Structure-function studies of Bubalus bubalis lingual antimicrobial peptide analogs

Antimicrobial peptides expressed on different epithelial lining are major components of the innate immune system. Based on the deduced amino acid sequence of Bubalus bubalis lingual antimicrobial peptide (LAP) cDNA (Accession No. DQ458768), five overlapping peptides LAP^23–55, LAP^42–64, LAP^21–64, LAP^1–26 and LAP^1–64 were synthesized using solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry. Circular Dichroism spectroscopy of synthesized peptides revealed predominantly β-structure for LAP^23–55, LAP⁴²⁻⁶⁴ and LAP^21–64 with less α-helix in different solutions. Quantitation of secondary structure indicated the highest β-structure for all these three peptides in membrane mimetic SDS solution. The helicogenic solvent TFE could not induce helix in LAP^23–55 however TFE induced helical propensity was observed in LAP^42–64 and LAP^21–64. The quantitation of secondary structure indicated the highest ordered structure for LAP^23–55 followed by LAP^42–64 and LAP^21–64. The antibacterial activity of LAP^23–55 was found to be more potent against Staphylococcus aureus, Listeria monocytogens, Escherichia coli and Salmonella typhimurium followed by LAP^42–64 and LAP^21–64. Minimum inhibitory concentration (MIC) also showed similar trend with lowest value for LAP^23–55 followed by LAP^42–64 and LAP^21–64. Haemolysis and cytotoxicity was observed above 3 fold for LAP^21–64, above six fold for LAP^23–55 and LAP^42–64 of their MIC. The LAP^1–26 and LAP^1–64 could not produce any characteristic CD spectra and did not show any antimicrobial activity, indicating that N- terminal of the peptide negates the antimicrobial activity.     

微小牛蜱一个抗菌多肽编码基因的克隆和表达

为了进行抗蜱及蜱传病疫苗的研究,本研究根据微小牛蜱巴西株报道的一种抗菌多肽核苷酸序列设计引物,从微小牛蜱中国安徽株克隆到该抗菌多肽基因,全长383bp,编码110个氨基酸残基,该蛋白预测的分子量为12.2ku,等电点为4.87.经同源性比较,该微小牛蜱巴西株抗菌多肽基因有100%的相同性.经RT-PCR分析表明,该基因在微小牛蜱卵、幼蜱、半饱血雌蜱、饱血雌蜱和雄蜱这几个阶段均有表达.将该基因亚克隆到pET-28a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,可成功表达.重组融合蛋白大小为15ku左右,与预期大小一致.初步体外抗菌试验表明,重组蛋白具有一定的抗菌活性.Western-blot显示,兔抗微小牛蜱唾液抗体能够识别重组表达蛋白.     

Molecular cloning and characterization of cDNA encoding CD11b of cattle

CD18, the common β subunit of β₂-integrins, associates with four distinct chains to give rise to four different β₂-integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. Previously, we and others showed that CD18 of LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin (Lkt). Level of expression of Mac-1 is higher than that of LFA-1 and other β₂-integrins on polymorphonuclear leukocytes (PMNs), which constitute the leukocyte subset most susceptible to Lkt. Hence, it is likely that CD18 of Mac-1 also mediates Lkt-induced cytolysis. Co-expression of CD11b and CD18 of cattle on Lkt-resistant cells is necessary to irrefutably demonstrate the role of Mac-1 in Lkt-induced cytolysis. This approach is hindered by lack of availability of complete sequence of cattle CD11b. Therefore, in this study, we cloned and sequenced the full length cDNA encoding cattle CD11b. The 3459 bp cDNA of cattle CD11b encodes a polypeptide of 1152 amino acids. The deduced amino acid sequence of CD11b of cattle exhibits 75% identity to that of humans and chimpanzees, 74% identity to that of dogs, and 70% identity to that of mice and rats. Availability of cattle CD11b cDNA should facilitate the elucidation of Lkt-receptor interactions in cattle and other species.     

