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

中文关键词: 凡纳滨对虾  过敏原  原肌球蛋白  表达

英文关键词: Litopenaeus vannamei  Allergen  Tropomyosin  Expression

中文摘要:

      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901 bp，其中包含长度为852 bp的完整开放阅读框，编码原肌球蛋白分子量为32.8 kDa；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38.2 kDa；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。

英文摘要:

      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 including heart, hepatopancreas, stomach, gills, intestine and muscle with different expressing levels. 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 expressed TPMS could be purified with His Bind Resin and the purity was higher than 90%.