紫贻贝LC3B蛋白的原核表达及抗血清制备

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。

Recombinant expression and polyclonal antibody preparation of Mytilus galloprovincialis LC3B

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 ofMytilus 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.