表面展示GnRH重组T7噬菌体构建及其免疫效果

摘要

Three copies of gonadotrophin releasing hormone ( GnRH ) gene were artificially synthesized and then cloned into the T7 Select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-3GnRH. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was con-firmed by SDS-PAGE and Western-blot. The mice were intramuscularly injected with 1×1010 PFU phage T7-3GnRH, and sera samples were tested for the titer of anti-GnRH antibody by ELISA and testosterone concentration by radioimmunoassay kit. A recombinant T7-3GnRH phage was confirmed displaying GnRH peptides, and the fusion protein generated favorable immunogenicity. All mice developed a certain amount of the anti-GnRH antibody which could neutralize the endogenous GnRH so as to down-regulate testosterone. Testicular histological examination revealed that testis development and spermat-ogenesis were markedly inhibited. These results indicate that the recombinant T7 phage displaying GnRH peptides has a great potential to be developed into a novel vaccine for the immunocastration.%构建展示促性腺激素释放激素( GnRH)多肽的重组T7噬菌体,检测其对小鼠的免疫效果。人工合成GnRH多肽基因序列并用柔性Linker将其串联至3拷贝,然后克隆到T7 Select 415-1b噬菌体多克隆位点,构建重组噬菌体T7-3GnRH。经PCR鉴定并序列测定筛选阳性重组噬菌体,SDS-PAGE和Western-blot检测重组噬菌体表面GnRH多肽。重组噬菌体以每只1×1010 PFU剂量免疫小鼠,免疫后不同时间段采血通过ELSIA检测血清中抗GnRH抗体,检测血清中睾酮含量,免疫后12周称体质量,剖检小鼠计算睾丸指数,进行睾丸的组织学观察。结果表明,成功构建了重组噬菌体T7-3GnRH,插入拷贝GnRH基因获得表面展示。噬菌体疫苗免疫后产生高水平抗GnRH抗体,其抗体发挥中和效应显著降低血清中睾酮含量。 GnRH主动免疫抑制小鼠睾丸组织的发育,使睾丸萎缩,精子生成减少。获得了展示GnRH多肽的重组噬菌体,噬菌体疫苗免疫小鼠产生较高血清抗体并发挥良好的中和作用,为激素类小分子主动免疫做出有益探索。