺乳动物比较，鸡IFN-α的研究相对来说比较滞后。本研究利用体外表达技术成功研制重组IFN-α蛋白，为研制抗鸡IFN-α的多克隆抗体和单克隆抗体以及ChIFN-α蛋白的生物学特性的研究奠定基础。
Domestic infective diseases which cause the tremendous economic losses to poultry industry, provoke the serious threat of poultry industry. 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.Therefore the research on the chicken interferon has the important meaning.
One pair of primers were designed according to the sequences of chicken Interferon-alpha published in GenBank. Chicken interferon-alpha gene were amplified from genome DNA extracted from liver tissue of luoman chicken, hainan chicken and gushi chicken, respectively,and were linked to pGEM-T Easy vector, then transformed to competent cells of JM109. By blue-white spot screening , idenfication of plasmid PCR and enzyme digestion,positive strain was sent to Takara for sequencing. And sequencing results showed that chicken Interferon-alpha gene of luoman chicken, hainan chicken and gushi chicken were obtained.The amplified fragment of ChIFN-α was 582bp in length, which includes one open-reading frame, encoding 162 amino acid residues..The results of homology analysis with other chicken interferon-alpha gene published previously in GenBank by DNAStar analysis reveal there was identity of 99.3% at nucleotide homology and that of 97.9% at amino acid homology.
On this basis,another pair of primers were designed. Maturity protein gene of IFN-α was amplified from pGEM- ChlFN-α and was digested by SphI and SalI,then was inserted into pQE30 of the prokaryotic expression vector which was digested by SphI and SalI, the recombinant expressed plasmid pQEIFN-α was constructured ,then transformed to competent cells of JM109. pQEIFN-α was identificated by PCR, enzyme digestion and DNA sequencing conclusively. The result indicated that the gene of encoding ChIFN-α maturity protein was inserted correctly to the target site of
引文
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