汉坦病毒S基因在原核细胞中高效表达的研究

Study on high-level expression of Hantavirus S gene in prokaryocyte

目的通过基因工程的方法从鼠肺中扩增出汉坦病毒的S基因,并实现其蛋白的体外表达。方法应用RT-PCR方法扩增汉坦病毒汉城型(SEO)的YZG-Changchun株S基因,然后克隆到pMD18-T载体中,经序列分析后,定向克隆入原核表达载体pET-28a中,转化大肠埃希菌Rosetta用IPTG诱导表达后,将全菌裂解,用SDS-PAGE和Western-blot检测重组菌中外源蛋白的表达情况和免疫反应性的分析。结果S基因在原核细胞中得到了高效表达,表达的蛋白占菌体蛋白总量的37%,且具有良好的免疫反应性。结论汉坦病毒S基因蛋白可通过基因工程手段获得体外高效表达,这将对其功能的研究及为汉坦病毒疫苗的研究提供基础。

Objective To clone S gene from the lung of mice and to realize the expression of its protein in vitro. Methods The S genes of YZG-Changchun stains of Hantavirus were amplified by RT-PCR, and then the RT-PCR products were cloned into the pMD18-T vector. After sequencing analysis, the foreign gene in the recombinant plasmid was cut by restriction enzymes and cloned into the expression vector pET-28a. The recombinant plasmid was then transformed into E. coli Rosetta induced with IPTG. To detect the expression and the immunoreactive of exogenous protein in recombination plasmld by SDS-PAGE and Western-blot. Results The fusion protein was about 37% of the total proteins by gel thin scanner instrument CS-9000 and possessed favourable immunoreactive. Conclusion The gene of S protein can be expressed in a high efficiency in vitro by genetic engineering method, so it provides a good basis for further research on its function and the development of Hantavirus vaccine.