尼罗罗非鱼Ly75基因的克隆、原核表达和多克隆抗体制备

Cloning,prokaryotic expression and polyclonal antibody preparation of lymphocyte antigen 75 in Oreochromis niloticus

[目的]获得尼罗罗非鱼淋巴细胞抗原75(OnLy75)基因的cDNA序列和多克隆抗体。[方法]采用RACE技术克隆OnLy75基因的cDNA全序列,通过DNAMAN、MEGA等软件分析其序列特征;构建OnLy75的原核表达载体,并对其重组蛋白进行原核诱导表达;将纯化后的OnLy75重组蛋白免疫新西兰大耳白兔,并通过Western Blot技术检测多克隆抗体的效果。[结果]OnLy75的cDNA序列全长为6 632 bp,开放阅读框5 199 bp,编码1 732个氨基酸。其OnLy75重组蛋白主要存在于包涵体中;将重组蛋白纯化后免疫新西兰大耳白兔,成功获得了兔抗OnLy75多克隆抗体。用该多抗对尼罗罗非鱼不同器官进行Western Blot内源性检测,结果显示该蛋白在精巢中有丰富的表达。[结论]兔抗OnLy75多克隆抗体的成功制备,为进一步深入研究OnLy75在尼罗罗非鱼组织中的定位及功能研究提供了工具。

[ Objective ] To clone the Lymphocyte antigen 75 gene of Oreochromis nilotcus （ OnLy75 ） and obtain the polyclonal antibody of OnLy75. [ Methods] The full -length eDNA sequence of OnLy75 was cloned by RACE approach. The physieo- chemical properties of OnLy75 were analyzed by the software of DNAMAN and MEGA. Then a prokaryotic expression vector of OnLy75 was constructed and the OnLy75 recombinant protein was induced by IPTG. To obtain the polyclonal antibody, the puri- fied OnLy75 recombinant protein was used to immunize New Zealand white rabbits. In addition, the polyclonal antibody was tested by Western Blot in different organs of Nile tilapia. [ Results] The full length of OnLy75 cDNA sequence was 6 632 bp with an open reading frame （ORF） of 5 199 bp,encoding 1 732 amino acid residues. The OnLy75 recombinant protein was mainly obtained from the inclusion body. After the purified recombinant protein was immunized with New Zealand white rabbits, the rabbit anti - OnLy75 polyclonal antibody was successfully obtained. The polyclonal antibody was tested by Western Blot in different organs of Nile tilapia. The results showed that the polyclonal antibody was highly expressed in the testis. [ Conclusion] Rabbit anti - OnLy75 polyclonal antibody was successfully prepared to further study the localization and function of OnLy75 in Nile tilapia.