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Published on: April 29, 2020
[Cloning, expression and purification of the major surface antigen P30 of Toxoplasma gondii]
Da-li Zheng1, Qing-ling Huang, Tao Zhang
1Department of Biochemistry and Molecular Biology, Fujian Medical University, Fuzhou 350004.
Objective:
To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecular cloning.
Methods:
The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signal peptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence of P30. The recombinant plasmid was constructed using EcoR I, Xho I and was then transformed into E. coli Top10. The positive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTG and purified by affinity chromatography using ProBond Resin (a kind of nickel-charged sepharose resin) and was identified by SDS-PAGE and Western blotting.
Results:
The products of PCR, cleavage and link reaction were same as expected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy mutation. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography.
Conclusion:
Fusion protein containing Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.
Insights
Researchers successfully produced a fusion protein of the Toxoplasma gondii P30 antigen using molecular cloning techniques. This recombinant protein is now available for further scientific investigation.
Area of Science:
- Molecular biology
- Parasitology
- Protein expression
Background:
- Toxoplasma gondii is a significant protozoan parasite.
- The P30 antigen is a major surface protein of T. gondii.
- Understanding P30 is crucial for developing diagnostic and therapeutic strategies.
Purpose of the Study:
- To obtain the protein of the major surface antigen P30 of Toxoplasma gondii via molecular cloning.
- To generate a recombinant P30 protein for experimental use.
Main Methods:
- Polymerase chain reaction (PCR) was used to amplify the P30 gene.
- Recombinant plasmid construction and transformation into E. coli.
- Protein induction, purification via affinity chromatography, and identification by SDS-PAGE and Western blotting.
Main Results:
- The P30 gene was successfully amplified and cloned into a recombinant plasmid.
- A 58 kDa fusion protein was expressed and purified.
- Sequence analysis confirmed the integrity of the inserted fragment.
Conclusions:
- A fusion protein containing Toxoplasma gondii P30 was successfully produced.
- The recombinant protein serves as valuable experimental material for future research.
- This work facilitates further studies on T. gondii P30 antigen function and applications.
