Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants
Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants
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在不存在佐剂的情况下，通过粘膜施用 Hsp65 携带的 P277 重复序列诱导针对 P277 的持久特异性抗体。
DOI：
10.1016/j.vaccine.2006.11.052
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发表时间：
2007-03-01
期刊：
影响因子：
5.5
通讯作者：
Liu Jingjing
中科院分区：
文献类型：
--
作者：
Jin Liang;Wang Yu;Liu Jingjing
为提高P277肽的弱免疫原性，将PCR扩增的5 × P277插入到pET 28-Hsp 65-P277载体中，构建重组表达质粒pET 28-Hsp 65 -6 × P277。转化大肠杆菌BL 21（DE 3），经乳糖诱导后，融合蛋白（Hsp 65 -6 x P277）以可溶性形式得到高效表达。采用细胞破碎、硫酸铵沉淀、双蒸水透析、DEAE 52-纤维素柱层析等方法对融合蛋白进行纯化。在没有佐剂的情况下接种。每隔3周收集免疫小鼠的血清样品。用酶联免疫吸附试验（ELISA）和Western blot检测免疫小鼠血清中抗P277和HSP 65的抗体。经鼻内途径免疫动物，即使在无佐剂的情况下，也能成功诱导特异性抗体，并持续20周以上。(c)2006爱思唯尔有限公司版权所有。
To improve the weak immunogenicity of peptide P277, the recombinant expression plasmid pET28-Hsp65-6 x P277 was constructed by inserting 5 x P277 which was amplified by PCR into the vector pET28-Hsp65-P277. It was transformed into Escherichia coli BL21 (DE3) and the fusion protein (Hsp65-6 x P277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified by means of cell disruption, ammonium sulfate precipitation, double-distilled H2O dialysis, DEAE52-cellulose column chromatography, and then used to immunize female NOD mice with three i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at 3 weeks interval. Antibodies against P277 and HSP65 were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and Western blot. Specific antibodies were successfully induced and lasted for more than 20 weeks in animals immunized with the fusion protein via intranasal route even in the absence of adjuvants. (c) 2006 Elsevier Ltd. All rights reserved.