Analysis of sla class ii polymorphism to study disease resistance in pigs

Dissertation for Degree of Doctor Supervisor: Prof Chankyu Park Analysis of SLA class II polymorphism to study disease resistance in pigs Submitted by LE MINH THONG February, 2015 Department of Animal Biotechnology Graduate School of Konkuk University Analysis of SLA class II polymorphism to study disease resistance in pigs A Dissertation submitted to the Department of Animal Biotechnology and the Graduate School of Konkuk University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Biotechnology Submitted by LE MINH THONG October, 2014 This certifies that the Dissertation of Le Minh Thong is approved Approved by Examination Committee: Chairman 도정태 Member 전태훈 Member 김종주 Member 조쌍구 Member 박찬규 November, 2014 Graduate School of Konkuk University TABLE OF CONTENTS List of Tables iv List of Figures v List of Abbreviations vi Abstract ix Chapter Literature Review 1.1 Pig and infectious disease 1.2 Biology of swine leukocyte antigens 1.2.1 Structure and function 1.2.2 Disease resistance and susceptibility 1.2.3 Association with productive traits 10 1.2.4 Genetic diversity and fitness 10 1.3 Genetics of SLA 11 1.3.1 Mapping 11 1.3.2 Polymorphism and linkage disequilibrium 13 1.4 Nomenclature of SLA genes 14 1.5 Comparison of typing methods for swine leukocyte antigen 15 1.5.1 Serology 16 1.5.2 Cell-based typing 16 1.5.3 Sequence-based Typing 16 Chapter Development of simultaneous high resolution typing of three SLA class II genes, SLA-DQA, DQB1 and DRB1 using genomic DNA multiplex PCR and direct sequencing 19 i 2.1 Introduction 19 2.2 Materials and methods 21 2.2.1 Animals 21 2.2.2 Multiplex PCR for the simultaneous amplification of SLA-DQA, DQB1 and DRB1 21 2.2.3 Direct sequencing and allelic determination of SLA-DQA, DQB1 and DRB1 22 2.2.4 Determination of SLA class II haplotypes 22 2.3 Results and discussion 25 Chapter Association between the polymorphisms of SLA-DQB1and DRB1 and piglet survival 29 3.1 Introduction 30 3.2 Material and method 32 3.2.1 Animals 32 3.2.2 High resolution typing of SLA-DQB1 and SLA-DRB1 32 3.2.3 Statistic association test 33 3.2.3.1 Association of SLA-DQB1, DRB1 alleles and haplotypes 33 3.2.3.2 Association of exon polymorphisms 33 3.2.3.3 Impact of substituted amino acids 34 3.2.4 Prediction of peptide and SLA binding 34 3.3 Results 35 3.3.1 Typing and association of DQB1, DRB1 35 3.3.2 Identify associated polymorphism 41 3.3.3 Effect of amino acids 43 ii 3.3.4 In silico binding analysis 50 3.4 Discussion 53 Chapter Establishing immortalized cell panel with diverse SLA genotypes 60 4.1 Introduction 60 4.2 Materials and methods 62 4.2.1 Tissue and primary cells 62 4.2.2 SLA typing 62 4.2.3 Cell culture, transfection and selection 63 4.2.4 Cell growth characterization 63 4.2.5 Immune fluorescent analysis 64 4.3 Results 64 4.3.1 SLA genotypes 64 4.3.2 Characterization