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[Studies on in vitro expression for gI gene of Marek's disease in E. coli by pGEX vector]
Abstract:
Glycoprotein I Gene(gI) was amplified from genomic DNA of Marek's disease virus (MDV) 648 strain by polymerase chain reaction(PCR). PCR product was cloned into pGEX-6p-1 according to the right open reading frame(ORF). The expression of GST-gI fusion protein was studied in detail on many factors including temperature, timing and IPTG. The curve for OD600 and the growing time of the recombinant bacteria is also established., which is helpful to find the optimal inducing time. GST-gI fusion protein was identified by SDS-PAGE and Western-blotting., and the result shows that the best concentration of IPTG is 0.2-0.5 mmol/L and inducing time have great effects on expression while temperature has little. The fusion protein was injected into mouse five times to identify its antigenicity and the result is positive in indirect fluorescent assay IFA.
Insights
Marek
Area of Science:
- Veterinary Virology
- Molecular Biology
- Immunology
Background:
- Marek's disease virus (MDV) causes significant economic losses in the poultry industry.
- The glycoprotein I (gI) gene of MDV is a potential target for vaccine development.
- Understanding the expression and antigenicity of MDV gI is crucial for developing effective control strategies.
Purpose of the Study:
- To clone and express the Marek's disease virus glycoprotein I (gI) gene.
- To optimize the expression conditions for the GST-gI fusion protein.
- To evaluate the antigenicity of the expressed GST-gI fusion protein.
Main Methods:
- Polymerase chain reaction (PCR) was used to amplify the MDV gI gene.
- The amplified gI gene was cloned into the pGEX-6p-1 vector.
- Recombinant bacteria expressing GST-gI fusion protein were cultured and induced with IPTG.
- Protein expression was analyzed using SDS-PAGE and Western blotting.
- Antigenicity was assessed by injecting the fusion protein into mice and performing indirect fluorescent assays (IFA).
Main Results:
- The MDV gI gene was successfully amplified and cloned.
- Optimal expression of GST-gI fusion protein was achieved with 0.2-0.5 mmol/L IPTG and specific induction times.
- Temperature had minimal impact on protein expression.
- The expressed GST-gI fusion protein demonstrated positive antigenicity in mice via IFA.
Conclusions:
- The study successfully cloned and expressed the Marek's disease virus glycoprotein I gene as a GST-gI fusion protein.
- Optimized expression conditions were established for the recombinant protein.
- The results confirm the antigenicity of the MDV gI protein, supporting its potential as a vaccine candidate.