猪13号染色体q41区的3个功能基因STS多态性与产肠毒素大肠杆菌F4ab／ac易感性相关

由产肠毒素大肠杆菌（Escherichia coli）F4（ETECF4）引起的断奶前仔猪腹泻病是一种常见细菌感染病,对养猪业造成了巨大经济损失．编码ETECF4ab／ac受体的位点已被定位于猪（Susscrofa）13号染色体（SSC13）q41区域,S0075为最紧密连锁的标记之一．本研究从S0075侧翼染色体区域选择了SLC12A8,MYLK和KPNAI3个基因,建立猪特异性序列标签位点（STS）．利用白色杜洛克×二花脸资源家系群体中的ETECF4ab／ac侵染抗性和易感个体,通过比较测序法,建立了这3个基因的STS,并鉴别到7个单核苷酸多态位点．进一步检测了白色杜洛克×二花脸资源家系群体祖代、亲本代和755头F2个体在SLC12A8g．159A〉G,MYLKg．1673A〉G和KPNA1g．306A〉G多态位点的基因型．传递不平衡检测（TDT）分析表明：这些多态位点和相应的单倍型与ETECF4ab／ac（特别是F4ac）刷状缘黏附表型存在显著相关,表明这些多态位点和单倍型与ETECF4ab／ac受体的编码基因因果突变存在连锁不平衡．本研究进一步证实了SSC13q41存在ETECF4ab/ac侵染易感性的遗传位点,为ETECF4ab／ac受体基因的精细定位提供了新型多态标记．     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

大肠杆菌F18菌毛及其亚型的PCR鉴定

F18菌毛是产肠毒素大肠杆菌 (ETEC)与产vero细胞毒素大肠杆菌 (VTEC)的重要致病因子 ,可介导细菌对小肠细胞的黏附 ,并具有F18ab和F18ac 2个抗原亚型。根据已发表的F18ab菌毛A亚单位 (FedA ab)的基因 (fedA ab)设计 3条引物 ,建立了 2种聚合酶链式反应 (PCR)扩增方法。通过对F18ab 大肠杆菌、F18ac 大肠杆菌、K88 大肠杆菌、K99 大肠杆菌、987P 大肠杆菌、F4 1 大肠杆菌的试验 ,结果表明所建立的PCR方法可特异性鉴定F18 大肠杆菌并区别其亚型F18ab与F18ac     

Ⅴ型分泌系统菌体表面展示F18大肠杆菌黏附素及其受体结合位点的确定

自主转运蛋白(V型分泌系统)的β结构域已被证明可以将异源性多肽展示在细菌表面。运用DNA重组技术优化构建V型分泌系统MisL并在菌体表面展示F18大肠杆菌黏附素FedF及其受体结合域FedF1。含重组质粒pnirBMisL-fedF或pnirBMisL-fedF1大肠杆菌(E.coli)DH5α经厌氧诱导后,分别与兔抗F18ab菌毛FedF亚单位单因子血清和F18大肠杆菌黏附素受体易感性仔猪的小肠上皮细胞做玻板凝集试验和体外黏附试验,结果表明上述两株诱导表达重组菌与FedF抗血清发生明显的凝集反应,且能较好地黏附于F18大肠杆菌黏附素受体易感性仔猪小肠上皮细胞。而菌体表面展示F18大肠杆菌黏附素FedF突变体FedF(M)(黏附素受体结合域第88和89位组氨酸残基双突变为丙氨酸)的重组菌则失去上述凝集和黏附特性。以上试验结果说明,F18大肠杆菌黏附素FedF及其受体结合域FedF1在大肠杆菌表面得到了功能性表达,并进一步证明了位于FedF受体结合域内第88和89位组氨酸残基对FedF受体结合域的形成至关重要。     