of immortalization 67 4.3.3 Correlation of morphology and growth rate 70 4.3.4 Expression of SLA class I on cell surface 71 4.4 Discussion 72 References 75 Appendix 99 Abstract (in Korean) 154 iii List of Tables Table 1.1 Recent emerging pathogens in pigs Table 2.1 Primers used for multiplex PCR and direct sequencing of SLA-DQA, SLA-DQB1, and SLA-DRB1 24 Table 3.1 Distribution and statistic association of 16 SLA-DQB1 alleles to piglet survivability 36 Table 3.2 Distribution and statistic association of 18 SLA-DRB1 alleles to piglet survivability 38 Table 3.3 Distribution and statistic association of 20 major SLA-DQB1:DRB1 haplotypes to piglet survivability 40 Table 3.4 Interaction between phenotype associated amino acids to piglet survivability 48 Table 4.1 Typing results of panel comprised 26 cell lines 66 Table 4.2 Summary of typing results of cell panel 67 iv List of Figures Figure 1.1 Molecular structure of MHC class I and II Figure 1.2 Schematic molecular organization of the SLA genes Figure 1.3 Comparative genomic organization of the human and swine major histocompatibility complex (MHC) class I region 12 Figure 1.4 Comparative genomic organization of the human and swine major histocompatibility complex (MHC) class II region 12 Figure 1.5 Polymorphism of MHC genes in various species (adapted from the Immuno Polymorphism Database-MHC (IPD-MHC) 13 Figure 2.1 Successful amplification of SLA-DQA, SLA-DQB1, and SLA-DRB1 by using multiplex PCR 27 Figure 3.1 The association across the exon of SLA-DQB1and DRB1 42 Figure 3.2 Plot of the association to the phenotypes of healthy and illness/early died piglet of amino acid substitutions across DQB1 exon 44 Figure 3.3 Plot of the association to putative disease of amino acid substitutions across DRB1 exon 47 Figure 3.4 Number and proportion of unique pathogenic peptides bind to each SLADRB1 alleles with predictably strong affinity 51 Figure 3.5 Boxplot of difference of predicted affinity to peptides with strong binding (pIC50 < 50nM) among SLA-DRB1 alleles, 0101, 0201, 0603Q and 04kn05 53 Figure 4.1 Comparison of the cell morphology between primary cultures and permanent cell line by the transfection of hTERT and and SV40LT 68 Figure 4.2 A colony of cells which escaped from the crisis stage 69 Figure 4.3 Growth curves of selected cell lines 70 Figure 4.4 Expression of SLA class I on the cell surface of selected lines 71 v List of Abbreviations ACP Annealing control primer ASFV African swine fever virus ATCC American Type Culture Collection BLAST Basic Local Alignment Search Tool BSA Bovine serum albumin CD8, Cluster of differentiation 8, CFSV Classical swine fever virus DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco's Modified Eagle Medium DMSO Dimethylsulphoxide ELISPOT Enzyme-Linked ImmunoSpot assay FAO Food and Agriculture Organization of the United Nations FBS Fetal bovine serum FMD Foot-and-mouth disease