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One of the major histocompatibility complex (Major Histocompatibility Complex, MHC) is tightly linked to by a group of highly polymorphic loci on chromosome gene system. MHC in a vertebrate immune response has an important role, it is not only encode transplantation antigens, control graft rejection, and is also involved in immune response regulation and immune recognition, wherein the most important function of antigen presentation. MHC genes have a wealth of polymorphism, and has a very close relationship between the susceptibility to disease and animal production performance, is the candidate of the Origin of Species, evolution and animal breeding for disease resistance marker gene. The MHC, also known as swine leukocyte antigen (the the Swine Lymphocyte Antigen SLA), divided into Ⅰ, Ⅱ, Ⅲ class play immune function, and pig disease resistance strength of the SLA II antigens for exogenous antigen close contact. Currently, SLA II: DRA, DRB, DQA, DQB and other physical positioning of the gene or gene cluster. Human major histocompatibility antigen system is referred to as HLA, and in the mouse is called the H-2, the SLA with human HLA and H-2 have a certain similarity. Many foreign scholars in recent years, experimental pigs used the SLA their genetic background and characteristics of in-depth research, and has laid a theoretical foundation to elucidate the mechanism of the human immune system recognizes the porcine source dissimilar organizations antigen. Domestic research in the late start, and not for the the Chinese pigs applicable xenograft SLA gene structure, its pig - human xenotransplantation research reported. Experimental Animal Center of Southern Medical University since 2004, introduced from Tibet Gongbo'gyamda 50 original species of Tibetan miniature pigs, after more than four years of nurturing, not only to complete the process of acclimatization and experimental animals nurture the Science and Technology Department of Guangdong Province issued experiments with the general level of miniature pigs certificate (license number of experimental animals: SCXK (Guangdong) 2006-0015; Certificate of Conformity: Cantonese and Certificate word 2006A055). Currently, there are 500 head of population size, this study Experimental Animal Center of my school own cultivation experiments for the study of Tibetan miniature pig. Employed a random sampling method to obtain 60 Tibet small pig blood samples, the use of comparative genomics, and PCR's success to obtain a located pigs on the 7th chromosome of MHC Ⅱ class district SLA-DQA and SLA-DQB gene of the second outer significant sub the fragment (including the complete exon 2, Part 2 introns and small part of the first intron). Restriction endonuclease enzyme amplification products were digested and analyzed by electrophoresis imaging digestion with type two genes, identify and calculate the different genotypes and allele frequency. Chi-square distribution of restriction sites with the fit test, to determine whether the various sites on the gene frequency equilibrium reached Hardy-Weiberg. Simultaneously compare the test results reported in the literature of other varieties of miniature pigs to explore Tibetan miniature pig whether mutations in these loci or its unique genetic traits. The PCR products were purified clones, cloning the DNA fragments were directly sequenced MHC Ⅱ class area of ??Tibetan miniature pig SLA-DQA, SLA-DQB gene exon 2 nucleotide sequence in GenBank other breeds of pig, human, mouse, and dog SLA-DQA, SLA-DQB gene corresponding sequence alignment analysis, to explore the homology between them as well as the origin of the Tibetan miniature pig and genetic mechanism of evolution. In the present study, using EcoR Ⅰ and Alu Ⅰ SLA-DQA gene digested the results of each of the three genotypes (AA, AB and BB; MM, MN and NN) and two alleles (A and B; M and N). EcoR Ⅰ digestion, homozygous genotype BB mostly derived from AA, AB and BB three genotypes frequencies of 23.333%, 31.667% and 45.000%, respectively, where B is the dominant allele (60.833%); After Alu Ⅰ digested, the resulting three genotype frequency distribution for the MN-type (50.000%), respectively, higher than the MM type (30.000%) and NN-type (20.000%), where M is the dominant allele (55.000%). Comprehensive digested results, there were seven combinations with type are: AAMM, AANN ABMM ABMN ABNN BBMM and BBMN, which BBMN combination with type highest frequency (30.000%), yet found AANN and BBNN two combinations with type. Hae Ⅲ and Rsa Ⅰ SLA-DQB gene digested by Hae Ⅲ digested draw four alleles (A, B, C, D) and five genotypes (AA, BB, BC, DD AD). SLA-DQB gene exon 2 to