V型分泌系统菌体表面展示F18大肠杆菌黏附素及其受体结合位点的确定

自主转运蛋白（V型分泌系统）的β结构域已被证明可以将异源性多肽展示在细菌表面。运用DNA重组技术优化构建V型分泌系统MisL并在菌体表面展示F18大肠杆菌黏附素FedF及其受体结合域FedF1。含重组质粒pnirBMisL-fedF或pnirBMisL-fedF1大肠杆菌（E．coli）DH5α经厌氧诱导后,分别与兔抗F18ab菌毛FedF亚单位单因子血清和F18大肠杆菌黏附素受体易感性仔猪的小肠上皮细胞做玻板凝集试验和体外黏附试验,结果表明上述两株诱导表达重组菌与FedF抗血清发生明显的凝集反应,且能较好地黏附于F18大肠杆菌黏附素受体易感性仔猪小肠上皮细胞。而菌体表面展示F18大肠杆菌黏附素FedF突变体FedF（M）（黏附素受体结合域第88和89位组氨酸残基双突变为丙氨酸）的重组菌则失去上述凝集和黏附特性。以上试验结果说明,F18大肠杆菌黏附素FedF及其受体结合域FedF1在大肠杆菌表面得到了功能性表达,并进一步证明了位于FedF受体结合域内第88和89位组氨酸残基对FedF受体结合域的形成至关重要。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

不同猪种肠毒素大肠杆菌（ETEC）F4受体微卫星标记遗传差异性研究The

采用13号染色体上与K88ab和K88ac受体基因连锁的2对引物（S0223和S0068）,研究沙子岭猪和大约克猪的遗传差异性。结果表明,2个猪种在2个基因座均存在多态性,其基因杂合度和Shannon信息指数存在很大差异,而中外猪种的K88ab和K88ac受体基因也存在遗传差异,这2对引物可望作为K88ab和K88ac受体基因的遗传标记。Abstract: The genetic variation of ETEC F4 receptor in Shaziling and Yorkshire breeds were studied using two microsatellite markers(S0223 and S0068). The results showed that there were polymorphisms in the two markers, and there were great variations of the gene heterozygosity and Shannon information index in the two breeds. It was also reported that there were differences in K88ab and K88ac receptors in Chinese native breeds and foreign breeds, so the two markers might be the genetic markers of F4 receptor gene.     

猪a1-岩藻糖转移酶基因(FUT1)M857位点遗传变异分析

肠毒素大肠杆菌 (ETEC) F18是引起仔猪断奶后水肿和腹泻病的主要病原菌, a-1岩藻糖转移酶(FUT1)基因是ECEC F18侵染猪小肠的受体蛋白基因。利用PCR-RFLP方法检测了1个野猪以及20个中外家猪猪种(群)共696个个体在FUT1基因开放阅读框架的857核苷酸位点的遗传变异, 结果表明: 在所有猪种中, 均未检测到抗性的AA型纯合子, 在外来猪种杜洛克和约克夏、国内猪种临高猪和杂交猪种中检测到AG型杂合子, 外来猪种中的皮特兰、长白猪以及除临高猪外的所有国内猪种和野猪均表现为极端的单态分布, 只有易感的GG基因型。研究结果提示, 中国地方猪种不具备抵抗ETEC F18大肠杆菌的遗传基础, 与外来猪种确实存在差异, 这种差异可能与各自不同的起源有关, ETEC F18抗性基因可能起源于欧洲野猪; 并推测猪种的生长速度与ETEC F18大肠杆菌病的发生具有密切的关系。     

产肠毒素型大肠杆菌F41黏附素是染色体编码并与K88在遗传学上相关

<正> 产肠毒素型大肠杆菌(ETEC)寄居人类及动物宿主的小肠,产生肠毒素导致液体与电解质的分泌。是通过一种命名为黏附素(adhesin)的繖状结构的细菌蛋白介导寄居于小肠。人与动物ETBC黏附素有几种不同的抗原型。表达这些黏附素的菌体细胞有凝集各种动物红细胞的能力。并与黏附素抗原型有关。黏附素对确定菌株致病性起一定作用。黏附素表达的遗传学基础是与这些抗原型相关的质粒。 最近已确定F41是对牛及猪有致病性的ETEC黏附素。θ9及0101血清型ETEC菌株的另一种黏附素K99的产生与其有关,但最近提及的某些菌株不产K99也产F41。F41在甘露糖存在的条件下可介导凝集人红细     

Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of Escherichia coli-producing K88 pilus antigen

Summary. Brush borders, enterocytes, or both preparations obtained from the small intestine of 345 pedigreed pigs, carrying components of seven breeds, were tested by adhesion assay in vitro with 6–32 enteropathogenic Escherichia coli strains, each expressing one of the three K88 pilus antigens, K88ab, K88ac and K88ad. With few exceptions, all pigs were classified as belonging to one of four adhesion phenotypes: I – corresponding to K88ab(-),ac(-),ad(-); II – K88ab(-),ac(+),ad(+); III – K88ab(+),ac(+),ad(-); and IV – K88ab(+),ac(+),ad(+). The non-adhering phenotype I was found to be the most frequent among the pigs tested, with the exception of one commercial herd, and this phenotype seems to be inherited as a recessive trait. The remaining three phenotypes are adhering, or are susceptible to adherence by one K88 variant, K88ad (phenotype II), by two variants, K88ab,ac (phenotype III), or by all three K88 variants, K88ab,ac,ad (phenotype IV). Phenotype II was found to be at low frequency, whereas III and IV occurred with similar frequencies. While the prevailing phenomenon was the bacterial adhesion to all, or none, of the brush borders, some pigs exhibited both adhering and non-adhering brush borders, a mixed adherence phenotype. Preliminary segregation data, obtained from the F1 generation, seem to indicate that phenotypes III and IV correspond to two haplotypes with genes at two or three closely linked loci respectively. An alternative hypothesis is that the phenotypes [II and IV are expressions of alleles at a single locus, each allele specifying a receptor able to bind two or three different serological types of K88 E. coli.     

Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of Escherichia coli-producing K88 pilus antigen

Brush borders, enterocytes, or both preparations obtained from the small intestine of 345 pedigreed pigs, carrying components of seven breeds, were tested by adhesion assay in vitro with 6-32 enteropathogenic Escherichia coli strains, each expressing one of the three K88 pilus antigens, K88ab, K88ac and K88ad. With few exceptions, all pigs were classified as belonging to one of four adhesion phenotypes: I I--corresponding to K88ab(-),ac(-),ad(-); II--K88ab(-),ac(-),ad(+); III--K88ab(+),ac(+),ad(-); and IV--K88ab(+),ac(+),ad(+). The non-adhering phenotype I was found to be the most frequent among the pigs tested, with the exception of one commercial herd, and this phenotype seems to be inherited as a recessive trait. The remaining three phenotypes are adhering, or are susceptible to adherence by one K88 variant, K88ad (phenotype II), by two variants, K88ab, ac (phenotype III), or by all three K88 variants, K88ab,ac,ad (phenotype IV). Phenotype II was found to be at low frequency, whereas III and IV occurred with similar frequencies. While the prevailing phenomenon was the bacterial adhesion to all, or none, of the brush borders, some pigs exhibited both adhering and non-adhering brush borders, a mixed adherence phenotype. Preliminary segregation data, obtained from the F1 generation, seem to indicate that phenotypes III and IV correspond to two haplotypes with genes at two or three closely linked loci respectively. An alternative hypothesis is that the phenotypes III and IV are expressions of alleles at a single locus, each allele specifying a receptor able to bind two or three different serological types of K88 E. coli.     

Refined mapping of the Escherichia coli F4ab/F4ac receptor gene(s) on pig chromosome 13

Enterotoxigenic Escherichia coli expressing F4 fimbriae is the major cause of diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the loci controlling the F4ab/F4ac receptors are located on SSC13q41, between markers SW207 and S0283. In this study, we refined their positions in a two generation population containing 366 piglets of three breeds (Large White, Landrace, and Songliao Black). Nine microsatellite markers within this region were selected from the MARC (U.S. Meat Animal Research Center) porcine linkage map, and the pedigree disequilibrium test was employed for fine-mapping. The F4abR gene was located in the interval between S0283 and SW1833, a 4.8-cM region, and the F4acR gene was located in the interval between S0283 and SW1876, a 1.6-cM region. Our results also suggest that the F4ab/F4ac receptors might be controlled by two different but closely linked loci. The results of microsatellite-based haplotype analysis in the corresponding region show that some specific haplotypes were overwhelmingly present in the adhesive or non-adhesive animals, indicating that there are mutations within the identified regions that are strongly associated with the F4ab/ac phenotypes.     

