对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆...对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。展开更多
Genome-wide association studies with an Illumina Bovine50K chip have detected 105 SNPs associated with one or multiple milk production traits in the Chinese Holstein population.Of these,38 significant SNPs detected wi...Genome-wide association studies with an Illumina Bovine50K chip have detected 105 SNPs associated with one or multiple milk production traits in the Chinese Holstein population.Of these,38 significant SNPs detected with high confidence by both L1-TDT and MMRA methods were selected to further mine potential key genes affecting milk yield and milk composition.By blasting the flanking sequences of these 38 SNPs with the bovine genome sequence combined with comparative genomics analysis,26 genes were found to contain or be near to such SNPs.Among them,the C14H8orf33 gene is merely 87 bp away from the significant SNP,Hapmap30383-BTC-005848.Hence,we report herein genotype-phenotype associations to further validate the genetic effects of the C14H8orf33 gene.By pooled DNA sequencing of 14 unrelated Holstein sires,a total of 18 with seven novel SNPs were identified.Among them,nine SNPs were in the 5′regulatory region,one in exon 6 and the other in the 3′UTR and 3′regulatory region.A total of nine of these identified SNPs were successfully genotyped and analyzed by mass spectrometry for association with five milk production traits in an independent resource population.The results showed that these SNPs were statistically significant for more than two traits[P<(0.0001–0.0267)].In addition,mRNA expression analyses revealed that C14H8orf33 was ubiquitous in eight different tissues,with a relatively higher expression level in the mammary gland than in other tissues.These findings,therefore,provide strong evidence for association of C14H8orf33 variants with milk yield and milk composition traits and may be applied in Chinese Holstein breeding programs.展开更多
Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion mor...Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gam- maherpesviruses including Kaposi＇s sarcoma-associ- ated herpesvirus （KSHV） and Epstein-Barr virus （EBV） is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvi- rus-68 （MHV-68） is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion mor- phogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network （TGN）, a site where secondary envelopment takes place.展开更多
文摘对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。
基金supported by the National High-tech R&D Program of China(2013AA102504)the National Key Technology R&D Program(2011BAD28B02)+3 种基金the National Transgenic Major Project(2014ZX08009-053B)the Beijing Innovation Team of Technology System in the National Dairy Industry,the Beijing Research and Technology Program(D121100003312001)the earmarked fund for Modern Agro-industry Technology Research System(CARS-37)the Program for Changjiang Scholar and Innovation Research Team in University(IRT1191).
文摘Genome-wide association studies with an Illumina Bovine50K chip have detected 105 SNPs associated with one or multiple milk production traits in the Chinese Holstein population.Of these,38 significant SNPs detected with high confidence by both L1-TDT and MMRA methods were selected to further mine potential key genes affecting milk yield and milk composition.By blasting the flanking sequences of these 38 SNPs with the bovine genome sequence combined with comparative genomics analysis,26 genes were found to contain or be near to such SNPs.Among them,the C14H8orf33 gene is merely 87 bp away from the significant SNP,Hapmap30383-BTC-005848.Hence,we report herein genotype-phenotype associations to further validate the genetic effects of the C14H8orf33 gene.By pooled DNA sequencing of 14 unrelated Holstein sires,a total of 18 with seven novel SNPs were identified.Among them,nine SNPs were in the 5′regulatory region,one in exon 6 and the other in the 3′UTR and 3′regulatory region.A total of nine of these identified SNPs were successfully genotyped and analyzed by mass spectrometry for association with five milk production traits in an independent resource population.The results showed that these SNPs were statistically significant for more than two traits[P<(0.0001–0.0267)].In addition,mRNA expression analyses revealed that C14H8orf33 was ubiquitous in eight different tissues,with a relatively higher expression level in the mammary gland than in other tissues.These findings,therefore,provide strong evidence for association of C14H8orf33 variants with milk yield and milk composition traits and may be applied in Chinese Holstein breeding programs.
基金We thank Drs. Ren Sun and Tao Xu for kindly providing plasmids, Mr. Shufeng Sun at the Center for Biological Imaging, Institute of Biophysics, for help with EM sample preparation and members of the Deng laboratory for helpful discussions. This work was sup- ported by grants from the National Natural Science Foundation of China （Grant No. 81171582） and the National Basic Research Program （973 Program） （No. 2011CB504300）.
文摘Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gam- maherpesviruses including Kaposi＇s sarcoma-associ- ated herpesvirus （KSHV） and Epstein-Barr virus （EBV） is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvi- rus-68 （MHV-68） is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion mor- phogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network （TGN）, a site where secondary envelopment takes place.
