| 鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建 |
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| 引用本文： | 朱艳平,何勇,刘佳,邢瑞林,常乐凯,李润芷,岳锋,吴玉苹,李鹏,孙国鹏,张艳芳,王选年.鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建[J].中国畜牧兽医,2019,46(10):2860-2866. |
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| 作者姓名： | 朱艳平 何勇 刘佳 邢瑞林 常乐凯 李润芷 岳锋 吴玉苹 李鹏 孙国鹏 张艳芳 王选年 |
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| 作者单位： | 1. 新乡学院生命科学技术学院生物技术研究中心, 新乡 453003;2. 郑州大学生命科学技术学院, 郑州 450000;3. 河南科技学院动物科学学院, 新乡 453003 |
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| 基金项目： | 河南省科技攻关项目（182102110221）；新乡学院青年骨干教师资助计划项目（GGJS2016-06）；新乡学院科技创新团队项目（XXUTD20170106）；河南省教育厅自然科学重点研究项目（19A230009） |
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| 摘 要： | 为获得高活性的鸡传染性法氏囊病病毒（infectious bursal disease virus，IBDV）VP2蛋白的重组蛋白，本试验对IBDV VP2蛋白基因与能进行自我组装的幽门螺杆菌（Hp）铁蛋白基因进行融合PCR扩增，获得其融合基因。根据成熟VP2蛋白基因cDNA序列和Hp铁蛋白基因的碱基序列，设计合成2对引物，应用融合PCR技术扩增获得融合基因片段VP2-Fe。将目的基因克隆至pMD18-T载体筛选阳性重组克隆质粒，然后将目的基因亚克隆至真核表达载体pPICZaC后转化大肠杆菌DH5α感受态细胞，筛选获得阳性表达质粒。结果显示，通过两轮PCR扩增出长度为1 824 bp的融合基因VP2-Fe，亚克隆至pMD18-T载体，测序结果显示，融合基因无任何碱基的突变，筛选的阳性质粒命名为pMD-VP2-Fe。双酶切回收目的基因大小为1 824 bp，亚克隆至pPICZaC，PCR和双酶切鉴定出现预期大小的片段，将获得的阳性表达质粒命名为pPICZaC-VP2-Fe。本研究为后期利用真核表达载体获得其融合重组蛋白奠定基础。
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| 关 键 词： | 传染性法氏囊病病毒(IBDV) VP2基因 幽门螺杆菌 铁蛋白 融合PCR |
| 收稿时间： | 2019-04-29 |
Fusion PCR of Chicken Infectious Bursal Disease Virus VP2 and Helicobacter pylori Ferritin Fusion Gene and Construction of Its Eukaryotic Expression Vector |
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| ZHU Yanping,HE Yong,LIU Jia,XING Ruilin,CHANG Lekai,LI Runzhi,YUE Feng,WU Yuping,LI Peng,SUN Guopeng,ZHANG Yanfang,WAGN Xuannian.Fusion PCR of Chicken Infectious Bursal Disease Virus VP2 and Helicobacter pylori Ferritin Fusion Gene and Construction of Its Eukaryotic Expression Vector[J].China Animal Husbandry & Veterinary Medicine,2019,46(10):2860-2866. |
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| Authors: | ZHU Yanping HE Yong LIU Jia XING Ruilin CHANG Lekai LI Runzhi YUE Feng WU Yuping LI Peng SUN Guopeng ZHANG Yanfang WAGN Xuannian |
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| Institution: | 1. Center for Biotechnology Research, College of Life Science and Technology, Xinxiang University, Xinxiang 453003, China;2. School of Life Science, Zhengzhou University, Zhengzhou 450000, China;3. College of Animal Sciences, Henan Institute of Science and Technology, Xinxiang 453003, China |
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| Abstract: | In order to obtain the recombinant protein of VP2 protein of infectious bursal disease virus (IBDV) with high activity,the fusion of IBDV VP2 protein and self-assembled Helicobacter pylori (Hp) ferritin gene were amplified by fusion PCR.Two pairs of primers were designed and synthesized according to the DNA sequence of mature VP2 protein gene and the base sequence of Hp ferritin.The fusion gene fragment VP2-Fe was amplified by fusion PCR.The target gene was cloned into pMD18-T vector and the positive recombinant plasmid was screened.Then the target gene was subcloned into eukaryotic expression vector pPICZaC and transformed into competent cells of Escherichia coli DH5α,the positive expression plasmids were screened.The results showed that the fusion gene VP2-Fe with the length of 1 824 bp was amplified by two rounds of PCR and subcloned to pMD18-T vector.Sequencing results showed that there was no mutation in the fusion gene.The positive plasmid was named as pMD-VP2-Fe.The target gene (1 824 bp) was recovered by double digestion and subcloned to pPICZaC.The expected size fragment was identified by PCR and double digestion.The positive expression plasmid was named as pPICZaC-VP2-Fe.This study laid a foundation for the later use of eukaryotic expression vector to obtain its fusion recombinant protein. |
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| Keywords: | IBDV VP2 gene Helicobacter pylori (Hp) ferritin fusion PCR |
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