中国水牛遗传育种研究进展

我国水牛遗传育种的研究主要集中在染色体核型、蛋白质多态性、水牛胚胎体外生产与胚胎移植技术等方面。本文从中国水牛的类型和分布特征、遗传特性的研究现状、现代生物技术在水牛育种中的应用、中国水牛育种研究前景及展望等四个方面综述水牛的育种进展,认为以现代生物技术为核心的分子育种将成为水牛育种的总趋势,为我国水牛的产业化发展提供了理论依据。     

传染性法氏囊病病毒VP2-4-3 cDNA的分子克隆和序列分析

从法氏囊组织分离IBDV超强毒株HK46并提取基因组RNA。以RNA为模板进行反转录合成cDNA第一链。采用长PCR扩增技术获得VP2－4－3 cDNA全长片段。将PCR产物克隆到pcDNA3.1( )载体，得到重组质粒pPP1。对pPP1插入片段全长序列进行了测序并对其序列进行了分析。结果表明，VP2－4－3 cDNA阅读框架由3039bp组成，可编码1012个氨基酸组成的前体多聚蛋白。经比较得知，HK46超强毒株VP2－4－3氨基酸序列与经典毒株间存在19－28个氨基酸的差异；与Harbin强毒株相差32个氨基酸；而与超强毒株OKYM和UK661分别相差2和6个氨基酸，且它们的VP2序列完全相同。在HK46超强毒株所特有的9个氨基酸中，3个位于VP2可变区，显示超强毒株其抗原性存在着变异。     

Immunolocalization of lingual antimicrobial peptide (LAP) in the bovine mammary gland

Lingual antimicrobial peptide (LAP), a member of the β-defensin family in cows, is involved in the innate immune system and plays a crucial role in killing a large variety of microorganisms. The aim of the present study was to demonstrate the immunolocalization of LAP in the mammary glands of cows. A LAP antibody was raised in a rabbit by immunity with a synthetic 11 amino acid sequence out of a 42-amino acid sequence of the mature form of LAP. The specificity of the LAP antibody was checked using a competitive immunoassay and Western blotting. Paraffin sections of the mammary gland were immunostained with LAP antibody. In the competitive immunoassay, an increase of synthetic LAP concentration suppressed the optical density. Western blotting analysis for LAP revealed the presence of the LAP peptide in mammary alveolar tissue. When the mammary gland was immunostained with LAP antibody, epithelial cells of both infected and non-infected alveoli were immunopositive. These results indicate that LAP is localized in the epithelium of non-infected as well as infected alveolus in the mammary gland in cows.     

新型欧洲鳗鲡抗菌肽elecilin在毕赤酵母中的表达

前期采用差减杂交的方法筛选到新型欧洲鳗鲡抗菌肽elecilin,其基因全长为477 bp,编码159个氨基酸,理论分子量约17.4 kDa。为了获得具活性的大量表达的抗菌肽,本研究利用毕赤酵母表达了elecilin。首先将elecilin基因克隆至酵母分泌表达载体pPICZaA,构建了重组分泌表达质粒pPICZaA-elecilin,电击转化毕赤酵母X-33。经Zeocin筛选和PCR鉴定,获得重组酵母菌,通过甲醇诱导,SDS-PAGE和Western blot鉴定,证实诱导后的培养基中存在与预期分子量大小相一致的蛋白,能被His-tag单克隆抗体识别,表明利用酵母成功表达了欧洲鳗鲡抗菌肽elecilin。这为进一步研究该抗菌肽的功能及作为饲料添加剂的应用奠定了基础。     

Molecular characterization and expression profile of ghrelin gene during different reproductive phases in buffalo (Bubalus bubalis)