GGP Great grandparent GSBT Genomic sequence-based typing GWAS Genome-wide association study HIV Human immunodeficiency virus HLA Human leukocyte antigen hTERT Human telomerase IEBD immune Epitope Database IFN Interferon vi IMGT ImMunoGeneTics information system IPD Immuno Polymorphism Database ISAG International Society for Animal Genetics KIR Killer cell immunoglobulin-like receptors KNP Korean native pig MHC Major histocompatibility complex mRNA Messenger ribonucleic acid NIH National Institutes of Health NK Natural killer OR Odd ratio PBS Phosphate buffered saline PCR Polymerase chain reaction PCR-SSP PCR using sequence-specific primers PCV2 Porcine Circovirus Type PCVAD Porcine circovirus type 2-associated diseases PEF Porcine embryo fibroblast PMWS Post weaning Multisystemic Wasting Syndrome PRRS Porcine Reproductive & Respiratory Syndrome RFLP Restriction fragment length polymorphism RT-PCR Reverse transcriptase-PCR SLA Swine leukocyte antigen SNP Single-nucleotide polymorphism SNU Seoul National University SV40 LT Simian Vacuolating Virus 40 large T antigen vii 338 TEATAIRPAQVGYNT 15 Coat protein VP1 Porcine parvovirus 339 DEPNGAIRFTMGYQH 15 Coat protein VP1 340 TQNKVFDFIVIFEKI 15 hypothetical protein 341 KVFDFIVIFEKIIRE 15 hypothetical protein 342 DFIVIFEKIIREFVP 15 hypothetical protein 343 EFEDKFLKGELLEKV 15 hypothetical protein 344 DKFLKGELLEKVLEK 15 hypothetical protein Porcine parvovirus Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 345 KKNKKKNPEKPHFPLATE DDVRHHFTPS 28 N nucleocapsid protein 346 LKGELLEKVLEKNNI 15 hypothetical protein 347 YYLVSLGPDLIKDSQ 15 hypothetical protein 348 VSLGPDLIKDSQIYK 15 hypothetical protein 349 GPDLIKDSQIYKDGI 15 hypothetical protein 350 QKENSQKNDVVNSQN 15 conserved hypothetical protein 351 NSQKNDVVNSQNKTE 15 conserved hypothetical protein 352 KNDVVNSQNKTEKTE 15 conserved hypothetical protein 353 VLSLKDNKKITSSKK 15 hypothetical protein 354 LKDNKKITSSKKIRL 15 hypothetical protein 355 NKKITSSKKIRLLYT 15 hypothetical protein 356 LGKIIAQQNQSRGKGPGK KSKKKSPEKP 28 Nucleoprotein 357 AGENLNTSKFKKNEF 15 hypothetical protein 358 NLNTSKFKKNEFIKK 15 hypothetical protein 141 PRRSV VR2332 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Lelystad virus Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 232 Mycoplasma hyopneumoniae 7448 Mycoplasma hyopneumoniae 7422 Mycoplasma hyopneumoniae 359 TSKFKKNEFIKKIID 15 hypothetical protein 360 VIDSKKTKLPNTLIP 15 hypothetical protein 361 SKKTKLPNTLIPYSS 15 hypothetical protein 362 TKLPNTLIPYSSVEV 15 hypothetical protein 363 KKDVVNSQNKTEKTE 15 hypothetical protein 364 KKDVVNSQNKTDKTE 15 hypothetical protein 365 KNDVVNSQDKTEKTE 15 hypothetical protein Mhp 366 366 RIRKVKVEFWPCSPI 15 capsid protein 367 LGKIIAQQNQSRGKGPGKK IKNKNPEKP 28 nucleocapsid protein 368 KVEFWPCSPITQGDR 15 capsid protein Porcine circovirus Porcine respiratory and reproductive syndrome virus JA142 Porcine circovirus 