新疆多浪羊和中国美利奴羊MHC-DRB1、DQB1基因SSCP多态性与包虫病抗性研究
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摘要
绵羊主要组织相容性复合体(Major histocompitibility complex, MHC)又称为绵羊淋巴细胞表面抗原(Ovine Lymphocyte Antigen, OLA),是由紧密连锁、高度多态的基因位点组成染色体上的一个遗传区域,其中MHC-Ⅱ类分子的DRB和DQB基因所编码的MHC抗原在其免疫系统中发挥重要的作用,与许多动物疾病的抗性、易感性、免疫应答以及生产性能都有密切关系。本试验对新疆多浪羊和中国美利奴羊(新疆军垦型)MHC-DRB1、DQB1基因PCR-SSCP多态性与包虫病抗性进行了关联分析,在此基础上,探讨了中国美利奴羊不同单倍型个体在人工感染细粒棘球绦虫过程中的免疫应答反应,为绵羊抗病育种及免疫遗传研究提供参考依据。
1、本试验利用PCR-SSCP方法,对新疆多浪羊MHC-DRB]、DQB1基因第2外显子进行遗传多态性分析。分析表明多浪羊DRB1和DQB1基因第2外显子均存在丰富的多态性。DRB1基因中出现了50种基因型,由22种等位基因控制。DQB1基因中出现了42种基因型,由17种等位基因控制。
2、检测了包虫病阴性和阳性多浪羊中DRB1基因出现的等位基因,利用T检验进行差异显著性分析,结果表明等位基因B(p<0.01)、A和C(p<0.05)在阴性羊中的频率显著高于阳性羊,认为B、A、C是包虫病的抗性相关等位基因。等位基因G(p<0.01)、M和R(p<0.05)在阳性羊中的频率显著高于阴性羊,认为G、M和R是包虫病的易感性相关等位基因。分析包虫病阴性和阳性羊的基因型频率,发现基因型BB(p<0.05)在阴性羊中的频率显著高于阳性羊,提示BB是包虫病抗性相关基因型。基因型GG(p<0.01)和MM(p<0.05)在阳性羊中的频率显著高于阴性羊,说明GG和MM是包虫病的易感性相关基因型。
3、进行包虫病阴性和阳性多浪羊DQB1基因中等位基因的差异显著性分析,分析表明等位基因H(p<0.05)和L(p<0.01)在阴性羊中的频率显著高于阳性羊,认为H、L是包虫病的抗性相关等位基因。等位基因J和M(p<0.01)在阳性羊中的频率显著高于阴性羊,认为J和M是包虫病的易感性相关等位基因。分析包虫病阴性和阳性羊的基因型,将它们的基因型频率进行差异显著性分析,结果表明基因型LL和DF(p<0.05)与包虫病抗性紧密相关。基因型MM(p<0.01)在阳性羊中的频率显著高于阴性羊,认为MM是包虫病的易感性相关基因型。
4、为了进一步分析多浪羊DRB1、DQB1基因多态性与包虫病之间的关系,筛选出抗包虫病的遗传标记,本研究分析了不同单倍型与包虫病抗性或易感性的相互关系。结果表明：单倍型DRB1-BB/DQB1-DF (P<0.01)和DRB1-JJ/DQB1-DH (P< 0.05)与包虫病抗性相关,而单倍型DRB1-GGIDQB1-MM (P<0.01)和DRB1-MMIDQB1-JJ (P<0.05)与包虫病易感性相关。
5、利用PCR-SSCP方法,分析中国美利奴羊MHC-DRB1、DQB基因第2外显子的多态性。分析显示,DRB1和DQB1基因第2外显子亦存在丰富的多态性。DRB1基因中出现了53种基因型,由22种等位基因控制。DQB1基因中出现了47种基因型,由17种等位基因控制。
6、利用T检验进行包虫病阴性和阳性中国美利奴羊DRB1基因的等位基因差异显著性分析,结果表明等位基因K(p<0.05)、Q和T(p<0.01)在阴性羊中的频率显著高于阳性羊,表明K、Q、T与包虫病的抗性相关,等位基因A和L(p<0.01)在阳性羊中的频率显著高于阴性羊,认为与包虫病的易感性相关。分析包虫病阴性和阳性中国美利奴羊的基因型,发现基因型KK和TT(p<0.05)在阴性羊中的频率显著高于阳性羊,表明KK和TT是包虫病抗性相关基因型。同样分析表明,基因型AA和LL(p<0.01)、AL(p<0.05)是包虫病的易感性相关基因型。
7、分析包虫病阴性和阳性中国美利奴羊DQB1基因出现的等位基因的差异,发现等位基因D和E(P<0.01)在阴性羊的频率显著高于阳性羊,说明D、E与包虫病的抗性相关。同样,结果表明等位基因C和K(p<0.01)与包虫病的易感性相关。分析包虫病阴性和阳性中国美利奴羊的基因型,发现基因型DD和EG(p<0.01)、EE(p<0.05)在阴性羊中的频率显著高于阳性羊,认为DD、EG和EE是包虫病抗性相关基因型。同样分析表明,基因型CC、KK和CK(p<0.01)是包虫病的易感性相关基因型。
