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Human cytomegalovirus strain Towne pp65 gene: nucleotide sequence and expression in Escherichia coli
H Pande1, K Campo, B Tanamachi
1Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Abstract:
The human cytomegalovirus (HCMV) encodes a 65-kDa tegument protein (pp65), which has been reported to be a target of immune response during natural infection. We have cloned and sequenced the gene encoding pp65 of HCMV Towne strain (pp65Towne), and have expressed this gene in Escherichia coli in order to study certain antigenic and structural properties of this polypeptide. The pp65Towne gene had a 99% nucleotide similarity and 99.7% amino acid similarity to pp65 of HCMV AD169 strain (pp65AD169). However, unlike the pp65AD169 gene, the pp65Towne gene was found to be incapable of undergoing RNA splicing due to a base substitution in the critical 3' splice-acceptor site. Insertion of this protein coding sequence into the bacterial expression plasmids enabled synthesis in E. coli of an immunoreactive pp65-related polypeptide. The recombinant pp65 (rpp65) reacted strongly in immunoblot analysis with pp65-specific murine and human monoclonal antibodies as well as with anti-pp65 rabbit antiserum. In immunoblot analysis, the reactivity of rpp65 with a panel of human HCMV-immune sera indicated that some sera were reactive while other HCMV seropositive sera were nonreactive, a finding similar to that for native pp65.
Insights
Researchers cloned and expressed the human cytomegalovirus pp65 gene in E. coli, creating a recombinant protein (rpp65) that mimics native pp65 antigenicity for immune response studies.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Human cytomegalovirus (HCMV) tegument protein pp65 is a key target of immune responses during infection.
- Understanding pp65 antigenicity is crucial for developing diagnostic and therapeutic strategies against HCMV.
Purpose of the Study:
- To clone, sequence, and express the HCMV pp65 gene from the Towne strain (pp65Towne) in Escherichia coli.
- To investigate the antigenic and structural properties of the resulting recombinant pp65 (rpp65).
Main Methods:
- Gene cloning and sequencing of pp65Towne.
- Expression of pp65Towne in E. coli using bacterial expression plasmids.
- Immunoblot analysis using monoclonal antibodies, rabbit antiserum, and human HCMV-immune sera.
Main Results:
- The pp65Towne gene showed high similarity to pp65AD169 but lacked RNA splicing capability due to a splice-acceptor site mutation.
- Recombinant pp65 (rpp65) was successfully synthesized in E. coli and demonstrated immunoreactivity.
- rpp65 reacted strongly with pp65-specific antibodies and showed variable reactivity with human HCMV-immune sera, similar to native pp65.
Conclusions:
- Bacterial expression provides a viable method for producing immunoreactive HCMV pp65 antigen.
- The observed variability in human serum reactivity to rpp65 highlights the complexity of HCMV pp65 as an immunogen.