Aim: To explore preliminarily the mechanisms of immunodeficiency resulted from the CGT52TGT point mutation of mannan-binding lectin(MBL) gene.
Methods: The MBL gene containing CGT52TGT point mutation was amplified from the plasmid pMBLm52 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. After confirmed by DNA sequencing, the recombinant expression vector was transfected into Chinese-hamster ovary(CHO) cells by electroporation. Zeocin of 800 mg/L had been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain stable transfectant. The expression of mRNA was analyzed by RT-PCR, the recombinant protein was purified from the culture supernatant by Ni-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing condition and Western blot.
Results: The cDNA fragment amplified from pMBLm52 plasmid by PCR was about 750 bp and the recombinant plasmid pcDNA4/HisMax C-MBLm52 was constructed and transfected into CHO cells. The expression product purified from the culture supernatant appeared mainly at the site of M(r) 60,000, indicating a much lower oligomerization level than that of the recombinant human wild MBL and human plasma-derived MBL.
Conclusion: The CGT52TGT point mutation of MBL gene does not affect the secretion of its product, but a Cys introduced by the mutation could form another disulfide bond which may disrupt the structure of MBL molecule as well as its function.