Molecular Cloning And Expression Of α₂-macroglobulin From Cristaria Plicata

Cristaria plicata is one of the most important "Freshwater pearl mussel"in China. But Cristaria plicata is vulnerable to disease,which make a great influence to the ability of the pearl.α₂-macroglobulin(α₂M) was cloned and part sequence ofα₂M was expressed with Prokaryotic expression in the paper.A cDNA encodingα₂-macroglobulin was cloned by RT-PCR,PCR walk, 5'RACE and 3'RACE.The gene spans 5621 bp,which contains an encoding region 5226 bp,5' untranslated region 32 bp,3' untranslated region 363 bp(including a polyA tail of 31 bp).The open reading frame encodes a protein of 1741 amino acid residues.The first 16 amino acid residuesin the N-terminus were recognized as signal peptide.The deduced mature protein contains 1725 amino acid residues,the relative molecular weight is 195 kDa,isoelectric point is 5.12.The Cristaria plicataα₂M sequence is the same with putative functional domains including a bait region,an internal thiol ester site and a receptor-binding domain as other animalα₂Ms.Blast analysis showed that theα₂M was the highest similarity with Hyriopsis cumingii and Chlamys farreri.Expression ofα₂M in different tissues of Cristaria plicata was determined by RT-PCR.The result showed that no expression was detected in mantel,conductor muscle,hepatopancreas and sexual gland,except of hemocyte.After injecting by Aeromonas hydrophila 6 12 24h,α₂M expression level had a certain amount of rise in hemocyte,which fully proved the existence ofα₂M immune system in Cristaria plicata.The expression primers were designed according to part of cDNA segment which conclude the receptor-binding domain of Cristaria plicata.The constructed recombinant expression plasmids were induced by the chemical inducer IPTG.The expression products were analyzed by SDS-PAGE.The results indicated that recombinationα₂M protein was successfully expressed in Escherichia Coli.The expression products were 69 KDa protein.