| The SARS-CoV was identified as the etiological agent of severe acute respiratory syndrome (SARS).The primary mode of transmission appears to be through direct or indirect contact of mucous membrane (eyes, nose, or mouth) with infectious respiratory droplets or fomites. The SARS is highly contagious and acute nature and its mortality is up to 5%～10%. Within months after its emergence in Guangdong Province in mainland China, it had affected more than 8000 patients and caused 916 deaths in 29 countries on five continents. The main reasons of its seriously results are that SARS-CoV had not previously been endemic in humans, there are not effective diognosis and treatment method, and there is little understanding to the prevalence of SARS. Now SARS has been controlled effectively, but the current information suggests the greatest risk of the reemergence of the disease.So the prevention and control of SARS are still very important.In this article we assayed the seroepidemic of IgG in SARS patients and detected viral RNA in the fecal, serum, urine, sputum collected at least 21 days after the onset of symptoms. The summary of experiment methods and results are listed below:1. Gene cloning and Gene expression in prokaryotic systems of SARS-CoVThe virus of HT-1 was passaged into Vero E6 cells.When the cytopathogenic effect was observed, we collected the cell and extracted total RNA. The sequence-specific primers was designed according to the published cDNA sequences of SARS-CoV on internet and ensured the accuracy of ORF. The coding regions of SARS-CoV proteins M,N,E and truncated S (S1,S2,S3) protein was amplified by PCR.The PCR products were cloned into pcDNAII vector and confirmed by DNA sequencing.The accurate plasmid was clove by enzyme and linked with expression vector. The constructs examined by enzymatic cleavage were transformed into E. coli BL21, and the expression of recombinant proteins was induced by the addition of 0.1mM IPTG at 37℃. These protein were analyzed on SDS–page 15% polyacrylamide gel and stained with Coomassie blue.The results showed that recombinant protein expressed well. To further identify the protein expressed, we carried out Western blot analysis.The result show that the recombinent protein react with the specific antibody and the protein band appeared on corresponding site.2. assaying the Seroepidemic of SARS patientsBy western blot,we detect the response of the four recombint protein with the convalescent serum from patient with SARS.The result show that the proteinband of N and S1,S2,S3 had a strong reaction with the SARS-CoV positive serum whereas it was not recongnized by the SARS-CoV negative serum.Tworecombinant proteins M and E were not recognized in this SARS-CoV positive serum. So we collected a total of 54 sera specimens from 11 patients with SARS in different stage of illness including acute and convalescent serum and detected the antibody change of IgG to S1, S2, S3 and N protein by western blot.The result show that the IgG antibody to S1,S3,N protein could be detected on seventh day after the onset of symptom, and that the IgG antibody to S2 protein could be detected on nineth day.The positive rate of antibody to N and S3 protein is up to 100% in later stage of disease; In 54 serum samples,the antibody to three truncated S proteins is different. In summary, the positive rate of IgG antibody to S3 protein is the highest and S2 is the lowest among three truncated S proteins. However, this is not true of all SARS patients. For example,the IgG antibody to S2 protein is highest in patient NO. 82. The reason is possibly the mutation of S gene and the different immune reactivity of SARS patients to S protein. 3. The infectivity detection in convalescent patients with SARSThe method of PCR can detect the virus RNA of SARS-CoV directly and it is one of methods of virus diagnosis. It was demonstrated that respiratory and fecal, serum ,urine specimens are suitable for diagnosis with the method of RT-PCR. Long time surve... |
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