凝血因子ⅩⅢ A亚基mRNA大片段缺失所致遗传性凝血因子ⅩⅢ缺乏症的分子机制研究

OBJECTIVE: To investigate the mechanisms of DelCD11-279 of factor ⅩⅢ subunit A mRNA in the pathogenesis of hereditary factor ⅩⅢ deficiency. METHODS: The recombinant plasmids containing pET-22b(+)/FⅩⅢA of normal subject and proband's mother and pET-22b(+)/FⅩⅢA-Del of the proband were constructed and transformed into E. coli BL21. Expressing protein was analyzed by the SDS-PAGE and purified by Ni-NTA resin. Purified proteins were detected by the Western-blot. The activity of purified protein was detected by the incorporation test with EZ-LinkTM5-(Biotinamido) Pentylamine. RESULTS: The recombinant plasmids containing pET-22b (+)/FⅩⅢA and pET-22b (+)/FⅩⅢA-Del which constructed and identified successfully by enzyme digestion and PCR, were transformed into E. coli BL21 and efficiently expressed by IPTG induction. The molecular weights of expressing proteins are 83 200 and 51 900 by the SDS-PAGE. Expressing proteins were purified by Ni-NTA resin, and were proved to be human FⅩⅢA proteins by Western-blot. Purified protein activity of proband' s mother and proband was 95.87% and 0 of the purified F ⅩⅢ A protein activity from the normal subject, respectively. CONCLUSION: DelCD11-279 of FⅩⅢA mRNA which encoding a 464 amino acids of inactive FⅩⅢA protein is one of the molecular mechanisms resulting in FⅩⅢ deficiency in the patient.