藏猪IGF-1成熟肽基因的克隆及原核表达
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- 英文论文题名：Cloning of Tibetan pig IGF-1 Mature Peptide Gene and Its Prokaryotic Expression
- 论文作者：张飞燕 ; 王振华 ; 张钧利 ; 潘康成 ; 唐慧琴 ; 谷笑笑 ; 李伟
- 英文论文作者：Feiyan Zhang ; Zhenhua Wang ; Junli Zhang ; Kangcheng Pan ; Huiqin Tang ; Xiaoxiao Gu ; Wei Li ; Animal Microecology Institute ; College of Veterinary Medicine ; Sichuan Agricultural University ; Chengdu agricultural science and technology Career Academy ; Wuxi Arbaor Biology Engineering Co.LTD ; Jiangsu
- 年：2016
- 作者机构：四川农业大学动物医学院动物微生态研究中心;成都农业科技职业学院;江苏无锡阿尔宝尔生物工程有限公司;
- 论文关键词：藏猪 ; 胰岛素样生长因子1 ; 原核表达 ; 蛋白纯化 ; 蛋白质印迹法
- 英文论文关键词：Tibetan pig ; IGF-1 ; Prokaryotic expression ; Protein purification ; Western Blot
- 会议召开时间：2016-11-04
- 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
- 语种：中文
- 分类号：S828
- 学会代码：ZGXJ
- 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
- 会议地点：中国山东青岛
- 主办单位：中国畜牧兽医学会动物微生态学分会
- 学会名称：中国畜牧兽医学会
- 页数：1
- 文件大小：86k
- 原文格式：O
- 会议级别：全国
摘要
旨在克隆藏猪胰岛素样生长因子1(IGF-1)的成熟肽基因,并进行原核表达的研究。提取藏猪肝脏组织RNA,通过RT-PCR扩增出藏猪IGF-1基因,构建重组质粒pMD19-T-IGF-1,以pMD19-T-IGF-1质粒为模板,克隆IGF-1成熟肽序列并构建成熟肽pET-32a-IGF-1表达质粒,转入大肠杆菌BL21(DE3),对IPTG诱导剂浓度和诱导时间进行优化,Ni-NTA琼脂纯化融合蛋白后采用Western blot对其鉴定。结果显示,获得了藏猪IGF-1成熟肽基因序列(315bp)并成功构建原核表达载体pET-32a-IGF-1;重组大肠杆菌BL21-IGF1以包涵体形式表达出分子量约31KDa融合蛋白,最优IPTG浓度为0.5mmol/L,最佳IPTG诱导时间为10h;纯化获得了高纯度的融合蛋白,经鉴定IPTG诱导表达和纯化的蛋白为IGF-1融合蛋白。结果表明成功构建了表达质粒pET32a-IGF-1及获得重组大肠杆菌BL21-IGF1,表达了藏猪IGF-1成熟肽融合蛋白及该蛋白具有抗原抗体反应活性。
The research was amied to clone IGF-1 mature peptide gene from Tibetan pig and study the IGF-1 prokaryotic expression.The total RNA was extracted by using Trizol from the Tibetan pig liver and used as template to amplify IGF-I gene by RT-PCR,the amplified fragments was cloned into pMD19-T vector toconstruct pMD19T-IGF-I plasmid,which was identified by sequencing.The pMD19T-IGF-I plasmid was used as a template to amplify IGF-1 mature peptide fragment which was cloned into vector pET32 a to obtain recombinant plasmid pET-32a-IGF-l and transformed into E.coli BL21(DE3).The pET-32a-IGF-1fusion 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 pET-32a-IGF-l fusion protein with Ni-NTA Sefinose~(TM) Resin.Finally,the expressing culture and purified protein was identified with SDS-PACE analysis and Western Blot.The Tibetan pig IGF-1 mature peptide gene was 315 bp,Restriction enzvme mapping and sequencing showed that expression vector as constructed successfully.The pET-32a-IGF-l fusion protein induced in E.coli BL21(DE3) and could be expressed by IPTG induction with 0.5mmol/L IPTC induction 10 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 pET-32a-IGF-1 fusion protein with SDS-PACE and Western Blot analysis.IGF-1 mature peptide gene was cloned and expressed,The fusion protein has the antigen antibody reactivity.
The research was amied to clone IGF-1 mature peptide gene from Tibetan pig and study the IGF-1 prokaryotic expression.The total RNA was extracted by using Trizol from the Tibetan pig liver and used as template to amplify IGF-I gene by RT-PCR,the amplified fragments was cloned into pMD19-T vector toconstruct pMD19T-IGF-I plasmid,which was identified by sequencing.The pMD19T-IGF-I plasmid was used as a template to amplify IGF-1 mature peptide fragment which was cloned into vector pET32 a to obtain recombinant plasmid pET-32a-IGF-l and transformed into E.coli BL21(DE3).The pET-32a-IGF-1fusion 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 pET-32a-IGF-l fusion protein with Ni-NTA Sefinose~(TM) Resin.Finally,the expressing culture and purified protein was identified with SDS-PACE analysis and Western Blot.The Tibetan pig IGF-1 mature peptide gene was 315 bp,Restriction enzvme mapping and sequencing showed that expression vector as constructed successfully.The pET-32a-IGF-l fusion protein induced in E.coli BL21(DE3) and could be expressed by IPTG induction with 0.5mmol/L IPTC induction 10 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 pET-32a-IGF-1 fusion protein with SDS-PACE and Western Blot analysis.IGF-1 mature peptide gene was cloned and expressed,The fusion protein has the antigen antibody reactivity.
引文
[1]Tse M C L,Chan K M,Cheng C H K.Cloning,characterization and promoter analysis of the common carp IGF-II,gene[J].Gene,2008,412(1-2):26-38.