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采用RTPCR技术从SARS冠状病毒基因组扩增编码S蛋白的S2基因片段(第2170到2814位碱基),克隆到pMD18T载体并测序。用限制性内切酶消化后,S2基因亚克隆至表达载体pGEX4T2,转化大肠杆菌JM109,筛选鉴定阳性菌落。扩增培养含pGEXS2质粒的JM109大肠杆菌,经IPTG诱导,超声破菌,GSHSepharose亲和层析纯化目的蛋白,Westernblot检测SARS患者血清可以识别纯化的蛋白。用此蛋白免疫NIH小鼠,获得了高滴度抗GSTS2抗体的血清,为进一步研究SARS冠状病毒的亚单位疫苗奠定基础。
The S2 gene fragment encoding the S protein (bases 2170 to 2814) was amplified from SARS coronavirus genome by RTPCR technique, cloned into pMD18T vector and sequenced. After restriction endonuclease digestion, the S2 gene was subcloned into the expression vector pGEX4T2 and transformed into E. coli JM109 to screen for positive colonies. The JM109 Escherichia coli containing pGEXS2 plasmid was amplified and cultured. The target protein was purified by IPTG induction, sonication and GSHepharose affinity chromatography. The purified protein was detected by Western blot in sera of SARS patients. NIH mice were immunized with this protein to obtain sera of high titer anti-GSTS2 antibody, which laid the foundation for further research on the subunit vaccine of SARS coronavirus.