369 PCSPITQGDRGVGSS 15 capsid protein Porcine circovirus 370 TQGDRGVGSSAVILN 15 capsid protein Porcine circovirus 371 GVGSSAVILNDNFVT 15 capsid protein Porcine circovirus 372 AVILNDNFVTKATAL 15 capsid protein Porcine circovirus 373 DNFVTKATALTYDPY 15 capsid protein Porcine circovirus 374 KATALTYDPYVNYSS 15 capsid protein 375 LLDTKGGLYRWRSPV 15 GP5 glycosylated envelope protein 376 SRKKKAAAAIEEEDI 15 Envelope protein p54 377 RYLASLHKKALPTSV 15 polyprotein 378 LGKIIAQQSQSRVKGPGRK NKKKNPEKP 28 nucleocapsid protein 379 VPQSGSITHPKFAGK 15 ORF1 Porcine circovirus Porcine reproductive and respiratory syndrome virus African swine fever virus BA71V Classical swine fever virus Porcine respiratory and reproductive syndrome virus MD-001 Torque teno sus virus 1a 142 380 TTSSCTSKAVTLSSL 15 major surface antigen Toxoplasma gondii 381 DAQSCMVTVTVQARA GQAKKKKPEKPHFPLAAED DIRHHLTQT 15 major surface antigen Toxoplasma gondii 28 Nucleoprotein Lelystad virus 28 ORF 1ab polyprotein 28 ORF7 28 Major capsid protein 27 envelope protein 46 glycoprotein Classical swine fever virus 27 Nucleoprotein Lelystad virus 27 Nucleoprotein Lelystad virus 27 Genome polyprotein 27 Polyprotein pp220 26 SLA-2 histocompatibility antigen, class I 382 383 384 385 386 387 388 389 390 391 392 TSNETLSGADPEEFVELK RPRFSAQALM GAMIKSQRQQPRGGQAK KKKPEKPHFPL LCNIHDLHKPHQSKPILTD ENDTQRTCS EMEPPFGDSYIVVGRGDKQ INHHWHKA IHTPAFNVNVRTGGGIHTPAFN VNVRTGGGIHTPAFNVNVRTGKKK VNQLCQLLGAMIKSQR QQPGGQAKKKK GAMIKSQRQQPGGQAKK KKPEKPHFPL NGTSKYAVGGSGRRGDM GSLAARVVKQ MSRIFRGDNALNMGR PKFLSDQIFNKV FDSDAPNPRMEPRAP WIEKEGQEYWD Porcine respiratory and reproductive syndrome virus 111/92 Porcine reproductive and respiratory syndrome virus African swine fever virus BA71V Japanese encephalitis virus strain SA-14 Foot-and-mouth disease virus (strain A24 Cruzeiro) African swine fever virus BA71V Sus scrofa Porcine transmissible gastroenteritis coronavirus strain Purdue Porcine respiratory and reproductive syndrome virus 111/92 393 CYTVSDSSFFSYGEIPFG VTDGPRYC 26 Spike glycoprotein precursor 394 HTFKTNGDYAWSHADDWQ GVAPVVKV 26 Replicase polyprotein 1ab (ORF1ab polyprotein) 26 GP5 envelope protein PRRSV VR2332 26 ORF1 Torque teno sus virus k2 395 396 ANASNDSSSHLQLIYNLTL CELNGTD KSEQDIKKEAHSAEISREY TRDPKSK 397 VRGPQAILILLSGALALTGTWAGPH 25 SLA-2 histocompatibility antigen, class I Sus scrofa 398 QQSFMEKWLEYEGGGQQSFMEKWL EYEGGGQQSFMEKWLEYEGKKK 46 glycoprotein Classical swine fever virus 143 Foot-and-mouth disease virus C3 Foot-and-mouth disease virus Classical swine fever virus Shimen Classical swine fever virus Shimen Porcine respiratory and reproductive syndrome virus 111/92 Porcine respiratory and reproductive syndrome virus Lelystad virus Foot-and-mouth disease virus C-S8c1 PRRSV VR2332 Foot-and-mouth disease virus (strain O1) (O1BFS) 399 TGTTTYTTSARRGDLAHLATAHARH 25 capsid protein P1 400 RHKQKIVAPVKQTLPNLRGDLQVLA 25 VP1 protein 401 