Evidence for linkage between the swine L blood group and the loci specifying the receptors mediating adhesion of K88 Escherichia coli pilus antigens

Brush borders or enterocytes obtained from the small intestine of 248 pedigreed pigs were tested by adhesion assay in vitro with enterotoxigenic Escherichia (E.) coli strains, each expressing one of the three K88 pilus variants K88ab, K88ac and K88ad. All pigs were classified as belonging to one of the four adhesion phenotypes: I--K88ab(-), ac(-), ad(-); II--K88ab(-), ac(-), ad(+); III--K88ab(+), ac(+), ad(-); and IV--K88ab(+), ac(+), ad(+). Serum or red cells were typed for 15 blood group systems: A-O, B, C, D, E, F, G, H, I, J, K, L, M, N and O; for 11 biochemical polymorphisms: PI1, PI2, PO1A, A1BG, GPI, PGD, TF, HPX, ADA, PGM and AMY; the polymorphism at the IGHG1 locus. Linkage analysis was performed between the alleles at the locus (loci) specifying K88 receptors able to bind one or more different serological types of K88 E. coli and alleles for markers at other loci. Linkage was demonstrated between the locus for the L blood group system and the locus (loci) for K88 E. coli receptors (Z = 3.24), adding one locus (loci) to the previously identified linkage group IV (LGIV) [L-SLB]. The maximum likelihood estimate of the recombination fraction (theta) was 0.23. No evidence was found for linkage between any of the other biochemical and immunogenetic markers and the receptor locus (loci) of K88 E. coli.     

Polymorphisms of three gene-derived STS on pig chromosome 13q41 are associated with susceptibility to enterotoxigenic Escherichia coli F4ab/ac in pigs

Neonatal diarrhea caused by enterotoxigenic Escherichia coli(ETEC)F4 is a common and serious disease,resulting in significant economical loss in the pig industry.The locus encoding ETEC F4 receptor has been mapped to pig chromosome(SSC)13q41,and one of the most significantly linked markers is S0075.In this study,we selected three genes including SLC12A8,MYLK and KPNA1 from a chromosomal region flanking S0075 on SSC13 to develop pig specific sequence tagged sites(STS). Seven single nucleotide polymorphisms were identified in the three pig STS using DNA of four full-sib susceptible and resistant animals in a White Duroc×Erhualian intercross.All grandparents,parents and 755 offspring in the intercross were genotyped for three polymorphisms,including SLC12A8 g.159A>G,MYLK g.1673A>G and KPNA1 g.306A>G.Family-based transmission disequilibrium test(TDT) revealed that all polymorphisms and the corresponding haplotypes are significantly associated with ETEC F4ab/ac(especially F4ac)brush border adhesion phenotypes,indicating that these polymor- phism are in linkage disequlibrium with causal mutation(s)of the gene encoding ETEC F4ab/ac receptor. Our results strengthen the evidence for the involvement of SSC13q41 in high acquiring risk of ETEC F4ab/ac infection,and provide novel polymorphic markers for fine mapping of the ETEC F4ab/ac receptor locus.     

Fine-mapping of the intestinal receptor locus for enterotoxigenic Escherichia coli F4ac on porcine chromosome 13

