紫贻贝LC3B蛋白的原核表达及抗血清制备

Recombinant expression and polyclonal antibody preparation of Mytilus galloprovincialis LC3B

LC3B是检测自噬程度的标志性分子,但是在低等动物体内缺少特异性强的LC3B抗体。为研究贝类的自噬性细胞死亡,本研究通过将紫贻贝的LC3B编码序列克隆到pET-32a原核表达载体中构建重组表达载体,进而对IPTG诱导浓度、诱导表达时间进行摸索;采用亲和层析对重组蛋白进行纯化,并采用SDS-PAGE检测及Western blot进行验证;利用获得的重组蛋白免疫新西兰兔,制备其多克隆抗体。结果显示,在20℃,转速为150 r/min条件下,当IPTG浓度为0.6 mmol/L,诱导表达10 h后,可以得到高表达量、可溶性的重组MgLC3B-His融合蛋白,亲和层析纯化后,获得单一条带的MgLC3B-His可溶性重组蛋白;采用纯化后的MgLC3B-His融合蛋白对新西兰兔进行多次免疫,采集分离兔抗血清并利用protein A纯化,获得紫贻贝的LC3B多克隆抗体,效价为25 600。紫贻贝LC3B多克隆抗体的成功制备,为今后深入开展紫贻贝及相近物种的细胞自噬研究奠定了基础。

Autophagy is a cellular process for degradation of damaged proteins and organelles via forming autophagosome. Microtubule-associated protein 1 light chain 3 B（LC3B） is a marker protein for autophagy detection. However, in many lower animals, autophagy detection is limited for a lack of available specific LC3B antibody. In the present study, the coding sequence of LC3B of Mytilus galloprovincialis was cloned and inserted into a pET-32a prokaryotic expression vector. To obtain a high-level and soluble expression of recombinant MgLC3B-His protein, the IPTG concentration and induction time were investigated. Then, the purification conditions of this recombinant protein were also studied. Our results showed that MgLC3B was inserted in the pET-32a prokaryotic expression vector successfully. The recombinant MgLC3B-His protein was induced at 20 ℃,150 r/min with IPTG concentration of 0.6 mmol/L after 10 h culture. A single-band recombinant protein was obtained after affinity purification. After immune injection, and the rabbit polyclonal antibody was also obtained,with a titer of 25 600. Therefore, our results will be helpful for the investigation of autophagy in mussels and similar species.