凡纳滨对虾原肌球蛋白基因表达模式与重组表达

Expression pattern and recombinant expression of tropomyosin gene of Litopenaeus vannamei

根据相近物种的同类基因设计引物,从凡纳滨对虾Litopenaeus vannamei肌肉组织中克隆获得原肌球蛋白基因(TPMS),长度为901bp,其中包含长度为852bp的完整开放阅读框,编码原肌球蛋白分子量为32.8kDa;RT-PCR结果显示,原肌球蛋白在心、肝胰腺、胃、鳃、肠和肌肉组织中均有表达,在肌肉中表达量最高,在鳃中表达量最低;将原肌球蛋白基因构建原核重组表达载体TPMS-pET30a,转化受体菌株BL21(DE3)并利用IPTG诱导后能够大量表达,蛋白分子量为38.2kDa;经优化,IPTG的最适诱导浓度为0.05mmol/L,最适诱导时间为4h;经过亲和纯化后,能够得到纯度90%以上的重组表达蛋白。

A 901 bp fragment of tropomyosin gene （TPMS） was cloned from muscle of Litopenaeus vannamei using primers designed on the basis of homologous genes of other shrimps. A complete open reading frame of 852 bp coding a 32.8 kDa protein was found in the fragment. Results of RT-PCR indicated that the gene expressed in all examined tissues inclu- ding heart, hepatopancreas, stomach, gills, intestine and muscle with different expressing lev- els. The strongest signal was found in muscle and the weakest signal was in gills. TPMS was sub-cloned into pET-30a and expressed in E. coli BL21 （DE3）. After induction of IPTG, a 38.2 kDa protein was largely expressed and formed inclusion bodies. Five hours inducing period and 0.05 mmol/L IPTG were the best conditions for recombinant expression. Recombinant ex- pressed TPMS could be purified with His Bind Resin and the purity was higher than 90 %.