菜豆荚斑驳病毒外壳蛋白基因的克隆与原核表达

Cloning and Prokaryotic Expression of Coat Protein Gene from Bean Pod Mottle Virus

[目的]克隆菜豆荚斑驳病毒(BPMV)外壳蛋白(CP)基因,并对其进行原核表达。[方法]利用RT-PCR方法克隆BPMV CP基因,将其连接至pMD19-T Simple载体后对阳性克隆进行测序,检测其与已知病毒外壳蛋白基因序列的同源性;将CP基因定向插入双酶切的pET30a,构建其原核表达载体并转化大肠杆菌BL-21表达重组蛋白。[结果]试验克隆得到的CP基因大小为1 122 bp,与NCBI中目标基因的相似度达99%以上;试验成功构建了原核表达载体pET30a-BPMV CP,发现重组蛋白在37℃、1.0 mmol/L IPTG诱导4 h条件下表达量最高。[结论]该研究为BPMV抗血清的制备与液相芯片检测方法的建立奠定了基础。

[Objective] The paper was to clone the coat protein（CP） gene from bean pod mottle virus（BPMV） and express it in prokaryote.[Method] The BPMV CP gene was obtained by using RT-PCR method,and it was connected to the pMD19-T Simple vector to sequence the positive clones and detect the homology between the CP gene and the known virus coat protein gene sequence.The CP gene was inserted into the double enzyme digestive pET30a to construct the prokaryotic expression vector,and the recombinant protein was expressed after transformed into Escherichia coli BL-21.[Result] The size of the obtained CP gene was 1 122 bp,and the similarity with the NCBI target gene exceeded 99%;the prokaryotic expression vector pET30a-BPMV CP was constructed successfully,and it found that the recombinant protein had highest expression level when it was induced at 37 ℃ for 4 h with 1 mmol/L IPTG.[Conclusion] The study laid the foundation for the preparation of BPMV antiserum and construction of liquid phase chip detection method.