CKEDYRYAISSTNEIGLLGAGGLT 24 polyprotein 402 FDGTNPSTEEMGDDFGFGLCPFDT 24 polyprotein 403 QLPAQWDKELDVAPPPKPFGLAPE 24 ORF 1ab polyprotein 404 CARPHGASEESQSVTFNKPLNTPQ 24 GP4 protein 405 FADGNGDSSTYQYIYNLTICELNG 24 ORF5 406 TTTYTASARGDLAHLTTTHARHLP 24 polyprotein 407 ANASNDSSSHLQLIYNLTLCELNG 24 GP5 envelope protein 408 RYSRNAVPNLRGDLQVLAQKVART 24 polyprotein 46 glycoprotein Classical swine fever virus 24 ORF1 Torque teno sus virus 1a Classical swine fever virus Shimen Classical swine fever virus Shimen PRRSV VR2332 410 VHNMQKDMVLLQGGGVHNMQKDM VLLQGGGVHNMQKDMVLLQGKKK RKADQENPKVSTWPIEGTWNTQDT 411 SHDLQLNDGTVKAICVAGSFKVT 23 polyprotein 412 CPFDTSPVVKGKYNTTLLNGSAF 23 polyprotein 413 SSHLQLIYNLTLCELNGTDWLAN 23 envelope protein GP5 414 SSTYQYIYNLTICELNGTDWLSS 23 ORF5 Lelystad virus 415 NDSSSHLQLIYNLTLCELNGTDW 23 envelope protein GP5 416 RCAWEETKNIINDFLEIPEERCT 23 Structural protein A137R 417 VTDNPGNMATTFKASGGQHPDAI 23 ORF1 418 FRREKPFPHRMDCVTTTVENED 22 polyprotein 419 SSTKNLIYNLTLCELNVTGFQQ 22 major structural glycoprotein GP5 PRRSV VR2332 African swine fever virus BA71V Torque teno sus virus k2 Classical swine fever virus Shimen Lactate dehydrogenaseelevating virus 420 MHTNYCRTCFLSGGGMHTNYCRT CFLSGGGMHTNYCRTCFLSGKKK 46 glycoprotein 409 144 Classical swine fever virus 421 LKEEEKEVVRLMVIKLLKKNKL 22 Phosphoprotein p30 422 SLHKGALLTSVTFELLFDGTN 21 polyprotein 423 YTASARGDLAHLTTTHARHLP 21 polyprotein 424 CTAVSPTTLRTEVVKTFRREK 21 polyprotein 425 LDELSGTSSQTHDAIRRKDME 21 paramyosin African swine fever virus BA71V Classical swine fever virus Shimen Foot-and-mouth disease virus C-S8c1 Classical swine fever virus Shimen Taenia solium 426 SSNYTTTGSDQNSGGSTSAIQ 21 ORF1 Torque teno sus virus k2 427 PFEPGDRFHSGIQDPSKVQNT 21 ORF1 Torque teno sus virus k2 428 HPAAVSVRFVEGFAVCDGLC 20 envelope glycoprotein C Suid herpesvirus 429 CHIEKAKGTDQQNKEYCSKE 20 putative Rep protein (ORF1) Porcine circovirus 430 20 putative Rep protein (ORF1) Porcine circovirus 46 glycoprotein Classical swine fever virus 432 VTFVRNFRGLAELLKVSGKM HPTDYSLSYMYPGGGHPTDYSLSYM YPGGGHPTDYSLSYMYPGKKK AELLKVSGKMQKRDWKTNVH 20 putative Rep protein (ORF1) Porcine circovirus 433 SKWAANFADPETTYWKPPRN 20 putative Rep protein (ORF1) Porcine circovirus 434 ETTYWKPPRNKWWDGYHGEE 20 putative Rep protein (ORF1) Porcine circovirus 435 KWWDGYHGEEVVVIDDFYGW 20 putative Rep protein (ORF1) Porcine circovirus 436 MPSKKNGRSGPQPHKRWVFT 20 putative Rep protein (ORF1) Porcine circovirus 437 EEGRTPHLQGFANFVKKQTF 20 putative Rep protein (ORF1) Porcine circovirus 438 RSQGQRSDLSTAVSTLLESG 20 putative Rep protein (ORF1) Porcine circovirus 439 TAVSTLLESGSLVTVAEQHP 20 putative Rep protein (ORF1) Porcine circovirus 440 SLVTVAEQHPVTFVRNFRGL 20 putative Rep protein (ORF1) Porcine circovirus 441 VIVGPPGCGKSKWAANFADP 20 putative Rep protein (ORF1) Porcine circovirus 431 145 K Predicted ability of SLA-DRB1 alleles in binding to unique pathogenic peptides with strong