大肠杆菌Ee株SLT-IIeB与FedF基因的融合表达及表达产物的免疫原性研究

根据猪水肿病大肠杆菌Ee株主要免疫原性片段SLT-IIeB和FedF的基因序列,利用PCR技术克隆目的片段,将SLT-IIeB和FedF基因依序串联于谷胱甘肽-S-转移酶(GST)表达系统的GST下游,在大肠杆菌中成功获得了大小约为63kDa融合蛋白GST-SF,Western blot检测证实表达的融合蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原免疫新西兰兔制备血清,体外活性试验表明,所制备的抗血清能中和Ee株水肿毒素对Vero细胞的病变效应;黏附抑制试验证实抗血清能抑制Ee株对猪小肠刷状缘细胞的黏附。将融合蛋白免疫小鼠,结果表明,GST-SF能够诱发较好的免疫反应,并能提供对Ee株5LD50致死剂量的70%保护率。研究表明GST-SF融合蛋白具有良好的免疫原性,具有良好的应用前景。

Fusion expression of SLT-IIeB gene and FedF gene of Ee in Escherichia coli and its immunogenicity

The DNA fragment encoding the truncated SLT-IIeB and FedF of Ee strain were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKSF. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-SF fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with mouse anti-SLT-IIeB antiserum, mouse anti-FedF antiserum and moue anti-GST monoclonal antibody respectively. The fusion protein was further purified and used as an antigen for preparation of immune serum. The anti-sera of GST-SF were able to restrain the toxicity of SLT-IIe to Vero-E6 cells and inhibit the adhesin of F18 fimbriae to brush borders of swine in vitro. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-SF, GST-B, GST-F and challenged intraperitoneally with volume of 5 LD50 Ee strain. The results show the fusion protein GST-SF had more shrong immunogenicity and better protection against Ee strain.