臺灣博碩士論文加值系統

在人體中，免疫球蛋白A (IgA) 存在著兩種次類型，IgA1和IgA2，這兩種IgA同時存在著分泌型IgA (secretory IgA) 和膜鑲嵌型IgA (membrane-bound IgA, mIgA)； mIgA是由兩條膜?捙 (m??nchain) 結合兩條?袺 (kappa chain) 或?傶 (lambda chain) 所組成，在mIgA的膜?捙錀端胜肽的構造中，包含有細胞外 (extracellular)、穿膜 (transmembrane)、和細胞內 (intracellular) 三種區段；在過去的研究發現，m??存在著兩種長度的異構型 (isoforms)，長型的m?? (m??L) 在膜?捙鴘慢H3區域 (domain) 和穿膜區段之間，比短型 (m??S) 多出6個胺基酸殘基，序列為GSCSVA。透過研究三十個捐血者的周邊血液淋巴球(peripheral blood lymphocytes)的染色體DNA和mRNA的定序比對，我們發現了一條m??的對偶基因 (allele)，這條新的對偶基因會產生兩個不同構型的m??，和原對偶基因存在的兩種異構型相似，長異構型比短異構型多出6個胺基酸，序列為GSCCVA。新發現的對偶基因與原對偶基因的差異在第456號胺基酸，因此這兩個對偶基因分別命名為m??(456C)與m??(456S)。此外，從m??的DNA序列分析得知，m??只有以短型的異構型出現。
由於分泌型IgA的?捙鴩S有這段膜鑲嵌胜肽片段的存在，且這條胜肽片段在胺基酸序列上具有獨一性，因此我們提出這條mIgA的膜?捙錀端胜肽的細胞外片段 (稱為migis-?? 適合用來作為抗體標的表現mIgA的B細胞的抗原 (antigen)，希望能藉由抗體來調控表現mIgA的B細胞的活性，例如抑殺B細胞或降低IgA的產量。在這份研究裡，我們發展出幾個能夠專一性地辨識migis-?悀軉q的單株抗體，例如抗體29C11；在酵素免疫法的分析顯示，這些抗體可以很強地結合至含有migis-?悀軉q的胜肽或重組蛋白；然而，使用流式細胞儀來研究抗體結合至B細胞表面的膜型免疫球蛋白A的情形時，我們發現這些抗體無法有效地結合膜型免疫球蛋白A；在使用除去膽固醇的藥物來破壞B細胞上的脂筏後，這些抗體便能夠強烈地結合至B細胞表面的膜型免疫球蛋白A；此外，免疫沉澱法分析的結果指出，當膜型免疫球蛋白A的B細胞被很強的清潔劑溶解後，如SDS，或是利用藥物將細胞膜上的脂筏破壞後，這些抗體便能夠結合膜型免疫球蛋白A並將其沉澱下來，然而在一般的條件下，如使用中性的detergents溶解去細胞或不使用藥物處理細胞，大部分的抗體都無法將B細胞上的膜型免疫球蛋白A沉澱下來。綜合以上的結果顯示，膜型免疫球蛋白A的migis-?悀軉q在B細胞上會與脂筏結合，這樣的結合導致脂筏阻礙了辨識migis-?悛漣凗曀畢X至B細胞上的膜型免疫球蛋白A的能力。未來將發展高親和力的抗體29C11來增強標的表現膜型免疫球蛋白A的B細胞的能力與研究其發展的可行性。

In humans, secretory IgA exists as two subclasses, IgA1 and IgA2, which contain distinct ?? and ?? heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding m?? and m?? heavy chains, which differ from ?? and ?? by an additional “membrane-anchor” peptide segment extending from the CH3 domain of ?? and ??. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain m?? exists in short and long isoforms, referred to as m??S and m??L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of m?? and m?? from 30 individuals residing in Taiwan, we have found that, in addition to the known m?? allele, referred to as m??(456S), m?? also has a previously unknown allele, referred to as m??(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to m??(456S), and appears to exist only as the long isoform, i.e. m??L, rather than the short isoform, m??S. We also confirmed that m?? exists only as the short isoform.
Because the membrane-anchoring peptide segment which is only present on mIgA is unique in amino acid sequences we proposed that the extracellular segment, referred to as the mIg isotype-specific (migis-?? segment of m? could be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. We rationalized that the migis-??specific antibodies could bind to, and in turn, regulate IgA-expressing B cells, by mechanisms such as inducing apoptosis or decreasing the production of IgA. In the study, we developed several anti-migis-? monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-?? The mAbs bound strongly to synthetic peptides of migis-? and to various recombinant proteins containing migis-? as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-? region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-??specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.

中文摘要 I
Abstract II
中文摘要 I
Abstract II
誌謝 III
Chapter 1 Background and Significance 1
1. Biochemistry of membrane-bound Igs on B cell surfaces 1
2. Biological function of mIgA 3
3. IgA and IgA-mediated diseases 3
4. Significance of developing anti-migis-? mAbs 5
Chapter 2 Structural Design and Experimental Results 7
A. Alleles and isoforms of human membrane-bound IgA1
1. Cloning of m?? and m?? from mRNA of peripheral blood lymphocytes 7
2. Analyzing clones of PCR-amplified migis-?? and -?? from 30 individuals 8
3. Analysis of RNA splicing sites in the membrane exon of ? chain 9
4. RNA splicing in m?? and m?? gene-transfected cell lines 9
B. Preparing monoclonal antibodies specific for migis-? peptides
5. Generation of anti-migis-? mAbs 10
6. Examination of binding activity with migis-? on B cells 10
7. Assessing the relative affinity of anti-migis-? mAbs?n 11
8. Epitope mapping of mAbs using synthetic migis-? peptides 12
C. The Effects of membrane environments on antibodies binding to mIgA-expressing
B cells
9. Staining of M?毧D-treated B cell lines by anti-migis-? mAbs 13
10. Immunoprecipitation of mIgA on B cell lines 14
11. Diminished binding of 29C11 to cross-linked mIgA on B cell lines 16
12. Staining of mIgA-expressing B cells with peptide-immunized mouse antisera 16
Chapter 3 Discussion 18
1. The splicing efficiency among m? allelic variants 18
2. Binding properties of anti-migis-? mAbs 20
3. The influences of BCR environment on binding of anti-migis-? mAbs to mIgA 22
Chapter 4 Materials and Methods 25
1. Isolation of peripheral blood lymphocytes 25
2. RNA, cDNA and genomic DNA preparation 25
3. Amplification of m?? and m?? by PCR 25
4. Cloning and sequencing of PCR-amplified fragments 26
5. Cell lines and transfection 27
6. Western blotting analysis of membrane proteins 27
7. Antibodies 28
8. Expression and purification of recombinant proteins 28
9. Preparation and purification of mAbs 29
10. Generation of stable cell lines 30
11. Flow cytometry 31
12. Peptide synthesis 31
13. Biotinylation of mAb and ELISA 31
14. Disruption of lipid rafts by cholesterol extraction 32
15. Immunoprecipitation of mIgA by mAbs 32
Tables 34
Figures 37
References 53
Publications 59

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