affinity Allele 0101 0102 0201 0401 0402 0403 0404 0501 0502 0603Q 0701 0801 0901 1001 1102 1301 1401 04kn05 FMD 13 0 17 0 0 20 ASFV 2 1 0 1 1 CSFV 6 0 11 1 11 PCV2 4 0 14 0 0 16 146 PRRSV 13 2 0 18 2 0 0 21 other 5 0 12 0 15 Total 26 43 11 19 15 0 76 10 14 86 L Heterozygosity in SLA-DQB1 and DRB1 between successfully marketed and died during/after weaning piglets Population Case Control Total No of chromosome Locus Observed homozygosity 0.1194 0.0995 Observed heterozygosity 0.8806 0.9005 Expected homozygosity 0.1305 0.0986 Expected heterozygosity 0.8695 0.9014 DQB1 402 DRB1 Mean 0.1095 0.8905 0.1146 DQB1 0.1925 0.8075 0.1584 374 DRB1 0.1604 0.8396 0.1104 Mean 0.1765 0.8235 0.1344 DQB1 0.1546 0.8454 0.1315 776 DRB1 0.1289 0.8711 0.0936 Mean 0.1418 0.8582 0.1126 Note: Expected homozygosty and heterozygosity were computed using Levene (1949) 147 0.8854 0.8416 0.8896 0.8656 0.8685 0.9064 0.8874 M Comparison of binding ability to PCV2 and PRRSV specific antigens between DRB1*0603Q and 0101 Pathogen PCV2 PRRSV Allele Bind Non-bind 0603Q 0101 0603Q 0101 14 18 52 62 66 77 148 P value OR 95% CI of OR 0.02 4.13 1.2 - 18.3 0.03 3.0 1.1 - 8.99 N Alignment of amino acid chains encoded by exon from SLA-DRB1 alleles typed in this study The figure showed the difference in amino acid sequence among alleles, specifically in the strongest locations associated to the phenotypes (red outline) as well as the pockets as showed 149 O Alignment of amino acid chains encoded by exon from SLA-DQB1 alleles typed in this study The figure showed the difference in amino acid sequence among alleles, specifically in the strongest locations associated to the phenotypes (red outline) as well as the pockets as showed 150 P Linkage disequilibrium partern of significant associated SNPs across SLA-DQB1 and DRB1 exon Boxes represent linkage disequilibrium (LD; range 0–1) between pairs of SNP markers as generated by Haploview Red, strong LD (no number means score 1); gray, uninformative; white, strong evidence of recombination, triangle point down outlines indicate haplotypes (16 blocks) Note: Some significant association SNPs from SLA-DRB1 exon could not be visualized in this figure because those SNPs are tri- or quadrallelic SNPs that Haploview program have limitation 151 in calculation Q Linkage disequilibrium partern of significant associated SNPs across SLA-DQB1 and DRB1 exon Boxes represent linkage disequilibrium (LD; range 0–1) between pairs of SNP markers as generated by Haploview Red, strong LD (no number means score 1); gray, uninformative; white, strong evidence of recombination, triangle point down outlines indicate haplotype blocks Blue circle, synonymous DQB1 SNPs significant associated to the putative disease and in complete LD with DRB1 SNPs or non-infomative (DQ-206); Red circle, non-synonymous DQB1 SNPs with significant association; Blue oulines indicate LD partern of corresponding SNPs