神經胺酸酶(Neuraminidase)為流感病毒膜上的醣蛋白，其功能為切割病毒顆粒與宿主細胞受體間的連結唾液酸來促進病毒的釋放及感染，也可以透過破壞於纖毛、黏膜以及細胞醣複合物之誘餌受體進而促使病毒顆粒的感染傳播。神經胺酸酶抑制抗體可以限制流感病毒的散播以及減緩臨床症狀程度。在本研究中，我們利用昆蟲桿狀病毒表現系統來構築以及純化pandemic H1N1 (pH1N1) (A/Texas/05/2009), H3N2 (A/Udorn/307/1972), H5N1 (A/Vietnam/1203/2004) and H7N9 (A/Shanghai/02/2013)重組神經胺酸酶蛋白。我們挑選位於酵素活性區周圍之344、365與366胺基酸位置。並利用點突變來產生H3N2與H7N9的突變神經胺酸酶蛋白。透過免疫N2帶有N344Y, N344H, E365I/D366S, E365T/D366N與E365T/D366A突變蛋白，N9帶有H344N, H344Y, T365I/A366S, T365E/A366D 與 T365T/A366N突變蛋白，進而剖析對於神經胺酸酶抑制抗體之影響。我們結果顯示H7N9帶有T365T/A366N突變重組神經胺酸酶可以誘發更好的神經胺酸酶抑制抗體產生對抗pH1N1病毒株。我們希望這個發現能夠對於未來之廣效性神經胺酸酶疫苗發展提供有用的資訊。

Neuraminidase (NA) is an influenza virus envelope glycoprotein that can remove sialic acid from receptors for virus release from infected cells. NA also contributes to viral transmission and infection by destroying decoy receptors on cilia, mucins, and cellular glycocalyx. NA-inhibiting (NI) antibodies are known to limit virus spreading and to mitigate clinical symptoms of influenza A virus infection. In this study, we used a baculovirus-insect cell expression system to construct and purify recombinant NA (rNA). We targeted the NA epitopes at residues at 344, 365, and 366, all located close to NA enzyme active sites. Site-directed mutagenesis at these three residues produced the H3N2 and H7N9 rNA proteins. Cross-reactive NI antibodies were dissected via N2 mutant proteins of N344Y, N344H, E365I/D366S, E365T/D366N and E365T/D366A, N9 mutant proteins of H344N, H344Y, T365I/A366S, T365E/A366D and T365T/A366N immunizations. Our result shows the N9 mutant protein of T365T/A366N elicited higher NI antibody titers against heterosubtypic pH1N1 virus than that elicited by N9 wild-type proteins. It is our hope that these findings provide useful information for the development of an NA-based universal influenza vaccine.

中文摘要 I
Abstract II
致謝 III
Content IV
1. Introduction 1
1.1. Influenza viruses 1
1.2. Influenza A viruses 2
1.3. Avian influenza viruses 3
1.4. NA antigen of influenza A viruses 5
1.5. NA-inhibiting antibodies 6
1.6. Study goals 8
2. Material and Methods 10
2.1. Cell lines 10
2.2. Bac-to-Bac® Baculovirus expression system 10
2.2.1. Construction of recombinant soluble Neuraminidase protein (rNA) 10
2.2.2. Site-directed mutagenesis PCR 11
2.2.3. Generating the recombinant Bacmids 14
2.2.4. Generating the recombinant Baculovirus 15
2.3. Production and purification of soluble recombinant neuraminidase proteins 15
2.3.1. generation of soluble recombinant neuraminidase protein 15
2.3.2. Purification of soluble recombinant neuraminidase proteins 16
2.4. Characterize recombinant neuraminidase proteins 17
2.4.1. Sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel preparation and electrophoresis 17
2.4.2. Western blotting 18
2.5. Mouse immunization regimens 18
2.6. Fetuin-based recombinant neuraminidase enzyme activity and Neuraminidase-inhibiting (NI) assay 19
2.6.1. Detection of enzyme activity of recombinant neuraminidase 19
2.6.2. Neuraminidase-inhibiting (NI) antibodies assay 20
2.7. Enzyme-linked immunosorbent assay (ELISA) 21
2.8. statistical analysis 21
3. Results 22
3.1. Expression, purification, and characterization of wild-type rNA proteins of pH1N1, H5N1, H3N2, and H7N9 viruses 22
3.2. NA amino acid sequence alignment analyses of pH1N1, H5N1, H3N2, and H7N9 viruses 24
3.3. Three dimensional structure of NA proteins of pH1N1, H5N1, H3N2, and H7N9 viruses based on homology modeling 25
3.4. Expression and purification of H3N2-rNA mutant proteins (H344N, H344Y, E365I/D366S, E365T/D366N, E365T/D366A) 26
3.5. NA-specific IgG antibodies elicited by H3N2 recombinant NAs immunization 27
3.6. NI antibodies elicited by wild-type or mutant H3N2-rNA protein immunizations 28
3.7. Expression and purification of H7N9-rNA mutant proteins (N344H, N344Y, T365I/A366S, T365E/A366D, T365T/A366N) 29
3.8. NA-specific IgG antibodies elicited by H7N9 recombinant NAs immunization 30
3.9. NI antibodies elicited by wild-type or mutant H7N9-rNA protein immunizations 31
4. Discussion 33
5. Reference 36
6. Figure 41
Fig. 1 Characterization of recombinant soluble Neuraminidase proteins (rNA) 42
Fig. 2 Amino acid sequences comparison between NA proteins of pH1N1, H5N1, H3N2 and H7N9 43
Fig. 3 Neuraminidase stuctures of pH1N1 NA, H5N1 NA, H3N2 NA and, H7N9 NA. 47
Fig. 4 Characterization of H3N2 mutant recombinant neuraminidase proteins. 48
Fig. 5 NA-specific IgG titers elicited by H3N2 recombinant neuraminidases 49
Fig. 6 N2 NA-inhibiting antibodies induced by wild-type or mutant recombinant neuraminidase proteins against homologous H3N2 virus, heterosubtyupic pH1N1 virus, H5N1 VLP and H7N9 VLP 50
Fig. 7 Characterization of H7N9 mutant recombinant neuraminidase proteins. 52
Fig. 8 NA-specific IgG titers elicited by H7N9 recombinant neuraminidases 53
Fig. 9 N9 NA-inhibiting antibodies induced by wild-type or mutant recombinant neuraminidase proteins against homologous H7N9 VLP, heterosubtyupic pH1N1 virus, H3N2 virus and H5N1 VLP 54
Table 1. The enzyme kinetics of wild-type Rna proteins evaluated by Lineweaver-Burk plots 56
Table 2. The immunization regimens of H3N2- rNA and H7N9-rNA 57

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