Expression of SLA-3 in Escherichia coli and refolding the protein with peptides derived from foot-and-mouth disease virus

Abstract:

[Objective] To study the refolding between SLA-3-YDY protein derived from Yorkshire swine and peptides derived from foot-and-mouth disease virus in vitro. [Methods] A pair of primers was designed to amplify the extracellular domain of SLA-3-YDY and then the PCR product was cloned into pMD19-T Simple Vector. After cleaved by Nde I and Xho I, the positive clones were selected to be sequenced. Analyzed by biological soft, the cloned product with correct sequences was selected to be inserted into pET-21a(+) and transformed into BL21. After induction with IPTG, the interest of protein was detected by SDS-PAGE. The bacteria were broken ultrasonically, then inclusion body from the bacteria was isolated. The heavy chain of SLA-3-YDY, light chain sβ2m and the epitope-peptides Hu52 from foot-and-mouth disease virus (FMDV), were refolded at a ratio of 1:1:1 in diluted refolding buffer followed by purification in molecular sieve of superdex 200 to detect whether the complex was refolded. [Results] The PCR result shows that the SLA-3-YDY gene was amplified successfully. After sequencing and analysis, the sequence of the positive clone of SLA-3-YDY was consistent with the primary sequence. By cleavage, the interest of gene was proved to be successfully inserted into pET-21a(+) Vector. After induction with IPTG and SDS-PAGE detection, the interest of gene was expressed and the molecular weight was about 33 kD. The isolated inclusion body was also detected by SDS-PAGE, and it was shown that the molecular weight of the inclusion body was consistent with the interest of protein. By refolding in a dilution system, the heavy chains of SLA-3, peptides and light chain succeed to be refolded in vitro. Then, by using the molecular sieve column to separate and purify the refolded proteins and detection by SDS-PAGE, it was shown that the complex protein of SLA-3-Hu52-sβ2m were obtained (45 kD) finally. [Conclusion] The prokaryotic expressing vector of SLA-3 derived from Yorkshire swine was constructed successfully in this research, and the interest of protein was obtained so as to the SLA-3-Hu52-sβ2m complex was refolded finally, which will lay a base to study the structure and function of the SLA-3-YDY in future.