长角血蜱卵泡抑素相关蛋白的定性

Characterization of follistatin-related protein from the hard tick Haemaphysalis longicornis

摘要:

参照GenBank中长角血蜱致病性Okayama株卵泡抑素基因的核苷酸序列(GenBank Accession No. DQ248886)设计合成一对引物, 从本实验室保藏的单克隆洁净长角血蜱饥饿成蜱中快速提取总RNA, 通过RT-PCR扩增出814 bp的卵泡抑素基因, 序列比对结果显示:与长角血蜱致病性Okayama株的核苷酸序列及氨基酸序列一致性分别为97.8%和99%, 将其亚克隆到表达载体pGEX-4T-1中进行表达, GST融合重组蛋白预期分子量为57 kD。表达重组蛋白经MagneGSTTM蛋白纯化系统纯化后作为抗原分别与抗不同发育阶段长角血蜱(卵、幼蜱、若蜱、成蜱)多克隆抗体作为一抗进行免疫印迹, 结果表明: 与长角血蜱卵制备的多克隆抗体有很强的免疫反应, 而与其他发育阶段(幼蜱、若蜱、成蜱)饥饿长角血蜱制备的多克隆抗体反应性很弱。以上结果表明: 长角血蜱卵泡抑素蛋白在长角血蜱产卵及卵成熟发育时期的表达水平较其他发育阶段(幼蜱、若蜱、成蜱)的蛋白表达水平高。

Abstract:

We designed the primers based on the sequence of the follistatin-related protein from Haemaphysalis longicornis Okayama strain accessed in GenBank. We cloned a gene encoding follistatin-related protein by RT-PCR, and the length cDNA is 814?bp, encoding a deduced protein of 289 amino acids. The alignment with the sequence of follistatin-related protein from the H. longicornis Okayama strain showed that the percent of nucleotide sequence and amino acid sequence is 97.8% and 99%, respectively. The expected size of GST-fused recombinant protein was 57 kD. We purified the recombinant protein through MagneGSTTM protein purification system. Western blotting revealed that stronger reaction happened with the antiserum against eggs, but not clear with antisera against other developmental stages.