BC Hae Ⅲ-RFLP loci genotypes majority (33.333%), B is the dominant gene (33.333%); digested by Rsa Ⅰ draw three alleles (E, F , G) and five genotypes (EE, EF, EG, FF, GG). Rsa Ⅰ-RFLP site EF genotype majority (33.333%), E is the dominant allele (48.333%). This experiment, the SLA-DQB gene Tibetan miniature pig there are seven kinds of combination of PCR-RFLP genotype respectively: of AAEE, AAFF ADEG ADGG BBEE BCEF and DDGG, Hae Ⅲ-RFLP sites on the BC genotype EF genotype frequency corresponds exactly to the frequency with Rsa Ⅰ-RFLP sites, in combination with type BCEF the highest frequency (33.333%). Polymorphism information content (PIC) calculation results show that: Tibetan miniature pig showed moderate SLA-DQA and SLA-DQB gene polymorphism (0.25 DQA <0.5) and height polymorphism (PIC DQB > 0.5). Fit tested by chi-square, Tibetan miniature pig SLA-DQA EcoR Ⅰ restriction sites reached the Hardy-Weiberg equilibrium (x 2 = 4.300, P> 0.05, a = 0.05), while Alu Ⅰ restriction sites did not reach the Hardy-Weiberg equilibrium gene frequency x (x 2 = 8.400, P <0.05, a = 0.05); SLA-DQB gene Hae Ⅲ and Rsa Ⅰ restriction sites 2 value (7.200 and 2.933, respectively) in the a = 0.05 level did not reach a significant level, indicating that the DQB genes of the two sites in genetic equilibrium. Cloned and sequenced fat distribution obtained SLA-DQA, DQB gene exon 2 nucleotide sequences were detected in the resulting sequence to the base sequence of EcoR Ⅰ, Alu Ⅰ restriction sites: 5 '... G ↓ AATTC ... 3 'and 5' ... AG ↓ CT ... 3 ', and Hae Ⅲ, Rsa Ⅰ restriction sites base sequence: 5' ... GG ↓ CC ... 3 'and 5' ... GT ↓ AC ... 3 'sequencing results further show that the The obtained PCR amplification product is the objective gene fragment. DQB sequence of the second exon of GC content of 64.83%, while the lower DQA exon 2 of the GC content of only 45%. Will be included in the sequencing results with GenBank SLA-DQA the DQB gene sequence and the human HLA-DQA, DQB sequences BLAST comparison, the results included in the Tibetan miniature pig SLA-DQA sequences in GenBank accession number BX088590 The pig sequence homology of up to 99%, mutate only two bases in section 83 (C → T) and 131 points (T → A). The sequence (XM 0 01129369) and mice (NM 0 10378.2) the DQA corresponding sequence homology of 83% and 71%, respectively. Wuzhishan miniature pig research reports than Wu Qun (2004) as well as Yunnan Banna miniature pig SLA-DQA gene homologous to the corresponding the HLA high frequency allele percentage (respectively 80.87% and 81.96%) is slightly higher. Included in the Tibetan miniature pig SLA-DQB sequences in GenBank accession number AY769655 (Tibetan pig), AY769646 (Jinhua pig), and AY626114 (Yunnan wild boar) sequence homology of up to 98.2%, only one variable sites, of which the first both T → A mutation in the first 26 points, Yunnan wild boar G → C mutation in 67 points. The Miniature Pig homology of the sequence with the human HLA-DQB1 * 0305GR and HLA-DQB1 * 03011 allele was 82.4% and 81.7%, respectively, the results than Wu Qun research reports SLA-DQB gene HLA high frequency allele of the homologous percentage (79.31%) is slightly higher, but slightly lower than the Yunnan Banna miniature pig homology percentage (82.38%). In this study, the following conclusions: the center relative to the experimental Tibet mini pig populations SLA-DQB gene exon 2 of SLA-DQA richer polymorphism. Tibetan miniature pig dominant homozygous digested with type enzyme sites in these two genes, prompted two genes of small pig populations in Tibet has reached a high homozygosity as experimental animals used in life science research with unique advantages sex. Fit test, chi-square for each restriction sites genotype and the only Alu Ⅰ restriction sites genotype distribution in Hardy-Weinberg equilibrium, which may be long-term evolution of selected Tibetan miniature pig in specific climates whether their specific high stress resistance has a direct correlation, pending further evidence. PCR product was cloned and sequenced to obtain the SLA-DQA DQB gene exon 2 nucleotide sequence, and some other breeds than point mutations, and the corresponding sequence of human HLA high homology Wuzhishan pigs and Banna miniature pig, is also higher than that of mice. Experimental Tibet mini pigs more suitable for use as a xenograft. The phylogenetic tree clustering analysis showed that the Tibetan miniature pig and Taihu pigs and wild boar in Yunnan kinship is more recent than the other domestic breeds. This retrospective Tibetan miniature pig germline occurred, and to study the mechanisms of genetic evolution provides an important clue.
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