中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

利用单链构象多态性检测牛白细胞黏附缺陷症方法的建立

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响其生产性能的表现,严重影响了奶牛场的经济效益。该工作利用自行设计的特异性PCR引物,通过对PCR、PCR-SSCP条件的筛选和优化,并结合DNA序列测定,建立了PCR-SSCP检测BLAD 有害基因的新方法,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选,为奶牛BLAD有害基因的分型和筛选提供了新的方法和思路,为我国奶牛的分子选育提供了可靠的理论依据。     

非小细胞肺癌ROS1及其融合基因CD74-ROS1逆转录病毒载体的构建

目的:利用逆转录病毒载体pBaba-puro构建携带ROS1基因及其CD74-ROS1融合基因的重组载体pBaba-puro-ROS1,pBaba-puro-CD74-ROS1。方法:设计与合成引物,提取组织标本RNA,反转录和PCR扩增,经BamHI和TaqI双酶切,琼脂糖凝胶电泳,切胶回收进行连接转化,并再次酶切鉴定,测序分析。结果:成功构建携带ROS1基因及其CD74-ROS1融合基因的重组载体pBaba-puro-ROS1,pBaba-puro-CD74-ROS1,并通过双酶切与测序鉴定。结论:利用逆转录病毒载体基因重组技术能够成功构建出携带相应基因的逆转录病毒,可用于后续研究。     

牛脊柱畸形综合征检测方法的建立与应用

牛脊柱畸形综合征(Complex vertebral malformation, CVM)是近年来新发现的致死性牛常染色体隐性遗传缺陷病。由于编码UDP-N-乙酰葡糖胺载体的SLC35A3基因发生G→T的突变而引起本病的发生, 可引起胎牛死胎、流产、早产。为了解我国正常的荷斯坦牛(黑白花奶牛)的CVM携带和发生情况, 建立、应用创造酶切位点PCR(Created restriction site PCR, CRS-PCR)、等位基因特异性PCR(Allele-specific polymerase chain reaction, AS-PCR)检测方法检测了表型正常的436头荷斯坦母牛和93头荷斯坦公牛, 检测到3头CVM携带者, 其中杂合母牛1头, 杂合公牛2头, 携带率分别为0.60%、2.20%。此方法简便、可靠, 为奶牛CVM有害基因的分型和筛选提供了新的方法和思路, 为我国奶牛的分子选育提供了可靠的理论依据。     

荷斯坦奶牛中性粒细胞β-防御素cDNA的克隆与序列分析

为研发牛β-防御素生物制剂,防治奶牛乳腺炎。根据已发表的牛β-防御素基因序列设计一对引物。收集患乳腺炎奶牛乳静脉血液中性粒细胞,用试剂盒提取总RNA。经RT-PCR扩增牛β-防御素cDNA片段,回收纯化PCR产物,连接pMD18-T载体,转化感受态细胞JM109。在含X-Gal、Amp及IPTG的LB平板上筛选阳性克隆,经菌落PCR及双酶切鉴定后,对目的片段进行测序。结果表明RT-PCR扩增出的cDNA片段碱基数为189bp,含有一个完整的ORF,能编码63个氨基酸的多肽,多肽中含6个保守半胱氨酸残基。用Blast程序进行检索比较,表明扩增序列与GenBank中发表的BNBD-7编码区序列96.3%相同。结果成功克隆出奶牛β-防御素家族的成员BNBD-7基因,为重组牛β-防御素的开发奠定了基础。     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

郏县红牛DGAT2基因多态性与生长性状的相关性分析

利用PCR-RFLP技术对随机抽取的92头郏县红牛的DGAT2基因进行多态性分析。结果表明, DGAT2基因第6内含子的PCR扩增产物被限制性酶TaqⅠ酶切消化后表现多态性。经电泳检测后, 显出AA、AB两种不同基因型。该基因座处于Hardy-Weinberg平衡状态, 通过对郏县红牛DGAT2基因多态性与体重及体尺指标的方差分析显示：郏县红牛DGAT2基因不同基因型对郏县红牛体重和体长有显著影响(P<0.05), 而对其他体尺指标在统计学上无显著影响(P>0.05), 且初步认为AB基因型对选择有正向效应。     

奶牛IL-6基因克隆与原核表达

目的:克隆新疆地区奶牛il-6基因.获得原核表达IL-6蛋白.方法:根据GenBank上牛IL-6的序列,用premier 5.0软件设计一对引物.以牛肺巨噬细胞提取的RNA反转录产物(cDNA)为模板扩增出567bp的条带,克隆到pBS-T载体,测序正确后,提取质粒经BamH Ⅰ和EcoR Ⅰ双酶切回收目的条带,亚克隆到pGEX-4T-1原核表达载体,转化DE3菌,经IPTG诱导表达.以表达产物免疫小白鼠血清为一抗榆测表达产物的反应原性.结果:成功克隆了新疆地区奶牛il-6基因,克隆部分编码188个氨基酸与GenBank公布的序列100%同源,表达出46 kDa的融合蛋白,免疫印迹检测显示原核表达牛IL-6有免疫原性.结论:新疆地区奶牛在IL-6的基因序列上与其他地方牛无差异,原核表达奶牛IL-6蛋白为在奶牛乳房炎疫苗中的佐剂效果研究打下了基础.     