8、为了更全面地分析中国美利奴羊DRB1、DQB基因多态性与包虫病之间的关系,分析了中国美利奴羊不同单倍型与包虫病抗性或易感性的相关性。结果表明单倍型DRB-TT/DQB-EE (P＜0.05)是包虫病抗性相关单倍型,而单倍型DRB1-LL/DQB1-CK和DRB1-AA/DQB1-C1与包虫病易感性相关。
9、在DRB1和DQB1基因多态性分析的基础上,筛选中国美利奴羊抗性单倍型DRB1-TT/DQB1-EE个体作为抗性组,非抗性单倍型个体作为对照组进行人工感染攻虫试验,分析不同的单倍型个体感染包虫病过程中的免疫应答反应。在攻虫前7天(-7d)、攻虫当天(0d)、攻虫后第7d、21d、30d、60d用流式细胞仪分析外周血中CD4+T、CD8+T、B淋巴细胞亚群的变化,同时用ELISA方法定量分析不同感染时期血清中IgG、IgM、IgE、IFN-y含量,并利用瑞式染色法进行粒细胞计数。结果表明,CD4+T和B淋巴细胞两组之间差异不显著(P>0.05),CD8+T细胞在攻虫后第7d抗性组显著高于对照组(P<0.05)：IgG水平在攻虫后第7d两组均显著高于攻虫前7d(P<0.05)。攻虫后第30d抗性组IgM和IFN-y水平均显著高于对照组(P<0.05)。抗性组的白细胞、淋巴细胞分别在攻虫后第7d、21d显著高于对照组(P<0.05),嗜中性粒细胞和总蛋白在攻虫后第21d抗性组显著低于对照组(P<0.01)。结论,抗性单倍型绵羊对E.g的抵抗力显著高于非抗性单倍型绵羊;抗性组绵羊体内的CD8+T细胞、IgM、IFN-y和白细胞、淋巴细胞水平在攻虫后不同时期显著高于非抗性组绵羊。
10、抗性羊DRB1基因表达量在攻虫后第60d显著高于攻虫Od(P<0.05)和攻虫后第30d(P<0.05)；非抗性羊DRB1基因表达量在攻虫后第21d显著高于攻虫0d(P<0.01)和攻虫后第7d(P<0.05),攻虫后第30d极显著低于攻虫后第21d(P<0.01)。抗性羊在攻虫后第21d(p<0.01)、60d(P<0.05)DQB1表达量显著高于攻虫0d,第30d时DQB1表达量显著低于攻虫后第21d(P<0.05)；对照组DQB1表达量同抗性组的变化趋势基本一致,在攻虫后第21d的表达量显著高于攻虫0d(P<0.05),在攻虫后第30d显著低于攻虫后第21d(p<0.05)。抗性羊与非抗性羊之间两种基因的表达无显著差异。
The Major Histocompatibility Complex of sheep, also called Ovine Lymphocyte Antigen (OLA), is part of chromosomal region consisting of a group of closely linked and highly polymorphic loci, in MHC-Ⅱmolecules, the MHC antigen encoded by DRB and DQB genes in plays an important role in the immune system, which is closely related to many animal disease resistance, susceptibility, immune response and production performance. In this research, polymorphism of DRB1/DQB1 exon 2 and its resistance to Cystic Echinococcosis (C.E) were analyzed in Dolang sheep and Chinese Merino (Xinjiang Army Wasteland Reclamation) sheep, on the base of polymorphic analysis, to explore the immune response of different haplotypes in Chinese Merino sheep during the process of Echinococcus granulosus infection, which would provide an important basis and theoretical support for resist-breeding of sheep in the future..