Ghrelin, a novel motilin-related endogenous ligand for growth hormone secretagouge receptor, is implicated in various biological functions, including regulation of female reproduction. But the presence of ghrelin and its role in reproductive functions in buffalo, a species with poor reproductive efficiency, is not known. In the present study full-length ghrelin cDNA was isolated from bubaline abomasum, which encodes the entire prepropeptide of 116 amino acids. The deduced amino acid sequence of ghrelin of buffalo showed >95% and 31% identity with that of ruminants (cattle, sheep, and goat) and humans, respectively. Analysis of synonymous and nonsynonymous nucleotide substitutions in the coding region of ghrelin indicated that these sequences of different species have been under purifying selection. The 3995-bp amplicon of ghrelin gene consisting of 4 exons and 3 introns was cloned with genomic DNA from buffalo. Further, ghrelin expression was determined by quantitative real-time PCR, in situ hybridization, and immunohistochemistry in bubaline endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated the persistent expression of ghrelin mRNA and protein in the endometrium during stage I (day 3–5), stage II (day 6–15), and stage III (day 16–21) of the estrous cycle and also during early (∼day 30–40) pregnancy. Immunohistochemistry and quantitative real-time PCR experiments indicated the relatively higher expression of ghrelin in the endometrium during stage II (day 6–15) of the estrous cycle and early pregnancy than during stage I (day 3–5) and stage III (day 16–21) of the estrous cycle, but no statistically significant difference in ghrelin expression was observed among stages. To conclude, the results of the present study indicate the persistent expression of ghrelin in the uterine endometrium throughout the estrous cycle and in early pregnancy which might be helpful in determining its role in buffalo reproduction.     

抗菌肽BSN-37对大肠杆菌胞内物质泄露的影响

为了解抗菌肽BSN-37对大肠杆菌的作用机制,通过β-半乳糖苷酶试验测定了抗菌肽BSN-37对大肠杆菌CVCC1568的抑菌活性,以菌落计数法测定了抗菌肽BSN-37对大肠杆菌CVCC1568的抑菌动力学,用分光光度法测定了抗菌肽BSN-37对大肠杆菌CVCC1568细胞壁通透性,以及胞内紫外吸收物质、蛋白及核酸的泄露量。结果表明,抗菌肽BSN-37能有效抑制大肠杆菌CVCC1568的生长繁殖,随抗菌肽浓度增加,抑制活性越高,抗菌肽BSN-37作用后的大肠杆菌CVCC1568细胞壁通透性增加,胞内紫外吸收物质、蛋白和核酸的泄露量随时间延长而逐渐增加。说明抗菌肽BSN-37能破坏大肠杆菌CVCC1568的细胞壁和细胞膜,从而表现较好的抑菌活性。     

Molecular cloning and characterization of the full-length cDNA encoding the porcine CD28

CD28 is one of the most important co-stimulatory molecules required for effective activation of resting T cells in human and mouse. However, there are few studies on porcine CD28 (pCD28) until now. In the present study, we cloned and characterized the full-length cDNA of CD28 from the miniature?pig. The open reading frame (ORF) sequence of pCD28 gene was organized into four exons, which were predicted to be in correspondence with the signal sequence, immunoglobulin variable-like (IgV) domain, transmembrane domain and cytoplasmic tail, respectively. We also identified the putative ligand binding site of CD28 within the IgV domain and the consensus motifs (one "YMNM" motif and two proline-rich motifs) within the cytoplasmic domain. Porcine CD28 was confirmed to be expressed on the cell membrane as indicated by indirect immunofluorescence assay (IFA). The putative promoter region of pCD28 was also cloned by the modified nested PCR and the cloned region could successfully drive the expression of yellow fluorescent protein (YFP) expression in porcine peripheral blood mononuclear cells (PBMCs). The present study is the first report of cloning and characterization of CD28 in porcine. Our work provided fundamental information for further researches on the structure and function of CD28 in porcine.     

Dynamics of lingual antimicrobial peptide,lactoferrin concentrations and lactoperoxidase activity in the milk of cows treated for clinical mastitis

The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day ?6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days ?6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 10⁶ cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.     

