[Expression, purification and activity analysis of BCG HSP70.].

Area of Science:

• Molecular Biology
• Immunology
• Biochemistry

Background:

• Mycobacterium bovis BCG is a key component in vaccines against tuberculosis.
• Heat shock proteins (HSPs), like HSP70, play crucial roles in cellular stress response and immune modulation.
• Understanding BCG HSP70's function is vital for developing improved tuberculosis therapies.

Purpose of the Study:

• To express and purify recombinant BCG HSP70 protein with high biological activity using E. coli expression system.
• To confirm the identity and purity of the expressed BCG HSP70.
• To evaluate the immunomodulatory effect of purified BCG HSP70 on mouse splenocytes.

Main Methods:

• BCG HSP70 gene amplification via PCR and cloning into pMD18-T vector.
• Subcloning into pET28a expression vector and transformation into E. coli BL21(DE3).
• Protein expression using IPTG induction, followed by purification, SDS-PAGE, Western blot analysis, and splenocyte proliferation assays.

Main Results:

• Successfully amplified and sequenced the BCG HSP70 gene.
• Expressed a recombinant protein of approximately 70 kDa, confirmed by SDS-PAGE and Western blot.
• Achieved 96.5% purity for the recombinant BCG HSP70 protein.
• Demonstrated significant stimulation of mouse splenocyte proliferation by the purified protein.

Conclusions:

• Recombinant BCG HSP70 was successfully expressed and purified with high purity and significant biological activity.
• This provides a foundation for further research into the role of BCG HSP70 in tuberculosis.
• The study highlights the potential of BCG HSP70 as an immunomodulatory agent.