二种多管水母光蛋白基因的分离、表达及生物活性初步研究

分别从厦门东海域的大型多管水母和细小多管水母中分离到了新的光蛋白基因aeqxm和aeqxxm,并在大肠杆菌中进行了表达.aeqxm和aeqxxmDNA序列的编码区总长均为585bp,无内含子序列,推导的氨基酸序列总长均为195个氨基酸.aeqxm,aeqxxmDNA和AEVAQ440XcDNA之间的核苷酸序列同源性分别为80.7%,85.1%,aeqxm和aeqxxmDNA的核苷酸序列同源性为87.2%.原光蛋白apoaeqxm,apoaeqxxm与AEVAQ440X编码的氨基酸序列之间的同源性分别为84.7%,84.2%,apoaeqxm和apoaeqxxm的氨基酸序列之间的同源性为94.4%.分别将aeqxm和aeqxxm基因克隆至pTO-T7表达载体,apoaeqxm,apoaeqxxm在大肠杆菌中的表达量都达到40%左右.取菌体超声上清与腔肠动物荧光素f、巯基乙醇混合再生后,加入CaC_l2,用荧光全谱仪检测到470nm处的瞬时发光,表明表达的apoaeqxm,apoaeqxxm具有正常的生物学功能.

Cloning and expression of the aequorin genes from jellyfish aequorea and characterization of aequorins activities

Two new aequorin genes aeqxm and aeqxxm were isolated from fellyfish Aequorea macr odactyla and A.parva respectively,which are commonly found in the warmer waters on the coastal region of East China Sea.The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein.The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7% and 85.1% with AEVAQ 440X respectively,and the corresponding proteins share amino acid homologies of 84.7% and 84.2% with AEVAQ 440X.High amino acid homology was found between apoaeqxm and apoaeqx xm.The two genes were cloned into expression vector pTO-T7 respectively,and the expression yields were amounted to 40% of the total protein in E.coli BL21.The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f.In the presence of Caion,both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470 nm.