1. In this experiment, PCR-SSCP method was used to analyze the genetic polymorphism of MHC-DRB1/DQB1 gene exon 2 in Dolang sheep. The results showed that exon 2 of DRB1 and DQB1 gene had a high degree of polymorphism. While a total of 50 genotypes were identified in DRB1 gene, which were controlled by 22 alleles; 42 genotypes were identified in DQB] which were controlled by 17 alleles.
2. The frequency of allele and genotype of DRB1 in C.E-negative and-positive Dolang sheep were analyzed by T-test, to assess the relationship between different genotypes and C.E significance. Firstly, the allele frequencies of DRB1 were ananlyzed. Results were sumerized as follows:i) Sigficantly higher allele frequencies interms of B (P< 0.01), A and C (P< 0.05) occured in the negative sheep when compared with the positive sheep, implying that B, A and C ralated to hydatidosis resistance, ii) Sigficantly higher allele frequencies interms of G (P< 0.01), M and R (P< 0.05) occured in the positive sheep when compared with the negative sheep, implying that G, M and R ralated to hydatidosis susceptibility. In addition, the genotypes frequencies of DRB1 were also tested. Results were sumerized as follows:i) Genotype BB (P< 0.05) occured more often in the negative sheep when compared with the positive sheep, implying that genotype BB was related to hydatidosis resistance, ii) Genotypes GG (P< 0.01) and MM (P < 0.05) occured more often in the positive sheep when compared with the negative sheep, implying that genotypes GG and MM were related to hydatidosis susceptibility.
3. The allele frequemcy of DQB1 were analyzed which occurred in positive and negative echinococcosis of Dolang sheep, results showed that the allele frequency of H (P< 0.05) and L (P< 0.01) in the negative sheep were significantly higher than the positive sheep, to consider that the H and L is the resistance allele of hydatids disease, while the frequency of J and M (P< 0.01) in the positive sheep was significantly higher than negative sheep, that J and M are the susceptible to hydatid disease. To further analyze the genotype frequency of negative and positive hydatid disease of sheep, the results showed that the genotype frequency of LL and DF (P< 0.05) in the negative sheep were significantly higher than the positive sheep, that LL and DF genotype are associated echinococcosis resistance. The genotype frequency of MM(P< 0.01)in the positive sheep was significantly higher than negative sheep, that MM is hydatid disease susceptibility genotype.
4. In order to select the resistant genic mark, the relationship between different haplotypes and resistance or susceptibility to C.E in Dolang Sheep was analyzed.The results showed that individuals with the genic haplotype DRB1-BB/DQB1-DF (P< 0.01) and DRB1-JJ/DQB1-DH (P < 0.05) were relatively resistant to Echinococcus granulosus (E.g), while individuals with the haplotypes DRB1-GGIDQB1-MM (P<0.01) and DRB1-MM/DQB1-JJ (P<0.05) were more susceptible to E.g.
5. The polymorphism of MHC-DRB1/IDQB1 gene exon 2 in Chinese Merino sheep were analyzed by PCR-SSCP method, the results showed that polymorphisms in exon 2 of DRB1/IDQB1 gene are abundant there, emergence 53 genotypes in DRB1 gene, which controlled by 22 alleles; 47 genotypes in DQB1 gene, which controlled by 17 alleles.
