Construction Of CDNA Library Of Cristaria Plicata Expression The TIMP Gene In Cristaria Plicata

Cristaria plicata is one of the Freshwater Pearl mussel in China. It is ofen infected by pathogen, which make a great influence to the ability of the Pearl. Therefore, it is necessary and meaningful to study on the molecular immune.We were extracted Cristaria plicata total RNA with Trizol method,_Its blood cells full-length cDNA library were constructed according to SMART cDNA synthesis Kit description methods, and was ligated with PCR product (Sfi restriction) and the pBluescript Ⅱ SK carriers (Sfi restriction). The titer of cDNA library was1.07×106cfu/mL.The recombination percentage was96%, the average length of inserted fragment was about732bp. The library was constructed,297EST were obtained from500clone sequencing.The ESTs of known functions was123. The seven categories were divided into by biological function.Random screening of cDNA library of Cristaria plicata blood cells get a821bp of EST fragments. Through sequence alignment and preliminary analysis. we think it's Cristaria plicata TIMP genes. The maximum expression was in the adductor muscle, but it was much lower expression in blood cells, mantle, hepatopancreas and gonads. After the injection of Aeromonas hydrophila6,12,24,48h, the expression level was up to certain amount in blood cells, hepatopancreas and pancreas, gill by Real time Quantitative PCR.The expression primer was designed, and total RNA was extracted from blood cells of Cristaria plicata. TIMP gene was got and amplified with RT-PCR. The isolated fragment was sequenced, recombined with plasmid pET-30a(+) at KpnI and EcoRI sites and transformed into E.coli. Then the expression of TIMP was induced by IPTG. Expressed product of TIMP was analysed by SDS-PAGE. TIMP fusion protein was purified by N2+-NTA. Results showed that recombinant TIMP was induced in Escherichia coli. but it was inclusion bodies under the37℃, and it is about32KDa. Expressed product of N-Terminal TIMP was about20KDa. After optimization of expression conditions,1mM IPTG was added under15℃for34h. Expressed product of N-Terminal TIMP was soluble protein, and it was purified by N2+-NTA. TIMP fusion protein with bioactivity might be obtained by gene cloning and expression E. Coli.