Related Experiment Video
Updated: Feb 6, 2026
QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii
Published on: June 22, 2017
[Prokaryotic Expression, Purification and Immunological Characterization of Micronemal Protein 16 of Toxoplama
Objective:
To prokaryotically express three gene fragments of micronemal protein 16 （TgMIC16） of Toxoplasma gondii, and analyze the immunoreactivity of the three recombinant protein products.
Methods:
Primers were designed for three fragments of TgMIC16 gene which encode proteins within the functional domain. Reverse-transcription PCR was used to generate cDNA from RNA, and the three fragments were amplified on the cDNA by PCR using the designed primers. The PCR products were double-digested, inserted into the pET-32a（+） plasmid, and transformed into Escherichia coli TOP10 cells. Plasmids extracted from positive clones were confirmed by BamHⅠ/HindⅢ double digestion and sequencing, and further transformed into E. coli Rosetta cells. Protein expression was induced by IPTG, and confirmed by SDS-PAGE. The expressed recombinant proteins were purified with Ni-NTA affinity chromatography and their immunoreactivity analyzed with Western blotting.
Results:
The amplified three fragments were 1 806, 1 290 and 855 bp in size. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids. SDS-PAGE analysis showed successful expression of the three recombinant proteins (M(r) 88 000, 68 000 and 52 000, respectively）, in the form of inclusion bodies. Western blotting showed that the three purified recombinant proteins reacted with His monoclonal antibody and rabbit anti-T. gondii antibody.
Conclusion:
The three fragments within the functional domain of TgMIC16 are successfully expressed in prokaryotic expression system and show immunoreactivity.
Insights
Three gene fragments of Toxoplasma gondii micronemal protein 16 (TgMIC16) were successfully expressed in a prokaryotic system. The recombinant proteins demonstrated immunoreactivity, indicating potential for diagnostic applications.
Area of Science:
- Molecular Biology
- Parasitology
- Protein Expression
Background:
- Toxoplasma gondii micronemal protein 16 (TgMIC16) plays a role in parasite invasion.
- Understanding TgMIC16 function requires its recombinant protein expression.
Purpose of the Study:
- To achieve prokaryotic expression of three functional domain fragments of TgMIC16.
- To analyze the immunoreactivity of the expressed recombinant TgMIC16 proteins.
Main Methods:
- Designed primers for TgMIC16 functional fragments and amplified them via RT-PCR.
- Cloned fragments into pET-32a(+) plasmid, transformed into E. coli, and induced expression.
- Purified recombinant proteins using Ni-NTA affinity chromatography and confirmed immunoreactivity via Western blotting.
Main Results:
- Successfully amplified and cloned three TgMIC16 fragments (1806, 1290, 855 bp).
- Confirmed expression of recombinant proteins (88, 68, 52 kDa) in E. coli.
- Demonstrated immunoreactivity of purified proteins with anti-His and anti-T. gondii antibodies.
Conclusions:
- Prokaryotic expression of TgMIC16 functional fragments was successful.
- The recombinant TgMIC16 proteins exhibit immunoreactivity.
- These findings support further investigation of TgMIC16 in T. gondii.
Related Concept Videos
02:35Replication in Prokaryotes
01:32Replication in Prokaryotes
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
01:58Prokaryotic Transcriptional Activators and Repressors
Transcription of prokaryotic...
02:16Comparing Mitochondrial, Chloroplast, and Prokaryotic Genomes
01:23Immunological Memory
What is Immunological Memory?
Immunological memory is an integral function of the immune system that allows it to recognize and react more rapidly and effectively to pathogens previously encountered. This feature...
01:24Cattell's 16 Personality Factors
In contrast, source traits are the...