ABSTRACT
To determine the ratio of short fiber (sfiber) to long fiber (lfiber) in human adenovirus type 41 (HAdV-41) virions, sfiber and lfiber were expressed in E. coli, quantified, and used as loading standards in Western blot. Densitometric analyses of the standard and target bands indicated that the ratio of sfiber to lfiber in virions was 5.7±0.7. Sfiber-deleted HAdV-41, HAdV-41-DSF-GFP, was constructed, and Western blot analysis showed that the amount of lfiber in HAdV-41-DSF-GFP was about 7.3±1.9 times of that in HAdV-41 virions, confirming a ratio of approximate 6 for sfiber to lfiber in HAdV-41. In HAdV-41-infected cells, mRNAs of the sfiber and lfiber were comparable in quantity, while the expression at protein level was significantly different. Our results suggested an unequal number of short and long fibers, which might result from their differential protein expression during HAdV-41 packaging. The method used here could be extended to quantify other trace proteins.
Subject(s)
Adenoviruses, Human/genetics , Capsid Proteins/analysis , Virion/chemistry , Adenoviruses, Human/classification , Adenoviruses, Human/metabolism , Amino Acid Sequence , Blotting, Western , Capsid/chemistry , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line , Escherichia coli/genetics , Escherichia coli/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Virion/metabolismABSTRACT
Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.
Subject(s)
Adenoviruses, Human/classification , Adenoviruses, Human/genetics , Cloning, Molecular , DNA, Viral/genetics , Adenoviruses, Human/pathogenicity , Anti-Infective Agents/pharmacology , Drug Resistance, Viral/genetics , Genome, Viral , HEK293 Cells , Humans , Kanamycin/pharmacology , Plasmids , Virus Replication/physiologyABSTRACT
OBJECTIVE: To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus. METHODS: The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis. RESULTS: Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability. CONCLUSION: The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.
Subject(s)
Adenoviridae/genetics , Antigens, Viral/genetics , Capsid Proteins/genetics , Gene Expression , Genetic Vectors/genetics , Rotavirus/genetics , Adenoviridae/metabolism , Antigens, Viral/metabolism , Capsid Proteins/metabolism , Cell Line , Genetic Vectors/metabolism , Humans , Rotavirus/metabolism , Rotavirus Infections/virologyABSTRACT
Human adenovirus type 41 (HAdV-41) is difficult to cultivate under laboratory conditions. Tripartite leader sequence (TPL) of HAdV-41 was cloned, inserted into eukaryotic expression plasmid, and used to establish a HAdV-41 E1B55K-transduced cell line (293TE7). HAdV-41 E1B55K was expressed more abundantly in 293TE7 than in 293E12, an HAdV-41 E1B55K-expressing cell line developed previously. After being infected with E1-deleted HAdV-41 vector (HAdV-41-GFP), 293TE7 synthesized more viral genomic DNA and structural proteins, which led ultimately to a significant increase of the yield of progeny viruses. Typically, 293TE7 produced progeny viruses 3-15 times more than 293E12 did, depending on the amount of seed viruses and culture time. These data demonstrated that 293TE7 was an effective packaging cell line, and implied its application for wild-type HAdV-41 isolation, HAdV-41 virological study and recombinant HAdV-41 construction.