6. The allele and genotype frequency of DRB1 gene exon 2 in C.E positive and negative in Chinese Merino sheep was analyzed by T-test, the results showed that the allele K (P< 0.05),Q and T (P< 0.01) are related with resistance of C.E, while A and L (P< 0.01) are related with susceptivity of C.E. We further to analyze the genotype frequency found that KK and TT (P< 0.05) are the resistant to C.E, the genotype AA and LL (P< 0.01). AL (P< 0.05) are the susceptible to C.E.
7. The alleles of DQB1 were ananlyzed which occurred in positive and negative C.E in Chinese Merino sheep, results showed that the allele frequency of D and E (P< 0.01) in the negative sheep were significantly higher than the positive sheep, to consider that the D and E are the resistance allele to C.E, while the frequency of C and K (P< 0.01) in the positive sheep were significantly higher than negative sheep, that C and K are the susceptible to C.E. To further analyze the genotype frequency of negative and positive C.E of sheep, the results showed that the genotype frequency of DD and EG (P< 0.01) in the negative sheep were significantly higher than the positive sheep, that DD and EG genotype are associated with C.E resistance. The genotype frequency of CC and KK,CK (P< 0.01) in the positive sheep were significantly higher than negative sheep, that CC, KK and CK are associated with susceptivity to C.E.
8. In order to efficiently analyze the the polymorphism of DRB1/DQB1 gene and C.E, the relationship between different haplotypes and resistance or susceptibility to C.E was researched in Chinese Merino Sheep. The result found that individuals with the genie haplotype DRB1-TT/DQB1-EE (P< 0.05) are resist to C.E, while DRB1-LL/DQB1-CK and DRB1-AA/DQB1-CC (P<0.01) are susceptible to C.E.
9. Based on the polymorphism analysis of DRB1 and DQB1 gene exon 2 in Chinese Merino sheep and Dolang sheep,16 Chinese Merino sheep with different haplotypes were artificially infected with Echinococcus granulosus (E.g) at the identical raising condition,8 sheep with resistant haplotype were selected as the test group, while 8 individuals with nonresistant haplotype were selected as the control group. The CD4+T, CD8+T, B lymphocyte subpopulations of peripheral blood were analyzed by flow cytometry at day (-7),0,7,21,30 and 60 after the artificial infection, meanwhile, the content of IgG, lgM, IgE, IFN-y of the blood serum and granulocyte quantity in the blood were also tested. The results showed that the incidence of Cystic Echinococcosis in the test group was significantly lower than that of the control group (P< 0.05), there was no significant difference of CD4+T cells percentage between the two groups (P>0.05). CD8+T cells of the test group were significantly higher than that of the control group (P< 0.05) at 7th day after infection., IgG level of the two groups at 7th day after infection were significantly higher than that at the (-7)th day (P<0.05). IgM and IFN-y of the test group were significantly higher than that of the control group (P< 0.05) at 30th day after infection. Leukocytes and lymphocytes of the test group were significantly more than that of the control group (P< 0.05) at 7th,21st day after infection. Neutrophils and albumin of the test group were obviously lower than the control group (P<0.01) at 21st day. (Conclusion) Significantly higher resistance to Echinococcus granulosus occurred in the resistant haplotype sheep, when compared with the nonresistant haplotype individuals. CD8+T cells, IgM, IFN-y, leukocytes and lymphocytes in the resistant haplotype sheep were significantly higher than those of nonresistant haplotype individuals during the different period after infection.
