目前已有研究指出，細菌DNA中所含之未甲基化CpG motifs可活化脊椎動物免疫系統。在本實驗室先前研究中將E.coli DNA作為刺激物，使金目鱸魚(Lates calcarifer)頭腎細胞產生細胞增生因子(proliferation inducing factors; PIFs)促進細胞增生。本實驗主要目的為將鱸魚頭腎細胞經E.coli DNA刺激過後之上清液為抗原製作多株抗體，利用其抗原抗體親和力特性，試圖以抗體辨識細胞上清液中所含PIFs後，測試移除細胞上清液中PIFs對於頭腎白血球細胞增生影響。當抗血清可有效移除PIFs時，應會抑制上清液促進細胞增生之作用。本研究結果顯示，在雙向免疫沉澱試驗中可發現，抗血清可有直接效辨識上清液抗原出現沉澱線。抗血清中之多株抗體可辨識細胞表面分子以及成奶予M細胞所分泌之PIFs。後續研究可以延伸針對上清液製備多株抗體單株化後，以單株抗體各別篩選分離其蛋白質並進行定序，了解PIFs中主要影響細胞生長之蛋白質為何。 Bacterial genimic DNA contains unmethylated CpG motifs can activate the vertebrate immune system, and it has been reported CpG oligodeoxyribonucleotides may activate the osteichthyes immune system effectively. Previous study in our laboratory showed E.coli DNA was able to induce sea bass(Lates calcarifer) head kidney cells to proliferate and produce proliferation inducing factors (PIFs).
In this study, polyclonal antibodies were produced to neutralize the PIFs produced by E.coli DNA treated sea bass head kidney cells. When PIFs were depleted, the proliferation responses induced by PIFs supposed to be suppressed. The polyclonal antibodies(pAbs) obtained from immunized mice could form a clear precipitation line in double immunodiffusion. Antibodies also recognized head kidney cell surface markers and successfully neutralize the effect of PIFs. PIFs are able to be purified and sequenced by monoclonal antibodies for identification in the future.
