凡纳滨对虾精氨酸激酶的多克隆抗体制备及组织特异性表达分析

精氨酸激酶（arginine kinase，AK）在调节无脊椎动物磷酸精氨酸与ATP之间能量平衡的过程中具有重要作用。在前期工作基础上，设计特异引物，以凡纳滨对虾肌肉组织cDNA为模版，克隆得到了1 071 bp的凡纳滨对虾精氨酸激酶的完整开放阅读框；将它克隆到原核表达载体pGEX-4T-2上，转化BL21 (DE3) 菌株，经诱导后表达出GST-AK融合蛋白。以纯化的融合蛋白作为抗原免疫小鼠，制备多克隆抗血清，经检测该抗原与抗体识别良好。利用得到的抗体对凡纳滨对虾AK在蛋白质水平上的特异性表达进行分析发现，精氨酸激酶在对虾的肌肉、心脏、神经、血细胞和胃组织中具有高的表达量，而在眼柄、肝胰腺、鳃和皮肤组织中表达量较低。为进一步研究精氨酸激酶在对虾体内的功能奠定了基础。

Preparation of polyclonal antibody and analysis of tissue specific expression of arginine kinase protein in Litopenaeus vannamei

Arginine kinase (ATP: arginine N-phosphotransferase, AK) in invertebrates catalyzes the reversible phosphorylation of arginine by MgATP to form phosphoarginine and MgADP. The conventional view is that phosphoarginine functions as ATP buffers, permitting maintenance of high ATP values during periods in burst of cellular activity in invertebrates. In this paper, the open reading frame (ORF) was cloned from shrimp, Litopenaeus vannamei, based on the full-length cDNA sequence that was obtained in our previous study. The ORF encoding 356 amino acids of AK was cloned and inserted to a prokaryotic expression vector pGEX-4T-2. The recombinant protein was expressed as glutathione S-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. The anti-AK polyclonal antibody was prepared by immunization of mice using this purified AK protein. The specific recognition of anti-AK antibody was further verified. The AK protein expression in various tissues was then analyzed using this newly prepared antibody by Western-blotting. The results revealed a strong expression of AK in heart, nerve, hemocyte, stomach and muscle, weak expression in eyestalk, hepatopancrease, gill and skin. The present study may be very useful for studying the function of AK in shrimp.