with other SNPs Note: Some significant association SNPs from SLA-DRB1 exon could not be visualized in this figure because those SNPs are tri- or quadrallelic SNPs that Haploview program have limitation in calculation 152 R Boxplot of difference of predicted affinity to peptides only derived from PCV2 and PRRSV with strong binding (pIC50 < 50nM) among SLA-DRB1 alleles, 0101, 0201, 0603Q and 04kn05 Significant difference (P value) in peptide binding affinity (pIC50) was indicated between couple of alleles X axis, SLA-DRB1 alleles, Y axis, the half maximal inhibitory concentration (pIC50) P values were conculated using ANOVA analysis with method of Tukey‘s multiple comparisons of means (in R) 153 Abstract (in Korean) 돼지의 질병저항성을 연구하기 위한 SLA class II 다형성 분석 레민통 동물생명공학과 건국대학교 대학원 면역체계의 주요 요소들 중 하나인 주조직적합성 복합체는 포유동물과 같은 고등동물에서 내인성, 외인성 위험 인자들에 대한 숙주의 면역반응을 경정하 는 중요한 역할을 수행한다 돼지 주조직적합성 복합체는 돼지 백혈구 항원 유전자로도 불리는데, 양돈산업에서 중요하게 판단되는 생산형질과의 연관 성이 보고되었으며, 의생명과학 연구에서는 돼지의 대동물모델로서의 잠재 성에 과 관련하여 중요하게 인식되고 있다 다른 포유동물 종의 주조직적합 성 복합체와 마찬가지로, 돼지 백혈구 항원 유전자 또한 돼지게놈 상에서 가 장 큰 다형성 및 복잡성을 가진다 현재까지 MHC 분석을 위한 다양한 분석 방법들이 다양한 포유동물에서 개발되었으나, 동물의 MHC 시스템의 변이와 그 생물학적 의미를 분석하기위에서는 사람과 생쥐를 제외하고는 주조직적 합성복합체에 포함되는 유전자들에 대해서 해상도와 정확도를 향상시키기 위해서는 기술적인 발전이 필요하다 그러므로 본 연구에서는 genomic DNA에 기반한 SLA 좌위특이적 PCR을 개 발하고 클로닝 방식이 아닌 직접염기서열분석(direct sequencing) 기법과 연 결하여 SLA class II 유전자 중 가장 높은 다형성을 가지는 DQA, DQB1, DRB1 세개의 좌위에 대하여 고해상도 타이핑을 성공적으로 수행하였다 새 로 개발된 다좌위 동시증폭법은 각각의 좌위를 따로 증폭하는 방법과 비교 하였을 때 비용의 감소, 소량의 DNA를 이용가능, 여러 좌위의 동시 분석을 통한 분석실수의 감소 등에 있어 단일 좌위분석법에 비해 장점을 가진다 잠재적인 위험이 될 수 있는 병원균의 감염에 의한 소모성 질병이 돼지자돈 의 생존율에 영향을 미친다는 가설을 검증하기에 앞서, 우리는 이유 후 돼지 154 자돈 소모성 질병 같은 표현형의 발현에 대한 SLA class II의 베타체인 유전 자인 DQB1과 DRB1의 유전적 차이 및 효과를 확인하기 위한 첫 시도로서 케 이스 컨트롤 연구를 수행하였다 유전적 차이 즉 반수체형, 대립유전자, 유전 자별 영향등에 따른 강건성 및 질병감염 및 사망과 높은 연관성을 나타내는 변이들을 발견하였고, 펩티드 결합력에 대한 in silico 분석을 통해 상기 가설 이 부합됨을 설명할 수 있었다 In vitro 상태에서 SLA와 특정 질병 항원 사이 의 관계를 분석하기 위하여 SV40LT와 hTERT를 형질전환하여 돼지 영구세포 주를 구축하였다 이들 세포주는 SLA class I 과 class II 타이핑을 통해 다양 한 SLA 유전자형을 갖는 세포들로 이루어 졌으며, 기존에 SLA typing이 되어 있는 8개의 ATCC 세포주들을 비교 컨트롤로 사용하였다 구축된 세포는 40 번 이상의 계대를 거쳤으고, 세포 표현형이 매우 일정하고 비교적 빠른 세포 분열주기를 가지며, 지속적인 SLA class I 발현을 나타내었다 이렇게 구축된 SLA 세포 패널은 돼지의 면역유전학 연구에서 항원과 SLA와의 관계, SLA의 생물학적 다양성에 대한 연구 기초재료로 매우 중요하게 이용될 수 있을 것 으로 기대된다 주제어 : 돼지 백혈구 항원, 유전자형검사, 질병, 연관연구, 세포 패널 155 .. .Analysis of SLA class II polymorphism to study disease resistance in pigs A Dissertation submitted to the Department of Animal Biotechnology and the Graduate School of Konkuk University in. .. of innate immune responses relating to SLA haplotype also were reported Examining the response on three homozygous SLA- defined strains of miniature swine (SLAa, SLAc and SLAd) and one recombinant... improving pig health in general In attempt to dissert and verify in detail the influence of SLA to piglet survivability, in this study we carried out a case-control study with major SLA class II