猪γ干扰素基因的克隆表达及其抗原性的初步研究

Cloning and expression of porcine interferon-gamma gene and its antigenicity

为研究猪γ干扰素(poIFN-γ)的生物学活性及其在机体免疫应答中的作用机理,克隆并表达了猪IFN-γ基因。首先应用RT-PCR方法通过一对自行设计的引物从猪脾脏淋巴细胞中扩增了猪γ干扰素基因片段,分别将其定向插入原核表达载体pGEX-4T-1和pET-32 a中,经测序表明与已报道的核苷酸序列同源性为100%。接着将重组质粒pGEX-poIFN-γ和pET-poIFN-γ分别转化大肠杆菌BL21株,经IPTG诱导,得到高效表达。所表达的融合蛋白均以不溶性的包涵体形式存在,分子量分别为43.0和35.0 ku,与理论推算值相符。为研究IFN-γ在表达中结构的变化,采用上述2种融合蛋白分别作为免疫抗原和检测抗原。结果用GST-poIFN-γ免疫小鼠所诱导的抗体可与poIFN-γ产生特异性结合,表明不同原核质粒表达的猪IFN-γ仍保持其特有的结构。

In order to research the biological function and the mechanism of the porcine interferon-gamma （poIFN-γ） in immune response,poIFN-γ gene was amplified from spleen lymphocytes by RT-PCR with a pair of designed primers and then the gene was inserted into the prokaryotic expression vector pGEX-4T-1 and pET-32a. The results indicated that the nucleotide sequence of the target gene showed 100,% similarity to the corresponding gene in GenBank. The recombinant plasmid pGEX-poIFN-γ and pET-poIFN-γ were transformed into E. coli BL21strain and highly expressed by IPTG. The fusion protein solubility was identificated and the result indicated that the expressed proteins were located in inclusion bodies and the molecular weight were about 43.0 and 35.0 ku as expected. The GST-poIFN-γ fusion protein was injected into mice and the induced antibody was specific for poIFN-γ.