10. DRB1 gene expression of the test group at 60 d after infection was significantly higher than at the 0 d (P< 0.05) and 30 d (P< 0.05); the expression of the control group at 21 d was significantly higher than at the 0 d (P< 0.01) and 7d (P< 0.05), and the expression at 30 d significantly lower at 21 d (P< 0.01).To analyze the DQB1 expression, the result showed that the expression of the resistant sheep at 21 d (P< 0.01) and 60 d (P< 0.05) were significantly higher than at the 0 d, the expression at the 30 d was significantly lower than at the 21 d (P< 0.05). The change tendency of DQB1 expression in the control group was similar to the test group, the expression at 21 d was significantly higher than at the 0 d (P< 0.05), and the expression at the 30 d was significantly lower than at the 21 d (P< 0.05). (Conclusion) The expression of DRB1 and DQB1 gene between the resistant and nonresistant sheep was no significant deviation.
1、本试验利用PCR-SSCP方法,对新疆多浪羊MHC-DRB]、DQB1基因第2外显子进行遗传多态性分析。分析表明多浪羊DRB1和DQB1基因第2外显子均存在丰富的多态性。DRB1基因中出现了50种基因型,由22种等位基因控制。DQB1基因中出现了42种基因型,由17种等位基因控制。
2、检测了包虫病阴性和阳性多浪羊中DRB1基因出现的等位基因,利用T检验进行差异显著性分析,结果表明等位基因B(p<0.01)、A和C(p<0.05)在阴性羊中的频率显著高于阳性羊,认为B、A、C是包虫病的抗性相关等位基因。等位基因G(p<0.01)、M和R(p<0.05)在阳性羊中的频率显著高于阴性羊,认为G、M和R是包虫病的易感性相关等位基因。分析包虫病阴性和阳性羊的基因型频率,发现基因型BB(p<0.05)在阴性羊中的频率显著高于阳性羊,提示BB是包虫病抗性相关基因型。基因型GG(p<0.01)和MM(p<0.05)在阳性羊中的频率显著高于阴性羊,说明GG和MM是包虫病的易感性相关基因型。
3、进行包虫病阴性和阳性多浪羊DQB1基因中等位基因的差异显著性分析,分析表明等位基因H(p<0.05)和L(p<0.01)在阴性羊中的频率显著高于阳性羊,认为H、L是包虫病的抗性相关等位基因。等位基因J和M(p<0.01)在阳性羊中的频率显著高于阴性羊,认为J和M是包虫病的易感性相关等位基因。分析包虫病阴性和阳性羊的基因型,将它们的基因型频率进行差异显著性分析,结果表明基因型LL和DF(p<0.05)与包虫病抗性紧密相关。基因型MM(p<0.01)在阳性羊中的频率显著高于阴性羊,认为MM是包虫病的易感性相关基因型。
4、为了进一步分析多浪羊DRB1、DQB1基因多态性与包虫病之间的关系,筛选出抗包虫病的遗传标记,本研究分析了不同单倍型与包虫病抗性或易感性的相互关系。结果表明：单倍型DRB1-BB/DQB1-DF (P<0.01)和DRB1-JJ/DQB1-DH (P< 0.05)与包虫病抗性相关,而单倍型DRB1-GGIDQB1-MM (P<0.01)和DRB1-MMIDQB1-JJ (P<0.05)与包虫病易感性相关。
5、利用PCR-SSCP方法,分析中国美利奴羊MHC-DRB1、DQB基因第2外显子的多态性。分析显示,DRB1和DQB1基因第2外显子亦存在丰富的多态性。DRB1基因中出现了53种基因型,由22种等位基因控制。DQB1基因中出现了47种基因型,由17种等位基因控制。
6、利用T检验进行包虫病阴性和阳性中国美利奴羊DRB1基因的等位基因差异显著性分析,结果表明等位基因K(p<0.05)、Q和T(p<0.01)在阴性羊中的频率显著高于阳性羊,表明K、Q、T与包虫病的抗性相关,等位基因A和L(p<0.01)在阳性羊中的频率显著高于阴性羊,认为与包虫病的易感性相关。分析包虫病阴性和阳性中国美利奴羊的基因型,发现基因型KK和TT(p<0.05)在阴性羊中的频率显著高于阳性羊,表明KK和TT是包虫病抗性相关基因型。同样分析表明,基因型AA和LL(p<0.01)、AL(p<0.05)是包虫病的易感性相关基因型。
