표제지
감사의 글
목차
I. 서론(Introduction) 12
I-1. HLA의 일반적 구조와 역할 12
I-1-1. HLA class I 12
I-1-2. HLA class II 14
I-2. HLA diversity의 주요 원인 16
I-2-1. HLA diversity와 질병과의 연관성 17
I-2-2. HLA diversity와 vaccine에 대한 반응성 차이와의 연관성 18
II. 가설 19
II-1. 이전의 연구결과 21
II-2. 연구의 목적(Specific aims) 22
III. 실험재료 및 방법(Materials and methods) 23
III-1. 실험재료(Materials) 23
III-1-1. Information of samples 23
III-1-2. Reagents 24
III-2. 실험방법(Methods)-HLA class II subtype cloning 과 SBT-(Fig. 4) 25
III-2-1. Sample LR 5, LR 6 그리고 KS 1, KS 2를 이용한 HLA class II subtype cloning 25
III-2-2. PCR (Ploymerase chain reaction) condition-(Fig. 5) 25
III-2-3. pGEM-T Easy Vector을 이용한 ligation 25
III-2-4. Competent cell (E.coli)을 이용한 transformation 25
III-2-5. Miniprep kit 을 이용한 plasmid 추출 26
III-2-6. Restriction enzyme cut을 이용한 vector 동정 26
III-2-7. Sequencing data analysis 26
III-3-1. SBT를 이용한 6개 cord blood sample genotyping 27
IV. 결과(Results) 28
IV-1. Hypothesis I 28
IV-1-1. HBsAg에 대한 HR 5, LR 5 그리고 LR 6의 HLA class II full sequencing 결과와 고 반응자와 저 반응자의 HLA class II genotype을 이용 한 Rankpep 및 phylogenetic 28
IV-2. Hypothesis II 32
IV-2-1. KS 1 과 KS 2 의 sequencing과 genotyping 결과 32
IV-2-2. 6개의 개의 cord blood HLA genotyping 결과 34
V. 고찰(Discussion) 60
VI. 인용문헌(References) 66
VII. 영문초록(Abstract) 72
Table. 1. Human leukocyte antigen class II associated with disease and it's risk factor. 51
Table. 2. Influence of HLA class II genes on vaccine response to HBsAg in man 52
Table. 3. Nomenclature for factors of the Human leukocyte antigen system update May 2006 53
Table. 4. Human leukocyte antigen class II genotype of high and low responder against HBsAg vaccine. 54
Table. 5. Human leukocyte antigen A, B, DR genotyping information of cord blood sample. 56
Table. 6. Primer list for Human leukocyte antigen class II amplification. 57
Table. 7. HLA class II subtypes that identified HLA class II cDNA of sample HR 5, LR 5 and LR 6 against HBsAg. 58
Table. 8. Genotyping result of 6 cord blood HLA class I and II. 59
Figure. 1. HLA genes in HLA loci. 36
Figure. 2. Structure of general HLA class I (upper-left) and binding antigen peptide in peptide binding cleft (upper-right) structure of general HLA class II (upper-left) and 37
Figure. 3. Immune response mechanism about HBsAg 38
Figure. 4. Entire HLA class II subtype cloning protocol. 39
Figure. 5. PCR condition (left) and pGEM-T Easy Vector used to ligation of PCR products (right). 40
Figure. 6. Confirmation of sample HR 5, LR 5 and LR 6 HLA class II PCR products using cDNA amplified primer through electrophoresis (upper) and confirmation of vector and 41
Figure. 7. Identified alternative splicing form (exon4 deletion) in samples HR 5, LR 5 and LR 6. 42
Figure. 8. Analysis of phylogenetic tree result using high responder and low responder against HBsAg. 43
Figure. 9. Prediction of binding peptide using a Rankpep. 47
Figure. 10. Confirmation of sample KS 1, KS 2 HLA class II PCR products using cDNA amplified primer through electrophoresis (upper) and confirmation of vector and 48
Figure. 11. Analysis of Sequence using Finch TV. 49
Figure. 12. Comparison of the exon 3 nucleotide sequences of HLA -B*070201, HLA-B*0731 and HLA-B*0743 (upper) and three dimension structure of novel sequence HLA-B* 50