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[Prokaryotic expression and identification of S-dsRNA gene from Cryptosporidium parvum virus]
Yu-mei Diao1, Peng-tao Gong, Wei Li
1College of Animal Science and Veterinary Medicine, Jilin University, Changchan 130062, China.
Objective:
To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant.
Methods:
Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis.
Results:
pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts.
Conclusion:
The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.
Insights
The S-dsRNA gene from Cryptosporidium parvum virus was successfully cloned and expressed in E. coli. The resulting recombinant protein demonstrated adequate reactogenicity, showing promise for diagnostic applications.
Area of Science:
- Molecular biology
- Virology
- Immunology
Context:
- Cryptosporidium parvum is a significant cause of diarrheal disease.
- Understanding the virus associated with C. parvum is crucial for disease control.
- The S-dsRNA gene is a component of the C. parvum virus.
Purpose:
- To clone and express the S-dsRNA gene of the Cryptosporidium parvum virus.
- To analyze the expression of the recombinant S-dsRNA protein.
- To investigate the reactogenicity of the expressed recombinant protein.
Summary:
- The S-dsRNA gene was amplified from Cryptosporidium parvum and cloned into the pET-28a(+) vector.
- The recombinant plasmid was expressed in E. coli BL21 (DE3), yielding a 37,000 M(r) protein.
- High-level expression was achieved under specific IPTG induction conditions, and the protein was recognized by immune sera.
Impact:
- Successful expression of the Cryptosporidium parvum virus S-dsRNA gene.
- Demonstration of the recombinant protein's reactogenicity.
- Provides a foundation for developing diagnostic tools and understanding C. parvum virus biology.
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