7、分析包虫病阴性和阳性中国美利奴羊DQB1基因出现的等位基因的差异,发现等位基因D和E(P<0.01)在阴性羊的频率显著高于阳性羊,说明D、E与包虫病的抗性相关。同样,结果表明等位基因C和K(p<0.01)与包虫病的易感性相关。分析包虫病阴性和阳性中国美利奴羊的基因型,发现基因型DD和EG(p<0.01)、EE(p<0.05)在阴性羊中的频率显著高于阳性羊,认为DD、EG和EE是包虫病抗性相关基因型。同样分析表明,基因型CC、KK和CK(p<0.01)是包虫病的易感性相关基因型。
8、为了更全面地分析中国美利奴羊DRB1、DQB基因多态性与包虫病之间的关系,分析了中国美利奴羊不同单倍型与包虫病抗性或易感性的相关性。结果表明单倍型DRB-TT/DQB-EE (P＜0.05)是包虫病抗性相关单倍型,而单倍型DRB1-LL/DQB1-CK和DRB1-AA/DQB1-C1与包虫病易感性相关。
9、在DRB1和DQB1基因多态性分析的基础上,筛选中国美利奴羊抗性单倍型DRB1-TT/DQB1-EE个体作为抗性组,非抗性单倍型个体作为对照组进行人工感染攻虫试验,分析不同的单倍型个体感染包虫病过程中的免疫应答反应。在攻虫前7天(-7d)、攻虫当天(0d)、攻虫后第7d、21d、30d、60d用流式细胞仪分析外周血中CD4+T、CD8+T、B淋巴细胞亚群的变化,同时用ELISA方法定量分析不同感染时期血清中IgG、IgM、IgE、IFN-y含量,并利用瑞式染色法进行粒细胞计数。结果表明,CD4+T和B淋巴细胞两组之间差异不显著(P>0.05),CD8+T细胞在攻虫后第7d抗性组显著高于对照组(P<0.05)：IgG水平在攻虫后第7d两组均显著高于攻虫前7d(P<0.05)。攻虫后第30d抗性组IgM和IFN-y水平均显著高于对照组(P<0.05)。抗性组的白细胞、淋巴细胞分别在攻虫后第7d、21d显著高于对照组(P<0.05),嗜中性粒细胞和总蛋白在攻虫后第21d抗性组显著低于对照组(P<0.01)。结论,抗性单倍型绵羊对E.g的抵抗力显著高于非抗性单倍型绵羊;抗性组绵羊体内的CD8+T细胞、IgM、IFN-y和白细胞、淋巴细胞水平在攻虫后不同时期显著高于非抗性组绵羊。
10、抗性羊DRB1基因表达量在攻虫后第60d显著高于攻虫Od(P<0.05)和攻虫后第30d(P<0.05)；非抗性羊DRB1基因表达量在攻虫后第21d显著高于攻虫0d(P<0.01)和攻虫后第7d(P<0.05),攻虫后第30d极显著低于攻虫后第21d(P<0.01)。抗性羊在攻虫后第21d(p<0.01)、60d(P<0.05)DQB1表达量显著高于攻虫0d,第30d时DQB1表达量显著低于攻虫后第21d(P<0.05)；对照组DQB1表达量同抗性组的变化趋势基本一致,在攻虫后第21d的表达量显著高于攻虫0d(P<0.05),在攻虫后第30d显著低于攻虫后第21d(p<0.05)。抗性羊与非抗性羊之间两种基因的表达无显著差异。
The Major Histocompatibility Complex of sheep, also called Ovine Lymphocyte Antigen (OLA), is part of chromosomal region consisting of a group of closely linked and highly polymorphic loci, in MHC-Ⅱmolecules, the MHC antigen encoded by DRB and DQB genes in plays an important role in the immune system, which is closely related to many animal disease resistance, susceptibility, immune response and production performance. In this research, polymorphism of DRB1/DQB1 exon 2 and its resistance to Cystic Echinococcosis (C.E) were analyzed in Dolang sheep and Chinese Merino (Xinjiang Army Wasteland Reclamation) sheep, on the base of polymorphic analysis, to explore the immune response of different haplotypes in Chinese Merino sheep during the process of Echinococcus granulosus infection, which would provide an important basis and theoretical support for resist-breeding of sheep in the future..
