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目的 构建日本血吸虫铁蛋白 (SjFer)原核表达质粒。以壳聚糖作佐剂 ,探讨重组日本血吸虫铁蛋白(rSjFer)粘膜免疫诱导小鼠抗血吸虫感染的保护力。 方法 用PCR扩增SjFer基因 ,亚克隆入原核表达载体pTWIN1上intein 2的N端 ,经PCR、限制性酶切筛选阳性重组子并经测序鉴定。将阳性重组子转化大肠埃希菌 ,在低温和低浓度的异丙基 β D 硫代半乳糖苷 (IPTG)条件下诱导表达可溶性重组融合蛋白。利用重组融合蛋白的壳聚糖结合功能域 (CBD)结合位点 ,使其与壳聚糖结合 ,经滴鼻免疫小鼠 ,于第 3次免疫后 2周攻击感染 ,用减虫率和减卵率表示保护力。感染前采血和唾液用ELISA检测抗体。 结果 成功克隆并表达了rSjFer。rSjFer以壳聚糖作佐剂经滴鼻免疫获得了 3 5 .5 1%的减虫率和 5 2 .17%的减卵率。免疫后小鼠血清IgG和IgA及唾液SIgA抗体水平与对照组比较均明显升高。 结论 获得了rSjFer ,该蛋白以壳聚糖作佐剂经粘膜免疫能诱导小鼠产生部分抗血吸虫感染的保护力。
Objective To construct the prokaryotic expression plasmid of Schistosoma japonicum ferritin (SjFer). To chitosan as an adjuvant, to explore the protection of recombinant schistosoma japonicum ferritin (rSjFer) mucosal immunity induced by anti-schistosoma infection in mice. Methods The SjFer gene was amplified by PCR and subcloned into the N terminus of intein 2 on the prokaryotic expression vector pTWIN1. The positive recombinant was screened by restriction enzyme digestion and sequenced. The positive recombinant was transformed into Escherichia coli and the soluble recombinant fusion protein was induced under low temperature and low concentration of isopropyl β D thiogalactoside (IPTG). The chitosan binding domain (CBD) of the recombinant fusion protein was used to bind to the chitosan. The mice were immunized intranasally and challenged 2 weeks after the third immunization. Egg rate indicates protection. Antibodies were detected by ELISA with blood and saliva prior to infection. Results The rSjFer was cloned and expressed successfully. rSjFer chitosan as an adjuvant intranasal immunization achieved 35.51% of the worm rate and 52.17% of the egg reduction rate. Immunized mice serum IgG and IgA and saliva SIgA antibody levels were significantly higher than the control group. CONCLUSIONS: rSjFer was obtained. The chitosan as an adjuvant can induce partial protection against infection by schistosomiasis in mice.