| 猪圆环病毒2型核衣壳蛋白基因原核表达载体的构建及表达 |
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| 引用本文： | 崔立,朱建国,芦银华,谈国蕾,陈溥言,华修国.猪圆环病毒2型核衣壳蛋白基因原核表达载体的构建及表达[J].上海交通大学学报(农业科学版),2005,23(4):353-359. |
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| 作者姓名： | 崔立 朱建国 芦银华 谈国蕾 陈溥言 华修国 |
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| 作者单位： | 上海交通大学,农业与生物学院,上海,201101;南京农业大学,动物医学院,南京,210095 |
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| 基金项目： | 上海市科技兴农重点攻关项目(农科攻字2001第3-5) |
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| 摘 要： | 用PK-15细胞增殖PCV-2病毒，提取病毒DNA，用PCR方法扩增核衣壳蛋白（Cap蛋白）全基因，克隆到pET-32a载体中，构建了表达载体pETORF2，转化入大肠杆菌BL21中，用胛G进行诱导表达，结果显示表达的蛋白大小与设计不符。同样的方法，又构建了表达栽体pETRF2，含有Cap蛋白的部分基因，结果显示表达的蛋白大小依然与设计不符。对Cap蛋白编码基因进行改造，在不改变原氨基酸序列的基础上，把其编码密码子全部换成大肠杆菌偏爱密码子，根据此序列，设计了4条长引物，参照SOE的原理，用降落PCR的方法扩增出含Cap蛋白表位基因的片段，并克隆到pET-32a栽体中，成功构建了重组表达栽体pETP1P4。SDS-PAGE电泳结果表明，表达的蛋白分子量与设计相符。
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| 关 键 词： | 猪圆环病毒2型 Cap蛋白 大肠杆菌 表达 |
| 文章编号： | 1671-9964(2005)04-0353-07 |
| 修稿时间： | 2005年4月26日 |
Construction and Expression of Prokaryotic Expression Vector of Porcine Circovirus Type 2 Nucleocapsid Protein |
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| CUI Li,ZHU Jian-guo,LU Yin-hu,TAN guo-lei,CHEN Fu-yan,HUA Xiu-guo.Construction and Expression of Prokaryotic Expression Vector of Porcine Circovirus Type 2 Nucleocapsid Protein[J].Journal of Shanghai Jiaotong University (Agricultural Science),2005,23(4):353-359. |
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| Authors: | CUI Li ZHU Jian-guo LU Yin-hu TAN guo-lei CHEN Fu-yan HUA Xiu-guo |
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| Institution: | CUI Li1,ZHU Jian-guo1,LU Yin-hua2,TAN guo-lei2,CHEN Fu-yan2,HUA Xiu-guo1 |
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| Abstract: | Inoculated PCV-2 in PK-15 cells,the Cap gene was amplified by polymerase chain reaction(PCR)from the extracted virus DNA and cloned into pET32-a.The recombinant plasmid pETORF2 was constructed and transformed into the BL21.A fusion protein was induced with IPTG and expressed,but the molecular weight of the fusion protein was not accord with the expected.In the same way,pETRF2 was constructed and the part of Cap gene was included.The result indicated the same situation.So the Cap gene was altered.On the basis of to not alteration the ammonia,the sequence of codons was replaced by the major codons of E.coli.According to the changed sequence and the principle of gene SOEing,four primers were designed and the gene including the epitopes of Cap was amplified by the touchdown PCR.Then it was cloned into pET32-a,the recombinant plasmid pETP1P4 was constructed.The result of SDS-PAGE indicated the correct molecular weights with the expected. |
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| Keywords: | porcine circovirus type 2 Cap protein E coli expression |
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