1. In this experiment, PCR-SSCP method was used to analyze the genetic polymorphism of MHC-DRB1/DQB1 gene exon 2 in Dolang sheep. The results showed that exon 2 of DRB1 and DQB1 gene had a high degree of polymorphism. While a total of 50 genotypes were identified in DRB1 gene, which were controlled by 22 alleles; 42 genotypes were identified in DQB] which were controlled by 17 alleles.
2. The frequency of allele and genotype of DRB1 in C.E-negative and-positive Dolang sheep were analyzed by T-test, to assess the relationship between different genotypes and C.E significance. Firstly, the allele frequencies of DRB1 were ananlyzed. Results were sumerized as follows:i) Sigficantly higher allele frequencies interms of B (P< 0.01), A and C (P< 0.05) occured in the negative sheep when compared with the positive sheep, implying that B, A and C ralated to hydatidosis resistance, ii) Sigficantly higher allele frequencies interms of G (P< 0.01), M and R (P< 0.05) occured in the positive sheep when compared with the negative sheep, implying that G, M and R ralated to hydatidosis susceptibility. In addition, the genotypes frequencies of DRB1 were also tested. Results were sumerized as follows:i) Genotype BB (P< 0.05) occured more often in the negative sheep when compared with the positive sheep, implying that genotype BB was related to hydatidosis resistance, ii) Genotypes GG (P< 0.01) and MM (P < 0.05) occured more often in the positive sheep when compared with the negative sheep, implying that genotypes GG and MM were related to hydatidosis susceptibility.
3. The allele frequemcy of DQB1 were analyzed which occurred in positive and negative echinococcosis of Dolang sheep, results showed that the allele frequency of H (P< 0.05) and L (P< 0.01) in the negative sheep were significantly higher than the positive sheep, to consider that the H and L is the resistance allele of hydatids disease, while the frequency of J and M (P< 0.01) in the positive sheep was significantly higher than negative sheep, that J and M are the susceptible to hydatid disease. To further analyze the genotype frequency of negative and positive hydatid disease of sheep, the results showed that the genotype frequency of LL and DF (P< 0.05) in the negative sheep were significantly higher than the positive sheep, that LL and DF genotype are associated echinococcosis resistance. The genotype frequency of MM(P< 0.01)in the positive sheep was significantly higher than negative sheep, that MM is hydatid disease susceptibility genotype.
4. In order to select the resistant genic mark, the relationship between different haplotypes and resistance or susceptibility to C.E in Dolang Sheep was analyzed.The results showed that individuals with the genic haplotype DRB1-BB/DQB1-DF (P< 0.01) and DRB1-JJ/DQB1-DH (P < 0.05) were relatively resistant to Echinococcus granulosus (E.g), while individuals with the haplotypes DRB1-GGIDQB1-MM (P<0.01) and DRB1-MM/DQB1-JJ (P<0.05) were more susceptible to E.g.
5. The polymorphism of MHC-DRB1/IDQB1 gene exon 2 in Chinese Merino sheep were analyzed by PCR-SSCP method, the results showed that polymorphisms in exon 2 of DRB1/IDQB1 gene are abundant there, emergence 53 genotypes in DRB1 gene, which controlled by 22 alleles; 47 genotypes in DQB1 gene, which controlled by 17 alleles.
6. The allele and genotype frequency of DRB1 gene exon 2 in C.E positive and negative in Chinese Merino sheep was analyzed by T-test, the results showed that the allele K (P< 0.05),Q and T (P< 0.01) are related with resistance of C.E, while A and L (P< 0.01) are related with susceptivity of C.E. We further to analyze the genotype frequency found that KK and TT (P< 0.05) are the resistant to C.E, the genotype AA and LL (P< 0.01). AL (P< 0.05) are the susceptible to C.E.
