Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRB...Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRBR2, of Chinese Holstein, Sanhe cattle, and Chinese Simmental was amplified by polymerase chain reaction （PCR）, respectively. The genetic polymorphisms in the three populations were detected by Single-Strand Conformational Polymorphism （SSCP） in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results showed that both alleles （A and B） of two loci were found in all the three populations and the value of polymorphism information content （PIC） indicated that these were a moderate polymorphism. Statistical results of X^2 test indicated that two polymorphism sites in the three populations fitted with Hardy-Weinberg equilibrium （P 〉 0.05）. After sequencing, A-G single nucleotide polymorphism （SNP） was identified at nucleotide 4,525 in intron 1 of TLR4 gene and C-T SNP was identified at nucleotide 1,397 in exon 3 of TLR4 gene. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score （SCS） was analyzed, the results indicated that the cattle with allele A in T4CRBR1 showed lower somatic cell score than that of allele B （P 〈 0.05）. In short, the allele A might play an important role in mastiffs resistance in bovine.展开更多
[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and lig...[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.展开更多
The objective of this research was to study the effect of ECS on cumulus expansion and rate of nucleus maturation of bovine oocytes. Media maturation were used： （1） TCM 199 ＋ FCS 10%; （2） TCM 199 ＋ FCS 10% ＋ E...The objective of this research was to study the effect of ECS on cumulus expansion and rate of nucleus maturation of bovine oocytes. Media maturation were used： （1） TCM 199 ＋ FCS 10%; （2） TCM 199 ＋ FCS 10% ＋ ECS 5%; （3） TCM 199 ＋ FCS 10% ＋ ECS 7, 5%; （4） TCM 199 ＋ FCS 10% ＋ ECS 10%; （5） TCM 199 ＋ ECS 10%. Supplementation of ECS had significantly difference （P 〈 0.05） on expansion of cumulus cells and rate of nucleus maturation. Supplementation of ECS 5% was the best result in expanded cumulus cells and metaphase II rate： 82% and 72% respectively. It was concluded that medium of TCM 199 ＋ FCS 10%o ＋ ECS 5% was the best maturation medium展开更多
The aim of the study was to evaluate the impact of organic selenium supplementation （selenium yeast） vs. inorganic selenium （selenium selenite） on the expression of selected defensin genes in milk somatic cells （...The aim of the study was to evaluate the impact of organic selenium supplementation （selenium yeast） vs. inorganic selenium （selenium selenite） on the expression of selected defensin genes in milk somatic cells （MSC） of dairy cows. Sixteen Polish Holstein-Friesian cows in second parity, with similar milk yield and milk composition were randomly divided into two equal groups. At the beginning of experiment, the animals were in the middle of their lactation curve （150, standard deviation （SD） = 26 d） and this experiment lasted 90 d. The basic diet, for both groups, consisted of corn silage, wilted grass silage and concentrates. The diet of control group was supplemented with commercial mineral and vitamin mixture with inorganic selenium and the for the experimental group daily selenium need was covered by the addition of Se-yeast （6 mg Se/cow/d）. Milk samples were collected three times during experiment （before experiment; on day 55; after the end the experiment）. Total RNA was isolated from milk somatic cells and the levels of transcripts of bovine β1-defensin （DEFB1）, β4-defensin （BNBD4）, β5-defensin （BNBDS）, β10-defensin （BNBD10） and lingual antimicrobial peptide （LAP） were measured with real time PCR, using glyceraldehyde 3-phosphate dehydrogenase （GAPDH） as a reference gene. The expressions of almost all studied genes were influenced by environmental conditions （very low and negative temperature）. However, BNBD4 （P 〈 0.05） and LAP （P ≤ 0.001 ） genes were influenced by selenium supplementation but in opposite ways, depending on the form of Se. These findings support that selenium is an important factor affecting the mRNA level in MSC, but the effect of the form of selenium might depend on genes.展开更多
基金This work was supported by the fifteenth "Five Years Key Programs" for Science and Technology Development of China (No. 2002BA518A14)the National High Science and Technology Foundation of China (863) (No. 2002AA242011).
文摘Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRBR2, of Chinese Holstein, Sanhe cattle, and Chinese Simmental was amplified by polymerase chain reaction （PCR）, respectively. The genetic polymorphisms in the three populations were detected by Single-Strand Conformational Polymorphism （SSCP） in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results showed that both alleles （A and B） of two loci were found in all the three populations and the value of polymorphism information content （PIC） indicated that these were a moderate polymorphism. Statistical results of X^2 test indicated that two polymorphism sites in the three populations fitted with Hardy-Weinberg equilibrium （P 〉 0.05）. After sequencing, A-G single nucleotide polymorphism （SNP） was identified at nucleotide 4,525 in intron 1 of TLR4 gene and C-T SNP was identified at nucleotide 1,397 in exon 3 of TLR4 gene. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score （SCS） was analyzed, the results indicated that the cattle with allele A in T4CRBR1 showed lower somatic cell score than that of allele B （P 〈 0.05）. In short, the allele A might play an important role in mastiffs resistance in bovine.
文摘[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.
文摘The objective of this research was to study the effect of ECS on cumulus expansion and rate of nucleus maturation of bovine oocytes. Media maturation were used： （1） TCM 199 ＋ FCS 10%; （2） TCM 199 ＋ FCS 10% ＋ ECS 5%; （3） TCM 199 ＋ FCS 10% ＋ ECS 7, 5%; （4） TCM 199 ＋ FCS 10% ＋ ECS 10%; （5） TCM 199 ＋ ECS 10%. Supplementation of ECS had significantly difference （P 〈 0.05） on expansion of cumulus cells and rate of nucleus maturation. Supplementation of ECS 5% was the best result in expanded cumulus cells and metaphase II rate： 82% and 72% respectively. It was concluded that medium of TCM 199 ＋ FCS 10%o ＋ ECS 5% was the best maturation medium
文摘The aim of the study was to evaluate the impact of organic selenium supplementation （selenium yeast） vs. inorganic selenium （selenium selenite） on the expression of selected defensin genes in milk somatic cells （MSC） of dairy cows. Sixteen Polish Holstein-Friesian cows in second parity, with similar milk yield and milk composition were randomly divided into two equal groups. At the beginning of experiment, the animals were in the middle of their lactation curve （150, standard deviation （SD） = 26 d） and this experiment lasted 90 d. The basic diet, for both groups, consisted of corn silage, wilted grass silage and concentrates. The diet of control group was supplemented with commercial mineral and vitamin mixture with inorganic selenium and the for the experimental group daily selenium need was covered by the addition of Se-yeast （6 mg Se/cow/d）. Milk samples were collected three times during experiment （before experiment; on day 55; after the end the experiment）. Total RNA was isolated from milk somatic cells and the levels of transcripts of bovine β1-defensin （DEFB1）, β4-defensin （BNBD4）, β5-defensin （BNBDS）, β10-defensin （BNBD10） and lingual antimicrobial peptide （LAP） were measured with real time PCR, using glyceraldehyde 3-phosphate dehydrogenase （GAPDH） as a reference gene. The expressions of almost all studied genes were influenced by environmental conditions （very low and negative temperature）. However, BNBD4 （P 〈 0.05） and LAP （P ≤ 0.001 ） genes were influenced by selenium supplementation but in opposite ways, depending on the form of Se. These findings support that selenium is an important factor affecting the mRNA level in MSC, but the effect of the form of selenium might depend on genes.
