| 抗凋亡融合蛋白PTD-Bcl-xL原核表达载体的构建及表达纯化 |
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| 引用本文： | 王晓晔,石博妹,王英群,李珣,刘徳玉,李铭,李芳芳,胡传活.抗凋亡融合蛋白PTD-Bcl-xL原核表达载体的构建及表达纯化[J].华北农学报,2016(1):1-7. |
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| 作者姓名： | 王晓晔 石博妹 王英群 李珣 刘徳玉 李铭 李芳芳 胡传活 |
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| 作者单位： | 1. 广西大学 动物科学技术学院,广西 南宁,530005;2. 广西畜禽品种改良站,广西 南宁,530001 |
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| 基金项目： | 广西科学研究与技术开发计划项目(桂科能1598022－2)；广西畜禽品种改良站横向科技项目(20140220) |
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| 摘 要： | 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。
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| 关 键 词： | Bcl-xL 蛋白 PTD 原核表达 蛋白纯化 |
Prokaryoti c Expression Plasmid Construction and Expression/Purification of Anti-a poptot ic Fu sion Protein PTD-Bcl-xL |
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| 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 pro-tein.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 I-dentified 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 recom-binant plasmid was identified by restriction enzyme and was transformed into E.coli BL21(DE3),PTD-Bcl-xL fu-sion protein was induced to express with different IPTG concentration and induction time.Then the expression cul-ture was analyzed for it′s solubility and was prepared to purify PTD-Bcl-xL fusion protein with Ni-NTA agarose un-der denaturing condition.Finally,the expressing culture and purified protein was identified with SDS-PAGE analy-sis,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 se-quencing 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. |
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| Keywords: | Bcl-xL protein PTD Prokaryotic expression Protein purification |
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