草鱼Mx蛋白基因的克隆与原核表达

Cloning of cDNA for grass carp Mx protein and its expression in prokaryocyte

根据已报道的Mx蛋白cDNA序列设计合成特异引物,应用逆转录 聚合酶链式反应(RT PCR)方法,从草鱼呼肠孤病毒(GCRV)诱导的草鱼(Ctenopharyngodonidellus)肝脏总RNA中扩增获得Mx蛋白cDNA,回收纯化后克隆到pGEM TEasyVector系统的T载体上。重组子的序列分析表明:所克隆的Mx蛋白cDNA长722bp,编码240个氨基酸,包括1个三联GTP结合区域和1个发动蛋白(dynamin)族特征的序列。与已知鱼类Mx蛋白基因序列比较表明,草鱼Mx蛋白基因序列与其它鱼类Mx基因相应序列具有较高的同源性,其中与斑马鱼Mx蛋白E型基因碱基序列比较同源性最高,为87.1%。将此基因改造后定向克隆至原核表达质粒pBV220,构建成重组草鱼Mx蛋白基因表达质粒pBVgcMx,并在大肠杆菌中获得高效表达,重组草鱼Mx蛋白的表达量占菌体总蛋白的26.6%。Q sephroseFF层析柱分离纯化的重组草鱼Mx蛋白纯度达95.6%。

The specific primers were designed referring to the reported Mx protein gene sequences of fishes. After the experimental fish were induced by grass carp Reovirus(GCRV) for 3 days,the cDNAs encoding grass carp (Ctenopharyngodon idellus) Mx protein gene (gcMx) was amplified by reverse transcription polymerase chain reaction (RTPCR) method using total RNA isolated from grass carp liver as template. The amplified cDNA fragments were purified and inserted into pGEMT Vector and sequenced. The sequencing results showed that the cloned cDNA was 722 bp in length, encoding 240 amino acid residues of the mature peptide, containing GTP binding region and dynamin family character sequence. The homology of the nucleotide sequences of Mx protein gene between grass carp and other fishes was more than 66.6%. The highest homology reached 87.1%, which was between grass carp Mx and Zebrafish MxE. The grass carp Mx cDNA was modified by PCR technique. The modified cDNA was cloned to the plasmid pBV220. The expression recombinant plasmid pBVgcMx was constructed. After temperature inducement and SDSPAGE analysis, the recombinant expression bacteria produced a special protein of about 26 kD in molecular weight. The proportion of the recombinant protein in total bacterial protein was 26.1%. After the anion chromatography column was purified, the purity of the recombinant protein reached 95.6%.