Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the e...Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.展开更多
A synthetic polypeptide, pt27, which is encoded by a cDNA clone with antloncogene activity, p14-6, is found to be able to reduce remarkably the soft agar colony formation ability of part of DT cells and to raise their...A synthetic polypeptide, pt27, which is encoded by a cDNA clone with antloncogene activity, p14-6, is found to be able to reduce remarkably the soft agar colony formation ability of part of DT cells and to raise their resistance to the ouabaln toxtcity. This shows that the pt27 peptide can affect the DT cells In a manner similar to the p14- 6 done and provides evidence that the reverting action of the p14-6 to DT cells may be exerted by the expression of its cDNA.展开更多
The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and...The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that展开更多
During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific express...During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from dif-ferent developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned andsequenced. This gene was specifically expressed during 4 days of induction, and had partial homologoussequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene whichis related to the LAA regulating function during the development of the gametophytes.展开更多
A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-l...A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides(nts)into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length cDNA was substituted with the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.展开更多
Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 st...Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.展开更多
The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propa- gated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and...The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propa- gated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and F114, Brescia, Alfort or C strain were 99.41%, 96.80%, 86.03%, 95.70% and 99.28%, 98.54%, 93.33%, 97.41% re- spectively. The cDNA fragments with correct sequence were ligated into a full-length cDNA and inserted into pMC18 plasmid (pMC12297). A full-length infectious viral RNA was synthesized by runoff transcription and transfected to PK15 cells. Viruses were recovered from transfected cells which wese titrated on PK-15 cells by endpoint dilution and indirect immunofluorescence with a CSFV-specific monoclonal antibody. The antigenicity and replication kinetics of the plasmid-derived virus (vM12297) were similar to the parental virus in vitro. The E01 or E2 gene was replaced with the genes from strain C and the pM/CE01 and pM/CE2 with chimeric full-length cDNA of cF114 were generated. The infectious viruses were obtained from pM/CE01 and pM/CE2. Both of the chimeric viruses can infect PK-15, SK- 6 and primary testicle cell of swine. The chimeric viruses can grow to a titer of 8?05 F-PFU/mL. These results are very important for understanding the genes related to the CSFV propagation and pathogenesis.展开更多
Vernalization is an essential factor which affects the flowering development in cold_requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To pr...Vernalization is an essential factor which affects the flowering development in cold_requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization , we examined mRNA populations in differently_treated plumules of winter wheat \%(Triticum aestivum \%L. \%cv\% Yanda 1817) using mRNA differential display. One vernalization_ related cDNA clone \%(VRC), VRC54\%, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that \%VRC54\% was a novel vernalization_related clone found in higher plant which not only might play an important role in the floral induction in vernalization_requiring plants but also was different from the cold_acclimatized genes.展开更多
TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulv...TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.展开更多
A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary...A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary development of the as say. Various combinations of human, sheep, rat, and mouse red blood cells (RBC) with human,rat,and mouse sera were tested on their effectiveness to detect PLA2.Complete hemolysis (b hemolysis) was evident in the plate with rat RBC mixed with mouse serum.No hemolysis was detected in plates containing human RBC and human serum. Human RBC mixed with mouse serum proved to be ideal, even though this combination displayed incomplete hemolysis(a hemolysis).Susceptible RBC, in conjunction with rat or mouse serum, are excellent indicators for the presence of PLA2.Mixtures of RBC and serum in combination with BB4(E. coli) cells,λbacteriophage,and IPTG on LB agar plates provide an excellent detection system for cDNA clones that express venonl PLA2. Hemolysis surrounding a plaque is identified as positive for PLA2.展开更多
基金This study was supported by grants from the National Key R&D Program of China(2018YFA0507201)。
文摘Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.
文摘A synthetic polypeptide, pt27, which is encoded by a cDNA clone with antloncogene activity, p14-6, is found to be able to reduce remarkably the soft agar colony formation ability of part of DT cells and to raise their resistance to the ouabaln toxtcity. This shows that the pt27 peptide can affect the DT cells In a manner similar to the p14- 6 done and provides evidence that the reverting action of the p14-6 to DT cells may be exerted by the expression of its cDNA.
文摘The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that
文摘During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from dif-ferent developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned andsequenced. This gene was specifically expressed during 4 days of induction, and had partial homologoussequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene whichis related to the LAA regulating function during the development of the gametophytes.
基金This work was supported by the National Natural Science Foundation of China(No.39893290).
文摘A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides(nts)into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length cDNA was substituted with the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.
基金Supported by the National Key Basic Research Program of China (Grant No. 2005CB523201)National High-Tech Research and Development Program of China (Grant No. 2006BAD06A03)
文摘Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.
基金This work was sup-ported by the Special Funds for the State Major Basic Research of China(Grant No.G 199901 1904)the National Natural Science Foundation of China(Grant No.301 70041)and the Ministry of Education of China.
文摘The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propa- gated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and F114, Brescia, Alfort or C strain were 99.41%, 96.80%, 86.03%, 95.70% and 99.28%, 98.54%, 93.33%, 97.41% re- spectively. The cDNA fragments with correct sequence were ligated into a full-length cDNA and inserted into pMC18 plasmid (pMC12297). A full-length infectious viral RNA was synthesized by runoff transcription and transfected to PK15 cells. Viruses were recovered from transfected cells which wese titrated on PK-15 cells by endpoint dilution and indirect immunofluorescence with a CSFV-specific monoclonal antibody. The antigenicity and replication kinetics of the plasmid-derived virus (vM12297) were similar to the parental virus in vitro. The E01 or E2 gene was replaced with the genes from strain C and the pM/CE01 and pM/CE2 with chimeric full-length cDNA of cF114 were generated. The infectious viruses were obtained from pM/CE01 and pM/CE2. Both of the chimeric viruses can infect PK-15, SK- 6 and primary testicle cell of swine. The chimeric viruses can grow to a titer of 8?05 F-PFU/mL. These results are very important for understanding the genes related to the CSFV propagation and pathogenesis.
文摘Vernalization is an essential factor which affects the flowering development in cold_requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization , we examined mRNA populations in differently_treated plumules of winter wheat \%(Triticum aestivum \%L. \%cv\% Yanda 1817) using mRNA differential display. One vernalization_ related cDNA clone \%(VRC), VRC54\%, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that \%VRC54\% was a novel vernalization_related clone found in higher plant which not only might play an important role in the floral induction in vernalization_requiring plants but also was different from the cold_acclimatized genes.
基金Supported by the National Key R&D Program of China(2017YFD0101900)China Agriculture Research System(CARS-23-A-16)the Science Foundation of Heilongjiang Province(C2017024)
文摘TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
文摘A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary development of the as say. Various combinations of human, sheep, rat, and mouse red blood cells (RBC) with human,rat,and mouse sera were tested on their effectiveness to detect PLA2.Complete hemolysis (b hemolysis) was evident in the plate with rat RBC mixed with mouse serum.No hemolysis was detected in plates containing human RBC and human serum. Human RBC mixed with mouse serum proved to be ideal, even though this combination displayed incomplete hemolysis(a hemolysis).Susceptible RBC, in conjunction with rat or mouse serum, are excellent indicators for the presence of PLA2.Mixtures of RBC and serum in combination with BB4(E. coli) cells,λbacteriophage,and IPTG on LB agar plates provide an excellent detection system for cDNA clones that express venonl PLA2. Hemolysis surrounding a plaque is identified as positive for PLA2.
