I SARS 21 SARS-CoV GeneBank SARS-CoV Tor2 PCR N PN422 N 40% SARS N 95% N C N 38 15%~30% SARS N N 39 Western N 30 60aa 160~170aa N Bal b/c 8 39 Western Blot 8 6 2 N N 30 184 301 360 2 2 IgG1 10 6 10 9 M 1 SARS-CoV N
II ABSTRACT Severe acute respiratory syndrome (SARS) was the first severe infectious disease in the 21 century,which was caused by a new coronavirus,named by WHO as SARS coronavirus (SARS-CoV). From the Genebank we got the genic sequences of Nucleocapsid (N) protein of SARS-CoV. Then according to the sequence of strain Tor2 we synthesized our genic sequence of N protein by the method of PCR. The N gene was cloned into a prokaryotic expression plasmid, named as PN422. The recombinant plasmid was expressed in E. coli. The expressed recombinant N protein account for more than 40% quantity of bacterial total protein, and most existed as soluble protein. The Western blot using a SARS patient acute phase serum showed that both soluble protein and insoluble protein had obvious biological activity. More than 95% purity can be obtained after purification, and the reactivity with serum remained similar as pre-purification. We designed a series of Nucleocapsid protein with its N or/and C terminal truncated, constructed the recombinant plasmid and expressed them in E.coli. The yields level of these thirty-eight truncated recombinant proteins were from 15% to 30%. The proteins were purified by metal chelate affinity chromatography or by electro-elution. Western Blot was used to detect the reactivity of these proteins against the convalescent sera from SARS patients. The results showed that the reactivity of the full length N protein was the best, which suggests that the integrality of N protein be important for the exposure of the prominent epitopes. After analysis of the Western Blot mode of the whole 39 recombinant proteins, we presume that the nitrogen terminal (30~60aa) and the middle region (160~170aa) might exist two important linear epitpes. The Bal b/c mice were immunized with the N proteins. Using the conventional technique of monoclonal antibody we filtrate out eight hybridomas. By the method of Western Blot and competitive inhibition assay, we categorized the eight monoclonal antibodies into two classes. The first class consisted of six monoclonal antibodies,and they recognized the immunity dominant epitope of N protein,which located between the 30th and the 184th amino acid. The second class consisted of two monoclonal antibodies, the epitope they recognized located between 301st and 360th amino acid. After identifed the subclass the titer and the affinity of the representative antibodies of the 2 class relatively, we found that they both belong to the IgG1 subclass, their ascites titers were 10 6 and their affinity were 10 9 M -1. Key word: SARS-CoV Nucleocapsid protein Monoclonal antibody
1. SARS 1 1. 2 2. 2 3. 2. SARS 3. SARS 4 1. 4 2. 5 3. 6 4. 6. SARS 7 1. 7 2. SARS 10 2.1 SARS-CoV 10 2.2 SARS-CoV 13 2.3 SARS-CoV 13 2.3.1 SARS-CoV RNA 13 2.3.2 SARS-CoV 14 2.4 SARS-CoV 14 3. SARS-CoV N 15 3.1 SARS-CoV N 15
4 3.2 SARS-CoV N 15 3.3 SARS-CoV N 16 3.4 SARS-CoV N 16 3.5 SARS-CoV N 17. 17 19 1. 19 2. 26 41. SARS-CoV N 41 1. N 41 2. N 43. SARS-CoV N 47 1. 47 2. PN422 49. N 51 1. 51 2. 39 N PCR 54 3. SARS-CoV N 54 4. 54 5. N 55 6. N 56 7. SARS 59. SARS-CoV N 60
1. 60 2. N 60 3. 64 4. 8 65 5. 4D4 4F4 67 6. 4D4 4F4 67 7. 4D4 4F4 68 8. 4D4 4F4 69 71 1. SARS-CoV N 71 2. N 72 3. N 73 76 77 82 83