Serological survey of Neospora caninum and Toxoplasma gondii in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in ten provinces of Brazil

The objective of this study was to determine the prevalence of antibodies to Neospora caninum and Toxoplasma gondii among 500 cattle (Bos indicus) and 500 buffaloes (Bubalus bubalis) using the indirect fluorescent antibody test (IFAT) technique. Blood samples from were collected from water buffalo and cattle in 10 municipalities in the northern region of Brazil. The frequency of cattle and water buffaloes seropositive for Neospora caninum in Pará state, Brazil, was 55% and 44%, respectively, and the frequency of cattle and water buffaloes seropositive for Toxoplasma gondii was 52% and 39%, respectively. Seropositivity for both N. caninum and T. gondii was detected in 10.6% of the cattle samples and 14.8% of the buffalo samples. The frequency of cattle positive for N. caninum and T. gondii was significantly (p < 0.05) higher than that of buffalo in two and three provinces, respectively. Buffaloes had a lower seroprevalence for N. caninum or T. gondii in all of the provinces studied. These results suggest that both species, when exposed to the same risks for N. caninum and T. gondii infection, have a high serological prevalence. Cattle showed a higher probability of being seropositive when exposed to the same risks for N. caninum and T. gondii. Our study, which included an extensive number of blood samples, provides important epidemiological information pertinent to buffalo production in tropical countries that can be used as a basis for disease-management practices in Latin America.     

Effects of age and season on conception rate of Mediterranean Italian Dairy Buffalo (Bubalus bubalis) following oestrus synchronization and fixed-time artificial insemination

The objective of this project was to report the effects of season and age on conception rate amongst Italian Mediterranean Buffalo subjected to an Ovsynch/Resynch (O/R) reproductive management protocol. The study utilized nulliparous (heifers), primiparous buffalo cows (PBC) and multiparous buffalo cows (MBC). The primiparous and multiparous groups were subjected to the synchronization protocol throughout the entire year, but heifers were synchronized and inseminated only during the spring/summer seasons. The conception rate obtained following the OvSynch oestrus synchronization protocol, applied during spring, was 68.4% for heifers, 83.3% for PBC and 67.7% for MBC. The overall total conception rates following the complete O/R protocol were 84.27%, 94.4% and 79%. Conception rates achieved during summer were heifers 52%, PBC 47.2% and MBC 49%, whilst overall conception rates following the full O/R protocol were 72%, 69.8% and 58.2% respectively. In the autumn seasons, PBC conceived 58.9% and MBC 52.1% following initial Ovsynch, which improved to total overall conception rates of 87.5% and 78.7% following the full O/R protocol. Similarly, in the winter season, PBC experienced a conception rate of 47.5% following Ovsynch and 72.5% after a follow-up Resynch. MBC experienced 60.0% and 74.4% conception following Ovsynch and full O/R, respectively, during winter. Total conception rates during all seasons were quite acceptable following the O/R protocol. There was a significant decrease from spring to summer in conception rate for all parity groups, but heifers were not a severely affected as older buffalo cows. This finding agrees with that of other investigators indicating that heifer fertility is not as negatively impacted by long photoperiod and higher ambient temperature as that of older animals. The O/R protocol as utilized in this study is an effective means of reproductive management for dairy buffalo cows and is effective for improving fertility during out-of-season breeding.     

Changes in the concentrations of somatic cell counts,lingual antimicrobial peptide and lactoperoxidase activity in milk at periovulatory period in dairy cows

The objective of the present study was to examine the changes in innate immune factors in milk during the estrous cycle in dairy cows. Milk was collected from cows with a normal ovulatory cycle and cows subjected to the OVSYNCH protocol, and somatic cell counts (SCC), lingual antimicrobial peptide (LAP) concentrations and lactoperoxidase (LPO) activity in milk were measured. In cows with a normal ovulatory cycle, there was no significant change in LAP concentrations and SCC during the ovulatory cycle. However, LPO activity at days 0 and 19 (day 0 = day of ovulation) were significantly higher than those at days 10, 12 and 15. In cows subjected to the OVSYNCH protocol, a significant increase in SCC was observed at day 9 (2 days after prostaglandin treatment) compared with that at day 11 (2 days after second administration of gonadotropin). There were no significant changes in LAP concentrations and LPO activity during the OVSYNCH protocol. These results indicate that LPO activity, an innate immune factor, was enhanced in the preovulatory period.