Construction Of Muscle Specific Receptor Yrosine Kinase And Mcherry Fusion Fluorescent Protein Gene

Objective:To construct the recombinant fusion protein gene of extracellular domain peptides fragment of muscle specific receptor tyrosine kinase (MuSK) and fluorescent protein mCherry, and late on expressed in mammalian cells and as antigen in the detection of antibodies against MuSK (MuSKAb) in myasthenia gravis (MG) patients with antibodies against acetylcholine receptor (AChRAb) negative.Methods:The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector. Furthermore, the mCherry gene, after digestion with restriction enzyme Age I and extracted from electrophoresis gel, was cloned into pMT/BiP/V5-His(MuSK) carrier, that contains MuSK extracellular domain22-452amino acids peptides fragment gene to construct the Eukaryotic expression vector of the recombinant fluorescent fusion protein gene pMT/BiP/V5-His(MuSK-mCherry). The recombinant vector was subsequently teansformed into E coli DH5a and checked by colony PCR. After isolation and purification, the fusion gene was digested by restriction enzyme BsrG I for identification. Additionally, the recombinant fusion protein gene was confirmed correctly by sequencing analysis.Results:The constructed recombinant fusion protein vector pMT/BiP/V5-His (MuSK-mCherry) was evaluated by electrophoresis and sequencing analysis. The result showed that a correct size of target fragment was found, and the recombinant gene of fusion protein MuSK-mCherry was right and correctly inserted in the open reading frame (ORF) of the vector.Conclusions:The Eukaryotic expression vector of the recombinant fluorescent fusion protein gene pMT/BiP/V5-His(MuSK-mCherry) has been successfully constructed. This experiment establishes a foundation for further expression of the fusion protein in eukaryotic cells, and used as antigen for the detection of MuSKAb in MG patients.