鸡TLR3胞外区基因克隆、表达及多克隆抗体的制备

Gene cloning and expression of the chicken toll-like receptor 3 extracellular domain and preparation of its polyclonal antibody

为了研究Toll样受体3(chTLR3)在病毒感染过程中的作用,试验采用RT-PCR技术从雏鸡法氏囊组织中扩增chTLR3胞外区部分基因片段(大小为1 317 bp),将其克隆于表达载体pET30a(+)中,经酶切鉴定后转化大肠杆菌Rosetta(DE3)感受态细胞,然后进行IPTG诱导表达;用纯化的重组蛋白免疫新西兰大白兔,获得抗血清;采用酶联免疫吸附试验(ELISA)、免疫印迹(Western-blot)及体外瞬时转染方法进行检测。结果表明:该基因片段得到较高表达,融合蛋白的分子质量约为55.8 ku,主要以包涵体形式存在,试验制备的兔抗chTLR3抗血清效价在1∶32 768以上,具有良好的免疫反应特性。

To study the role of chicken toll -like receptor 3 （chTLR3） in the process of viral infection, a 1 317 bp partial gent fragment was amplified from the bursa of fabricius in chicks by using RT - PCR, which was from the extracellular region of ehieken Toll - like rceeptor 3 se- quence, and then cloned into the expression vector pET - 30a （ ＋ ）. The reombinant plasmid was indentified by rcstrietion endonuclease anal- ysis,and transformed to E. coli Rosetta（DE3） competent cells, and then the expression was induced by IPTG. The purified recombinant protein was used to immunize New Zealand white rabbits to obtain the antisera, and then the detections were carried out by enzyme - linked immunosor- bent assay （ELISA） and Western - blot and in vitro transient transfection method. Ttle results showed that the gene fragment was highly expres- sion, and the fusion protein had a molecular weight of 55.8 ku, which was mainly in the form of inclusion body. The antisermn titer of rabbit antibody against chTLR3 was greater than about 1：32 768, which has a good eharacteristie of the immune response.