The aim of this study was to refine the localization of the receptor locus for fimbriae F4ac. Small intestinal enterocyte preparations from 187 pigs were phenotyped by an in vitro adhesion test using two strains of Escherichia coli representing the variants F4ab and F4ac. The three-generation pedigree comprised eight founders, 18 F1 and 174 F2 animals, for a total of 200 pigs available for the linkage analysis. Results of the adhesion tests on 171 F2 pigs slaughtered at 8 weeks of age show that 23.5% of the pigs were adhesive for F4ab and non-adhesive for F4ac (phenotype F4abR+/F4acR-; R means receptor). Pigs of this phenotype were characterized by a weak adhesion receptor for F4ab. No pigs were found expressing only F4acR and lacking F4abR. Receptors for F4ab and F4ac (F4abR+/F4acR+) were expressed by 54.5% of the pigs. Animals of this phenotype strongly bound both F4ab and F4ac E. coli. In the segregation study, the serum transferrin (TF) gene and 10 microsatellites on chromosome 13 were linked with F4acR (recombination fractions (theta) between 0.00 and 0.11 and lod score values (Z) between 11.4 and 40.4). The 11-point analysis indicates the F4acR locus was located in the interval S0068-Sw1030 close to S0075 and Sw225, with recombination fractions (theta) of 0.05 between F4acR and S0068, 0.04 with Sw1030, and 0.00 with S0075 and Sw225. The lack of pigs displaying the F4abR-/F4acR+ phenotype and the presence of two phenotypes for F4abR (a strong receptor present in phenotype F4abR+/F4acR+ and a weak receptor in phenotype F4abR+/F4acR-) led us to conclude that the receptor for F4ac binds F4ab bacteria as well, and that it is controlled by one gene localized between S0068 and Sw1030 on chromosome 13.     

Association of B3GNT5 Polymorphisms with Susceptibility to ETEC F4ab/ac in the White Duroc?×?Erhualian Intercross and 15 Outbred Pig Breeds

The B3GNT5 gene is a candidate for the F4ab/ac receptor conferring susceptibility to enterotoxigenic Escherichia coli (ETEC) F4ab/ac in pigs. In this study, we screened mutations in the complete coding region of the porcine B3GNT5 gene and identified four SNPs in the 3' untranslated regions. We genotyped the four SNPs across a large-scale White Duroc × Chinese Erhualian F2 resource population (total F2 = 755) and 292 purebred piglets representing 15 Chinese and Western breeds. We found that the g.1476G→A locus and haplotypes [A;T;G;T] and [A;G;G;T] had significant association with susceptibility to ETEC F4ac in the resource population. None of the B3GNT5 polymorphisms and haplotypes was associated with susceptibility to ETEC F4ab/ac in outbred piglets. This result, together with other reports, supports the conclusion that B3GNT5 is not the responsible gene encoding the ETEC F4ab/ac receptors.     

Investigation of the porcine MUC13 gene: isolation, expression, polymorphisms and strong association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac

Enterotoxigenic Escherichia coli (ETEC) F4ab and F4ac are major determinants of piglet diarrhoea. The locus for the ETEC F4ab/ac receptor has been mapped to SSC13q41. MUC13 is a transmembrane mucin expressed predominantly in the epithelial surface of the gastrointestinal tract and the MUC13 gene was assigned to SSC13q41, supporting it as a positional candidate gene for the ETEC F4ab/ac receptor. We herein determined the complete 2679-bp cDNA of pig MUC13, and proved that it was most highly expressed in the jejunum and moderately expressed in the trachea, stomach and liver. Furthermore, 13 MUC13 polymorphisms were identified in 19 founder animals of a White Duroc x Erhualian resource population, and a total of 727 F(2) animals with in vitro ETEC F4ab/ac adhesion phenotypes in this population were genotyped for three identified MUC13 polymorphisms including c.576C>T, c.908A>G and c.935A>C. The transmission disequilibrium test showed that the MUC13 alleles and haplotypes were significantly associated with susceptibility/resistance to ETEC F4ab/ac, especially between haplotype [C;G;A] and susceptibility to ETEC F4ac (P = 8.0e-18). Animals inheriting this haplotype were predominantly susceptible to ETEC F4ac (n = 291/303). Moreover, nearly all animals homozygous for haplotype [T;G;C] (n = 39/41) and a majority of those with the [C;A;A]/[T;G;C] haplotype pair (n = 79/88) were resistant to ETEC F4ab. Our results indicated that MUC13 is in strong linkage disequilibrium with the ETEC F4ab/ac receptor locus and provided potential markers for selection of ETEC F4ab/ac-resistant animals in the pig breeding scheme.