7. The alleles of DQB1 were ananlyzed which occurred in positive and negative C.E in Chinese Merino sheep, results showed that the allele frequency of D and E (P< 0.01) in the negative sheep were significantly higher than the positive sheep, to consider that the D and E are the resistance allele to C.E, while the frequency of C and K (P< 0.01) in the positive sheep were significantly higher than negative sheep, that C and K are the susceptible to C.E. To further analyze the genotype frequency of negative and positive C.E of sheep, the results showed that the genotype frequency of DD and EG (P< 0.01) in the negative sheep were significantly higher than the positive sheep, that DD and EG genotype are associated with C.E resistance. The genotype frequency of CC and KK,CK (P< 0.01) in the positive sheep were significantly higher than negative sheep, that CC, KK and CK are associated with susceptivity to C.E.
8. In order to efficiently analyze the the polymorphism of DRB1/DQB1 gene and C.E, the relationship between different haplotypes and resistance or susceptibility to C.E was researched in Chinese Merino Sheep. The result found that individuals with the genie haplotype DRB1-TT/DQB1-EE (P< 0.05) are resist to C.E, while DRB1-LL/DQB1-CK and DRB1-AA/DQB1-CC (P<0.01) are susceptible to C.E.
9. Based on the polymorphism analysis of DRB1 and DQB1 gene exon 2 in Chinese Merino sheep and Dolang sheep,16 Chinese Merino sheep with different haplotypes were artificially infected with Echinococcus granulosus (E.g) at the identical raising condition,8 sheep with resistant haplotype were selected as the test group, while 8 individuals with nonresistant haplotype were selected as the control group. The CD4+T, CD8+T, B lymphocyte subpopulations of peripheral blood were analyzed by flow cytometry at day (-7),0,7,21,30 and 60 after the artificial infection, meanwhile, the content of IgG, lgM, IgE, IFN-y of the blood serum and granulocyte quantity in the blood were also tested. The results showed that the incidence of Cystic Echinococcosis in the test group was significantly lower than that of the control group (P< 0.05), there was no significant difference of CD4+T cells percentage between the two groups (P>0.05). CD8+T cells of the test group were significantly higher than that of the control group (P< 0.05) at 7th day after infection., IgG level of the two groups at 7th day after infection were significantly higher than that at the (-7)th day (P<0.05). IgM and IFN-y of the test group were significantly higher than that of the control group (P< 0.05) at 30th day after infection. Leukocytes and lymphocytes of the test group were significantly more than that of the control group (P< 0.05) at 7th,21st day after infection. Neutrophils and albumin of the test group were obviously lower than the control group (P<0.01) at 21st day. (Conclusion) Significantly higher resistance to Echinococcus granulosus occurred in the resistant haplotype sheep, when compared with the nonresistant haplotype individuals. CD8+T cells, IgM, IFN-y, leukocytes and lymphocytes in the resistant haplotype sheep were significantly higher than those of nonresistant haplotype individuals during the different period after infection.
10. DRB1 gene expression of the test group at 60 d after infection was significantly higher than at the 0 d (P< 0.05) and 30 d (P< 0.05); the expression of the control group at 21 d was significantly higher than at the 0 d (P< 0.01) and 7d (P< 0.05), and the expression at 30 d significantly lower at 21 d (P< 0.01).To analyze the DQB1 expression, the result showed that the expression of the resistant sheep at 21 d (P< 0.01) and 60 d (P< 0.05) were significantly higher than at the 0 d, the expression at the 30 d was significantly lower than at the 21 d (P< 0.05). The change tendency of DQB1 expression in the control group was similar to the test group, the expression at 21 d was significantly higher than at the 0 d (P< 0.05), and the expression at the 30 d was significantly lower than at the 21 d (P< 0.05). (Conclusion) The expression of DRB1 and DQB1 gene between the resistant and nonresistant sheep was no significant deviation.
引文
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