6 Contents Abstract Chapter1 Preface 1 The epidemiology of SARS 1 1. The infectious source 2 2. Routes of transmission 2 3. Clinical symptom and death rate 2 Isolation of the pathogen of SARS 3 Special diagnosis of SARS 4 1. Serologic diagnosis 4 2. Nucleic acid diagnosis 5 3. Isolation of the virus from the culture cells 6 4. Electron microscopy(em) 6 SARS virus 7 1. Introduction to coronavirus 7 2. The biological properties of SARS-CoV 10 2.1 The genome sequence of SARS-CoV 10 2.2 The mutation of the genome of SARS-CoV 13 2.3 The Proteome of SARS-CoV 13 2.3.1 Replicase polyprotein of SARS-CoV 13 2.3.2 The structure protein of SARS-CoV 14 2.4 The resistibility and stability of SARS 14 3. The nucleocapsid protein of SARS-CoV 15 3.1 The bio-informational analysis of SARS N protein 15
3.2 The properties and functions of SARS-CoV N proteins 15 3.3 The immunogenic properties of SARS-CoV N proteins 16 3.4 Research of the epitopes of SARS-CoV N proteins 16 3.5 The monoclonal antibody research of SARS-CoV N proteins 17 The purpose and meaning of this research 17 Chapter2 Materials and Methods 19 1. Materials 19 2. Methods 26 Chapter3 Results and Analysis 41 Acquirement of the SARS-CoV N protein gene 41 1. Synthesis of the N protein gene 41 2. Analysis of the properties of N protein 43 Construction of the expression vector of SARS-CoV N protein and its expression and purification 47 1. Construction of the expression plasmids 47 2. Expression purification and activity identification 49 Acquirement of a series of truncated N proteins 51 1. Design of primers 51 2. Amplification of gene fragments by PCR 54 3. Construction of the recombinant plasmids containing the fragments 54 4. Identification of the recombinant plasmids by the small quantity expression 54 5. Large scale expression of the N recombinant proteins 55
8 6. Purification of the N recombinant proteins 56 7. WB assay of the recombinant proteins against the convalescence sera from SARS patients 59 Generation of N protein McAb and the initial research of its properties 60 1. Establishment of the hybridoma strains and induction of the ascites 60 2. The WB reactivity of the McAbs against the N recombinant proteins 60 3. The competitive inhibition assay of the McAbs 64 4. Classification of the 8 McAbs obtained 65 5. The titer of McAb 4D4 and 4F4 67 6. The purification of McAb 4D4 and 4F4 67 7. The subclass identification of McAb 4D4 and 4F4 68 8. The affinity determination of McAb 4D4 and 4F4 69 Chapter4 Discussion 71 1. Acquirement of SARS-CoV N proteins 71 2. Epitope study of N proteins 72 3. Research of McAbs to N protein 73 Brief summary 76 References 77 Acknowledgements 82 appendix 83
1 2002 11 Severe Acute Respiratory Sysndrome SARS [1, 2]. SARS SARS 30 1 2003 6 11 8437 813 [3] 1 SARS Fig 1 The globle distribution of SARS (http://www.who.int/csr/sars/map2003_05_14.gif)
2 1. SARS SARS 8 [4, 5] SARS SARS 10 [6] SARS 2. SARS SARS [7] [8, 9] 3. SARS [10] 10%~20% ARDS [11] SARS [6] WHO SARS 0 50 SARS
3 14 15 WHO 24 1 25 44 6 45 64 15 65 50. SARS [12, 13] 50 SARS [14] 40 RNA 200 CDC SARS SARS 3 2003 4 17 WHO Erasmus Albert Osterhaus [15] KM [16] SARS SARS-CoV
4 [17]. SARS SARS Roche SARS PCR SARS CDC SARS SARS PCR SARS - RNA 1. (enzyme linked immunosorbent assay ELISA) ELISA IgM IgG IgM IgG IgM SARS WHO SARS ( ) 21 d ( Immunofluorescence assay IFA) SARS SARS 10 d SARS
5 SARS SARS SARS SARS - - SARS SARS SARS 2. PCR SARS - ( reverse t ranscriptase polymerase chain reaction RT-PCR) SARS RNA 10 d SARS 20 90
6 SARS SARS (40 80nt) (5 30nt) 5 3 SARS SARS SARS SARS ( 9 h) 3. (VERO E6) SARS 24 h SARS 3 P3 SARS SARS P3 4. VERO E6 SARS SARS
7 SARS. SARS SARS [18, 19] SARS (SARS-CoV) (Order Nidovirales) (Family Coronaviridae) (Genus Coronavirus) RNA 1. 60 220nm 2 [20] Coronavirus 2. Figure 2 The electron micrograph of SARS-CoV Department of Microbiology, University of Leicester
8 S (Spike Protein) M (Membrane Protein) E (Envelope Protein) HE (Haemagglutinin-esterase) S - [21] M M M [22] E M E [23] 3. Figure 3 The particle structure of SARS-CoV ( N Engl J Med,2003,348:1948~1951)
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