| 摘要: |
| 【目的】克隆鸡传染性法氏囊病病毒(IBDV) BJ-10株VP2基因，构建其原核表达载体，并在大肠埃希菌中进行高效表达。【方法】根据IBDV VP2基因序列设计1对特异性引物，应用RT-PCR技术克隆IBDV BJ-10株VP2基因；将目的基因插入pMD18-T连接载体，筛选出阳性重组质粒；将该质粒克隆至原核表达载体pET-32α中，转化入JM109，经IPTG诱导表达，对产物进行SDS-PAGE电泳分析，确定IPTG的最佳诱导表达时间和浓度。【结果】克隆到了全长1 356 bp的IBDV BJ-10株VP2基因；PCR、酶切鉴定分析表明，成功构建了重组质粒pET-32α-VP2；SDS-PAGE电泳分析表明，在宿主菌JM109中成功表达了约为60 ku的VP2蛋白；IPTG诱导表达时间以4.5 h为宜，诱导最佳浓度为0.8 mmol/L。【结论】成功克隆了IBDV BJ-10株VP2基因，并在大肠埃希菌中表达了VP2蛋白。 |
| 关键词: 鸡传染性法氏囊病病毒 VP2基因 基因克隆 原核表达载体 |
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| 基金项目:渭南市基础研究计划项目(2011JH-6) |
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| Clone and expression of VP2 gene of infectious bursal disease virus BJ-10 isolate in Escherichia coli |
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| Abstract: |
| 【Objective】This study aimed to clone the VP2 gene of infectious bursal disease virus (IBDV) BJ-10 isolate,construct the prokaryotic expression vectors,and express them in Escherichia coli.【Method】A pair of specific primers were designed according to the sequences of IBDV BJ-10 strain,and the VP2 gene was cloned by RT-PCR.The IBDV VP2 gene was inserted into ligation vector pMD18-T and the recombinant plasmid was selected.The recombinant plasmid was cloned into the prokaryotic expression vector pET32α and transformed into E.coli JM109.Then it was highly expressed by inducing with IPTG and proved by SDS-PAGE electrophoretic analysis,to designate the best inducement time and concentration.【Result】The IBDV BJ-10 VP2 gene with 1 356 bp was cloned.From the PCR and identification by enzyme digestion analysis,the recombinant plasmid pET-32α-VP2 was constructed.SDS-PAGE analysis indicated that the VP2 protein 60 ku in size was expressed properly from E.coli JM109.The best IPTG inducement time was 4.5 h,and the best concentration was 0.8 mmol/L.【Conclusion】The IBDV BJ-10 isolate VP2 gene was successfully cloned,and the VP2 protein was expressed in E.coli. |
| Key words: infectious bursal disease virus (IBDV) VP2 gene gene cloning prokaryotic expression vector |
