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[Cloning of mouse TLR-2 gene and its expression in Pichia pastroris]
Wen-zhong Zhao1, Hai-yan Jiang, Yan-jun Liu
1Department of Immunology, The First Military Medical University, Guangzhou 510515, China. zhaowz@fimmu.edu.cn
Aim:
To clone the mouse TLR-2 gene and to express it in Pichia pastroris.
Methods:
Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot.
Results:
The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody.
Conclusion:
The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.
Insights
The mouse Toll-like receptor 2 (mTLR-2) gene was successfully cloned and expressed in Pichia pastoris. This research provides a method for producing recombinant mTLR-2 protein.
Area of Science:
- Molecular Biology
- Immunology
- Biotechnology
Context:
- Toll-like receptors (TLRs) are critical components of the innate immune system.
- Mouse Toll-like receptor 2 (mTLR-2) plays a significant role in pathogen recognition.
- Efficient expression systems are needed for studying TLR function and developing therapeutics.
Purpose:
- To clone the full-length mouse Toll-like receptor 2 (mTLR-2) gene.
- To establish a heterologous expression system for mTLR-2 in Pichia pastoris.
- To confirm the successful cloning and expression of the mTLR-2 gene and its protein product.
Summary:
- The full-length mTLR-2 gene was amplified using RT-PCR and cloned into the pPICZalphaC vector for Pichia pastoris expression.
- Recombinant Pichia pastoris strains were confirmed by PCR and RT-PCR.
- SDS-PAGE and Western blot analyses verified the expression of a ~97,000 M(r) recombinant mTLR-2 protein that reacted with anti-mTLR-2 antibodies.
Impact:
- This study successfully cloned the mTLR-2 gene and demonstrated its correct expression in Pichia pastoris.
- The developed expression system facilitates the production of recombinant mTLR-2 protein for further immunological research.
- This work contributes to the understanding of TLR-2 function and potential biotechnological applications.
