牛γ干扰素基因克隆和表达及多克隆抗体制备
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摘要
牛γ干扰素具有较强的抗病毒、抗肿瘤和免疫调节作用,作为生物药剂和疫苗佐剂具有广阔的应用前景。本试验成功地克隆、表达和纯化了BoIFN-γ,并制备了兔抗BoIFN-γ的多克隆抗血清,为BoIFN-γ生物学特性及其应用研究奠定了基础。
本研究采用刀豆蛋白A刺激牛脾脏淋巴细胞总RNA,设计合成特异引物,采用反转录聚合酶链式反应(Reverse Transcriptase-PCR,RT-PCR)技术扩增和克隆BoIFN-γ基因,与pMD18-T载体连接后进行酶切、PCR以及序列测定和分析。结果表明,所克隆的BoIFN-γ基因全长498bp,编码166个氨基酸,与GenBank发布的羊、猪、人、鼠和鸡的IFN-γ核苷酸序列进行比较,其同源率分别为81.3%、74.9%、73.6%、53.7%和50.7%。与GenBank上发布的其它BoIFN-γ基因核苷酸序列进行比较,其同源率为100%。
将BoIFN-γ基因亚克隆到表达载体pET-30a(+)中,构建重组表达质粒pET-BoIFN-γ并转化大肠杆菌BL21,于37℃经0.5mmol/L IPTG诱导表达6h,SDS-PAGE和Western blot分析表明BoIFN-γ基因在大肠杆菌中获得了高水平表达,表达的融合蛋白以分子量约为25ku,且表达产物占菌体总蛋白的35%。利用镍离子亲和树脂进行纯化得到纯化的目的蛋白浓度为1.6mg/ml,将纯化蛋白免疫新西兰白兔,制备了BoIFN-γ多克隆抗血清,经琼脂扩散实验和Western blot鉴定证明所得到的多克隆抗血清与表达的融合蛋白具有良好的免疫反应性。
Bovine Interferon-gamma (BoIFN-γ) has the characteristics of virustatic, antitumor and immunoloregulation function. This study succeeded in cloning, expression and purification of the recombinant Bovine IFN-γfusion protein, and preparation of rabbit polyclonal antibody against BoIFN-γ. This provides a solid foundation for further research on BoIFN-γbiological properties and its applications.
Bovine spleen lymphocytes were stimulated with ConA, then total RNA was extracted and used as template to amplify the IFN-γgene with the method of RT-PCR. The amplified fragment was subsequently ligated into the pMD18-T vector for sequencing. The results revealed that the IFN-γgene is 498bp longth, which can encode 166 amino acid. Comparison of the sequenced IFN-γwith the reported BoIFN-γsequence in GenBank revealed 100% identity at the nucleotide level. And shared 81.3%, 74.9%, 73.6%, 53.7%, 50.7% identities with goat, pig, human, mice, chicken IFN-γgene in nucleotide, respectively.
The BoIFN-γgene was subcloned into an expression vector pET-30a (+) and constructed the recombinant expression plasmid of pET-BoIFN-γ. Then it was transformed into E.coli BL21 cells, and induced by 0.5mmol/L IPTG at 37℃for 6 hours. The recombinant BoIFN-γwas expressed efficiently in the form of fusion protein with the yield accounting for 33% of total bacterial proteins. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight of approximate 25ku. After purification, the target protein was harvested with a concentration of 1.6mg/ml. And the polyclonal antibody against BoIFN-γwere generated by immunizing rabbits with the purified protein. The AGP and western blot experiment showed that the antibody could react with purified recombinant BoIFN-γprotein.
本研究采用刀豆蛋白A刺激牛脾脏淋巴细胞总RNA,设计合成特异引物,采用反转录聚合酶链式反应(Reverse Transcriptase-PCR,RT-PCR)技术扩增和克隆BoIFN-γ基因,与pMD18-T载体连接后进行酶切、PCR以及序列测定和分析。结果表明,所克隆的BoIFN-γ基因全长498bp,编码166个氨基酸,与GenBank发布的羊、猪、人、鼠和鸡的IFN-γ核苷酸序列进行比较,其同源率分别为81.3%、74.9%、73.6%、53.7%和50.7%。与GenBank上发布的其它BoIFN-γ基因核苷酸序列进行比较,其同源率为100%。
将BoIFN-γ基因亚克隆到表达载体pET-30a(+)中,构建重组表达质粒pET-BoIFN-γ并转化大肠杆菌BL21,于37℃经0.5mmol/L IPTG诱导表达6h,SDS-PAGE和Western blot分析表明BoIFN-γ基因在大肠杆菌中获得了高水平表达,表达的融合蛋白以分子量约为25ku,且表达产物占菌体总蛋白的35%。利用镍离子亲和树脂进行纯化得到纯化的目的蛋白浓度为1.6mg/ml,将纯化蛋白免疫新西兰白兔,制备了BoIFN-γ多克隆抗血清,经琼脂扩散实验和Western blot鉴定证明所得到的多克隆抗血清与表达的融合蛋白具有良好的免疫反应性。
Bovine Interferon-gamma (BoIFN-γ) has the characteristics of virustatic, antitumor and immunoloregulation function. This study succeeded in cloning, expression and purification of the recombinant Bovine IFN-γfusion protein, and preparation of rabbit polyclonal antibody against BoIFN-γ. This provides a solid foundation for further research on BoIFN-γbiological properties and its applications.
Bovine spleen lymphocytes were stimulated with ConA, then total RNA was extracted and used as template to amplify the IFN-γgene with the method of RT-PCR. The amplified fragment was subsequently ligated into the pMD18-T vector for sequencing. The results revealed that the IFN-γgene is 498bp longth, which can encode 166 amino acid. Comparison of the sequenced IFN-γwith the reported BoIFN-γsequence in GenBank revealed 100% identity at the nucleotide level. And shared 81.3%, 74.9%, 73.6%, 53.7%, 50.7% identities with goat, pig, human, mice, chicken IFN-γgene in nucleotide, respectively.
The BoIFN-γgene was subcloned into an expression vector pET-30a (+) and constructed the recombinant expression plasmid of pET-BoIFN-γ. Then it was transformed into E.coli BL21 cells, and induced by 0.5mmol/L IPTG at 37℃for 6 hours. The recombinant BoIFN-γwas expressed efficiently in the form of fusion protein with the yield accounting for 33% of total bacterial proteins. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight of approximate 25ku. After purification, the target protein was harvested with a concentration of 1.6mg/ml. And the polyclonal antibody against BoIFN-γwere generated by immunizing rabbits with the purified protein. The AGP and western blot experiment showed that the antibody could react with purified recombinant BoIFN-γprotein.
引文
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