YFP-CD18融合蛋白在U937细胞株中的表达与鉴定

目的：构建并表达Mac-1的β链CD18与黄色荧光蛋白（YFP）的融合蛋白YFP-CD18,为进一步直视研究Mac-1在白细胞内的分布、走向及归宿提供物质条件。方法：首先将CD18的全长cDNA与pEYFP-C1进行酶切,构建YFP与CD18的N末端连接的表达载体,并将其转染至U937细胞株中进行表达。通过荧光显微镜观察转染成功后的U937细胞黄色荧光是否存在,以及流式细胞术检测确定PMA刺激后YFP-CD18活化后可否由胞浆内转位至膜上和测定PMA活化前后转染的U937细胞与其配基ICAM-1粘附活性的变化等方面进行鉴定。结果：经酶切鉴定,pYFP-CD18构建正确,转染U937细胞株后,可见YFP-CD18融合蛋白发出的黄色荧光。结论：构建完成YFP-CD18融合基因并可以在U937细胞株中进行表达。     

牛β-乳球蛋白基因的克隆及部分序列测定

为了制备乳腺生物反应器所需要的乳腺特异性表达的调控序列,用ＰＣＲ法从奶牛染色体上分５段扩增出了全长的牛 ＢＬＧ基因约 ８ ｋｂ,包括 １．８ ｋｂ的 ５’侧翼区、１．７ ｋｂ的 ３’侧翼区及 ４．７ ｋｂ的ｇＤＮＡ区。扩增出的各片段克隆到Ｔ－Ｖｅｃｔｏｒ上,酶切鉴定及序列分析均证实了所扩增片段的正确性。     

Screening for bovine leukocyte adhesion deficiency,deficiency of uridine monophosphate synthase,complex vertebral malformation,bovine citrullinaemia,and factor XI deficiency in Holstein cows reared in Turkey

Background  

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.     

A missense mutation in the beta-2 integrin gene (ITGB2) causes canine leukocyte adhesion deficiency.

Canine leukocyte adhesion deficiency (CLAD) is a fatal immunodeficiency disease found in Irish setters. The clinical manifestations of CLAD are very similar to LAD in humans and BLAD in cattle, which are both caused by mutations in ITGB2 encoding the leukocyte integrin beta-2 subunit (CD18). Sequence analysis of the ITGB2 coding sequence from a CLAD dog and a healthy control revealed a single missense mutation, Cys36Ser. This cysteine residue is conserved among all beta integrins, and the mutation most likely disrupts a disulfide bond. The mutation showed a complete association with CLAD in Irish setters and was not found in a sample of dogs from other breeds. The causative nature of this mutation was confirmed by transduction experiments using retroviral vectors and human LAD EBV B-cells. The normal canine CD18 formed heterodimers with the human CD11 subunit, whereas gene transfer of the mutant CD18 resulted in very low levels of CD11/CD18 expression. The identification of the causative mutation for CLAD now makes it possible to identify carrier animals with a simple diagnostic DNA test, and it forms the basis for using CLAD as a large animal model for the development and evaluation of clinical treatments for human LAD.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Bovine Leukocyte Adhesion Deficiency in Danish Holstein-Friesian Cattle

A screening program for bovine leukocyte adhesion deficiency (BLAD) in Danish Holstein-Friesian cattle has been initiated. During the first months 1611 animals were tested by a PCR based assay. Of these animals 1256, 346, and 8 were assigned normal, BLAD carriers, and BLAD affected animals, respectively One bull, born as a co-twin, showed weak reaction for the BLAD allele on DNA isolated from leukocytes, but a normal genotype on DNA isolated from semen. Chromosome analysis showed that this bull was a blood chimaera. Estimation of the BLAD allele frequency upon the PCR test results showed that around 450 Danish calves born in 1991 might have been affected with the recessive disorder.     

Identification of bovine leucocyte adhesion deficiency (BLAD) carriers in Holstein and Brown Swiss AI bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101 bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation inherited to him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD-carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils

In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production.     

Identification of Bovine Leucocyte Adhesion Deficiency (BLAD) Carriers in Holstein and Brown Swiss AI Bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene, which is responsible for this disease, has recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101-bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation was inherited by him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI, and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

Simultaneous analysis of bovine K-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes

An improved and simplified method allowing simultaneous genetic typing of K-casein and CD 18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous amplification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the products by two restriction enzymes ( Pst I and Hae III) in the same incubation buffer. Digestion with Pst I distinguishes K-casein A and B alleles and does not cut within any of the BLAD alleles, while digestion with Hae III allows the differentiation between normal and mutant allele variants of the CD28 locus. All combinations of the known mutants of the two alleles, characterized to the regions amplified and resulting in phenotypic effect, could be detected by electrophoretic separation performed on the same agarose gel owing to the vast differences in the length of the restriction fragments.     

Identification of Novel Allelic Variants of Integrin Beta 2 (ITGB2) Gene and Screening for Bubaline Leukocyte Adhesion Deficiency Syndrome in Indian Water Buffaloes (Bubalus bubalis)

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).