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[Cloning and expression of transferrin protein from Culex pipiens pallens and a study of its antimicrobial activity]
Insights
Mosquito transferrin (Tsf) was successfully cloned and expressed in Pichia pastoris, demonstrating significant antibacterial activity against Escherichia coli. This research highlights the potential of insect-derived transferrin as a novel antimicrobial agent.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Transferrin (Tsf) is a key iron-binding protein involved in immune responses.
- Investigating insect transferrin offers potential for novel antimicrobial strategies.
Purpose of the Study:
- To clone and express transferrin from Culex pipiens pallens in Pichia pastoris.
- To evaluate the antibacterial activity of the recombinant transferrin.
Main Methods:
- Transferrin gene amplification using RT-PCR.
- Cloning into Pichia pastoris expression vector pGAPZalpha-A.
- Expression in P. pastoris GS115 and protein purification.
- Antibacterial activity assay against Escherichia coli.
Main Results:
- Successful amplification and cloning of the transferrin gene.
- Expression of recombinant transferrin with an approximate molecular weight of 80,200 Da.
- Demonstrated antibacterial activity against E. coli with a minimum inhibitory concentration of 0.25 mg/ml.
Conclusions:
- Recombinant transferrin from Culex pipiens pallens can be effectively expressed in Pichia pastoris.
- The expressed transferrin exhibits significant antibacterial activity against E. coli.
Objective:
To clone and express transferrin (Tsf) from Culex pipiens pallens in Pichia pastoris, and detect its antibacterial activity.
Methods:
The coding region of transferrin from Culex pipiens pallens was amplified by RT-PCR. The product of RT-PCR was inserted into the downstream of gene encoding a-factor signal sequence in a Pichia pastoris secreting expression vector pGAPZalpha-A. The recombinant pGAPZalpha-A-Tsf vector was transformed into P. pastoris GS115 by electroporation. Recombinant strains pGAPZa-A-Tsf/GS115 were screened by Zeocin resistance and PCR. Recombinant protein was detected by SDS-PAGE and Western blotting. The recombinant transferrin protein was purified by using Ni-NTA resin. The antibacterial activity of the purified transferrin against Escherichia coli was detected.
Results:
The transferrin gene with 2,100 bp was obtained by RT-PCR. The product of recombinant plasmid pGAPZalpha-A-Tsf was approximately 2 127 bp by double digestion with restriction enzymes, consistent with the anticipated fragment length. Sequencing results showed that the inserted sequence was correct. PCR result showed that the recombinant plasmid pGAPZalpha-A-Tsf/GS115 was constructed. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (Mr) of the protein was about 80,200. The recombinant transferrin protein showed antibacterial activity against Escherichia coli, and the minimum concentration was 0.25 mg/ml.
Conclusion:
The recombinant transferrin protein from Culex pipiens pallens has been expressed in P. pastoris, and shows antibacterial activity against E. coli.
