本地毛形线虫49 Ku ES蛋白结构基因的分子克隆及原核表达

Cloning and prokaryotic expression of 49 Ku ES protein structural gene for Trichinella nativa

提取Trichinella nativa(T.nativa)肌幼虫的总RNA,用RT-PCR方法扩增出了编码T.nativa 49 Ku ES蛋白的结构基因。基因克隆后测序,序列测定结果表明:目的基因TNPG长度为951 bp,核苷酸序列同已发表的Trichinella spiralis(T.spiralis)相应的序列P49同源性为97.68%,所推导的氨基酸序列同源性为95.24%。将目的基因TNPG插入到原核表达载体pET-30a的BamHⅠ酶切位点处,并转化到感受态表达菌中进行诱导表达。结果显示TNPG在原核表达菌BL-21中获得了高效表达,表达产物为40.8 Ku的融合蛋白,表达量达到菌体总蛋白的22.8%。通过Western blot分析,表达产物可以被小鼠T.nativa和T.spiralis阳性血清以及它们的中国地理株的小鼠血清特异性识别。

The gene TNPG encoding the 49 Ku ES protein of Trichinella nativa （T.nativa） was amplified by RT-PCR and sequenced. The gene consisted of 951 bp and shared 97.68 % nucleotide homology with the sequence of P49 from Trichinella spiralis （T.spiralis）, and 95.24 % homology of deduced amino acid between TNPG and P49. TNPG was cloned into the prokaryotic expression vector pET-30a and expressed in E.coli cells. The expressed product was 40.8 Ku in size and accounted for 22.8 % of the total protein. Western blot analysis showed that the expressed product reacted with sera of mice infected with T.nativa, T.spiralis and other geographical isolates in China.