抗凋亡融合蛋白PTD-Bcl-xL原核表达载体的构建及表达纯化

Prokaryotic Expression Plasmid Construction and Expression/Purification of Anti-apoptotic Fusion Protein PTD-Bcl-xL

人工抗凋亡蛋白PTD-Bcl-x L能保护多种因素引起的细胞异常凋亡,为了获得高纯度Bcl-x L与PTD(Protein transduction domains)的融合蛋白,首先采用TRIzol法提取SD大鼠肝脏总RNA,将RNA反转录为c DNA,设计引物以c DNA为模板,PCR扩增Bcl-x L基因,构建p UM19-T-Bcl-x L质粒,并对质粒双酶切鉴定和测序鉴定;其次设计包含PTD序列的Bcl-x L引物,以测序正确的p UM19-T-Bcl-x L质粒为模板,PCR扩增PTD-Bcl-x L序列,将扩增序列克隆入p ET28a载体,构建PTD-Bcl-x L蛋白的原核表达质粒p ET28a-PTD-Bcl-x L,并对p ET28a-PTD-Bcl-x L载体双酶切鉴定和测序鉴定;将p ET28a-PTD-Bcl-x L重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达,并对IPTG诱导融合蛋白表达的浓度和诱导时间进行了优化;SDS-PAGE分析表达蛋白的可溶性情况,在变性条件下用Ni-NTA琼脂纯化融合蛋白;最后用SDS-PAGE、Western Blot及质谱对融合蛋白进行鉴定。结果表明:双酶切p UM19-T-Bcl-x L质粒出现约774 bp大小条带,p UM19-T-Bcl-x L质粒测序结果与NCBI数据库比对序列一致,表明成功构建p UM19-T-Bcl-x L质粒;双酶切p ET28a-PTD-Bcl-x L质粒出现约744 bp大小条带,p ET28a-PTD-Bcl-x L质粒测序结果与预期序列一致,表明成功构建了p ET28a-PTD-Bcl-x L原核表达载体;在IPTG诱导下p ET28a-Bcl-x L重组质粒在大肠杆菌BL21(DE3)中表达出36 k Da大小蛋白,最优IPTG诱导浓度为0.1 mmol/L,最佳IPTG诱导时间为6 h;SDS-PAGE电泳显示融合蛋白主要出现在菌液超声后的沉淀里,以包涵体形式表达,经Ni-NTA琼脂纯化获得了高纯度的融合蛋白;Western Blot和质谱鉴定证明IPTG诱导表达蛋白和纯化的融合蛋白为PTD-Bcl-x L蛋白。纯化得到了PTD-Bc L-x L融合蛋白,推进了PTD-Bcl-x L蛋白在猪、牛等家畜精液冷冻保存的应用进程。

Artificial anti-apoptotic protein PTD-Bcl-x L can control abnormal apoptosis induced by a variety of factors. The present study was to obtain a high-purity Bcl-x L and PTD（ Protein transduction domains） fusion protein. SD rat liver total RNA was extracted by TRIzol and transcribed into c DNA. Bcl-x L gene was amplified by PCR with c DNA as a template and was cloned into p UM19-T vector to construct p UM19-T-Bcl-x L plasmid,which was Identified by restriction enzyme digestion and sequencing and the p UM19-T-Bcl-x L plasmid was used as a template to amplify PTD-Bcl-x L fragment which was cloned into vector p ET28 a to construct recombinant plasmid p ET28aPTD-Bcl-x L and PTD sequence were designed to be placed before the Bcl-x L by designing primers. Then the recombinant plasmid was identified by restriction enzyme and was transformed into E. coli BL21（ DE3）,PTD-Bcl-x L fu-sion protein was induced to express with different IPTG concentration and induction time. Then the expression culture was analyzed for it＇s solubility and was prepared to purify PTD-Bcl-x L fusion protein with Ni-NTA agarose under denaturing condition. Finally,the expressing culture and purified protein was identified with SDS-PAGE analysis,Western Blot and MS. The results showed that detected by sequencing and enzyme digestion plasmid p UM19-TBcl-x L was constructed; prokaryotic expression vector p ET28a-PTD-Bcl-x L was constructed with confirmed by sequencing and enzyme digestion; The fused protein PTD-Bcl-x L could be expressed by IPTG induction with 0. 1mmol / L IPTG induction 6 hours for well expression; The fusion protein expressed in an insoluble form of inclusion bodies and a high-purity fused protein was obtained with Ni-NTA agarose purification; the expressing culture and purified protein were proved to be the PTD-Bcl-x L fusion protein with SDS-PAGE,Western Blot and MS analysis.This study obtains purified PTD-Bc L-x L fusion protein and promotes the application process PTD-Bcl-x L protein in pork,beef and other livestock semen cryopreservation.