摘要： 人工抗凋亡蛋白PTD-Bcl-xL能保护多种因素引起的细胞异常凋亡,为了获得高纯度Bcl-xL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cDNA,设计引物以cDNA为模板,PCR扩增Bcl-xL基因,构建pUM19-T-Bcl-xL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-xL引物,以测序正确的pUM19-T-Bcl-xL质粒为模板,PCR扩增PTD-Bcl-xL序列,将扩增序列克隆入pET28a载体,构建PTD-Bcl-xL蛋白的原核表达质粒pET28a-PTD-Bcl-xL,并对pET28a-PTD-Bcl-xL载体双酶切鉴定和测序鉴定;将pET28a-PTD-Bcl-xL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pUM19-T-Bcl-xL质粒出现约774 bp大小条带,pUM19-T-Bcl-xL质粒测序结果与NCBI数据库比对序列一致,表明成功构建pUM19-T-Bcl-xL质粒;双酶切pET28a-PTD-Bcl-xL质粒出现约744 bp大小条带,pET28a-PTD-Bcl-xL质粒测序结果与预期序列一致,表明成功构建了pET28a-PTD-Bcl-xL原核表达载体;在IPTG诱导下pET28a-Bcl-xL重组质粒在大肠杆菌BL21(DE3)中表达出36 kDa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-xL蛋白。纯化得到了PTD-BcL-xL融合蛋白,推进了PTD-Bcl-xL蛋白在猪、牛等家畜精液冷冻保存的应用进程。

关键词: Bcl-xL蛋白, PTD, 原核表达, 蛋白纯化

Abstract: Artificial anti-apoptotic protein PTD-Bcl-xL can control abnormal apoptosis induced by a variety of factors.The present study was to obtain a high-purity Bcl-xL and PTD (Protein transduction domains) fusion protein.SD rat liver total RNA was extracted by TRIzol and transcribed into cDNA.Bcl-xL gene was amplified by PCR with cDNA as a template and was cloned into pUM19-T vector to construct pUM19-T-Bcl-xL plasmid,which was Identified by restriction enzyme digestion and sequencing and the pUM19-T-Bcl-xL plasmid was used as a template to amplify PTD-Bcl-xL fragment which was cloned into vector pET28a to construct recombinant plasmid pET28a-PTD-Bcl-xL and PTD sequence were designed to be placed before the Bcl-xL by designing primers.Then the recombinant plasmid was identified by restriction enzyme and was transformed into E.coli BL21(DE3),PTD-Bcl-xL fusion 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-xL 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 pUM19-T-Bcl-xL was constructed;prokaryotic expression vector pET28a-PTD-Bcl-xL was constructed with confirmed by sequencing and enzyme digestion;The fused protein PTD-Bcl-xL could be expressed by IPTG induction with 0.1 mmol/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-xL fusion protein with SDS-PAGE,Western Blot and MS analysis.This study obtains purified PTD-BcL-xL fusion protein and promotes the application process PTD-Bcl-xL protein in pork,beef and other livestock semen cryopreservation.

Key words: Bcl-xL protein, PTD, Prokaryotic expression, Protein purification

中图分类号: 

• Q78