| A novel coronavirus has been identified as the major cause of severe acute respiratory syndrome (SARS). Severe acute respiratory syndrome (SARS), referred to as atypical puenmoina, is an infectious disease of a kind of breath spread in the near future. An outbreak example appear in November of 2002 the region in Guangdong in the our country, and become the popular situation quickly, and become the popular situation quickly. Its contagion is strong, the infection rate is high, affecting the political economy development of the world-wide locations seriously. Have no special effect medicine and the treatment methods of cure SARS still currently, although after in time of the support treatment and to disease treatment, great majority the patient can recover from illness to recover from illness, its high infection rate, the high contagion and high possibility that patient relapse needs the medicine and the vaccine of the special effect urgently. But defend heavy in cure, the vaccine researches to manufacture to seem to be more important and urgent. According to the genome series of the virus of SARS research enunciation that measures the preface and former concerning the coronaviruses, the spike protein of the virus of SARS is the ideal antigen of target probably at keep virus from invade, moderating the virus aspect to rise the decisive function. The severe acute respiratory syndrome, (SARS) is main pathogenic body that causes SARS. The severe acute respiratory syndrome, (SARS) is a kind of coronaviruses. A sequence of SARS virus genes has already completed the measurement. The spike protein may be a second unit vaccine. The result of the SARS protein compared indicated that the N-terminal of spike protein has a evidence homology with the formerly MHV and BCV. S2 protein of MHV and BCV has counteracted antibody epitopes. So we presume that the S2 protein of SARS also has counteracted antibody epitopes. S2 protein gene was amplified fromthe genomic RNA of SARS-CoV by RT-PCR. The fragment was cloned into a prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-28a/S2. The Escherichia coli cells were transformed with the recombinant plamid and the transformants were induced with IPTG to express the recombinant protein and the target protein was identified. To study the key influence factors when lactose, the inducer substitute to the IPTG, was used in the processes of the recombinant gene's expression. The BL21(DE3)/S2 which expressed the fragment of the spike protein of the SARS virus was used as model Escherichia coli strain.Application of glucose/glycerol for carbon source and lactose/allolactose for induction of SARS/ S2 genes in BL21(DE3) was studied in shake flask. The result of the shake flask was verificated in 40 liters fermentor. Shake flask cultivations showed: 1. lactose induced gene expression is as efficiently as IPTG when concentration of the lactose was up to 0.5mM; 2. addition of glucose in the medium did inhibit the effect induced by the lactose but not the effect by allolactose; 3. addition of glycerol in the medium didn't inhibit the effect by the lactose or allolactose. The studying in fermentor has confirmed the same results. The lactose can be used to induce the lacI-based expression system. The medium must contain the non-glucose carbon source when the Escherichia coli was induced by lactose. The next in order, we carry on the first step to the target protein to purified, suspending the engineering germ in the broken up liquid(10mM Tris-HCl ,1mMEDTA),break the germ with a broken up instrument under the condition that ice bathe. Broken up evenly, centrifuge, 10000 rpm,10min. Leave up pure. After precipitate was washed away dirt by foundation buffer liquid (20mM PB, 8MUrea , PH 7.4) and fused. Indoor temperature 5-6h, centrifuge, 10000rpm,10min. Discarded the precipitate. Pass further analysis to the S2 protein property, We... |
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