日本血吸虫rSj14-3-3疫苗和rSjGST疫苗联合免疫的保护作用研究

发布时间：2019-05-08 22:44

【摘要】：目的利用成功构建好的含有日本血吸虫重组信号蛋白14-3-3(rSj14-3-3)和重组Mr26000谷胱甘肽S转移酶(rSj26GST)编码基因的菌株E.coli BL21/pET28a,诱导表达rSj14-3-3和rSj26GST这两种重组蛋白,并利用其免疫小鼠,观察日本血吸虫rSj14-3-3疫苗和rSj26GST疫苗联合免疫对小鼠的保护作用,同时观察日本血吸虫rSj14-3-3和rSj26GST疫苗联合免疫对小鼠抗病免疫效应的研究。 方法分别复苏本实验室-80℃低温保种的含有rSj14-3-3和rSj26GST编码基因的菌株E.coliBL21/pET28a,将其均匀涂布于LB/Kana/IPTG/ X-gal平板,培养、收集细菌、超声碎菌、分离、亲和层析法纯化重组蛋白,分别取5μl进行SDS-PAGE ,观察纯化结果,采用Bradford法测定重组蛋白rSj14-3-3和rSj26GST的浓度。将100只6～8周龄BALB/c雌性小鼠随机分为A、B、C、D、E5组,每组20只,将两种纯化后的蛋白分别皮下免疫BALB/c小鼠。A组(感染对照组),于第0周每鼠经背部皮下多点注射50μl PBS及等体积福氏完全佐剂(CFA),于第2及4周每鼠皮下多点加强注射50μl PBS加等体积福氏不完全佐剂(IFA)。B组(rSj14-3-3组),每鼠于第0、2、4周经皮下注射100μg(50μl)rSj14-3-3。C组(rSj26 GST组),每鼠于第0、2、4周经皮下注射100μg(50μl)rSj26 GST。D组(rSj14-3-3 + rSj26 GST联合免疫组),于第0、2、4周经背部皮下多点注射rSj14-3-3和rSj26 GST各50μg(25μl)。上述各组分别于末次免疫2周后每鼠经腹部皮肤感染日本血吸虫尾蚴(30±2)条。E组(对照组)未作任何处理。于感染45d后剖杀全部小鼠,眼眶取血,用于血清纤维化指标检测;计数成虫及肝内虫卵,计算各组间的减虫率和减卵率;将小鼠肝组织做成切片进行HE染色,测量各组单卵肉芽肿大小。 结果成功诱导表达rSj14-3-3和rSj26GST这两种重组蛋白,并对其进行纯化,经鉴定纯化结果较好。联合免疫组的减虫率为38.4%,显著高于rSj14-3-3(19.8%)和rSj26GST组(21.6%)(P值均0.01)。联合免疫组以及rSj14-3-3、rSj26GST组减卵率分别为48.8%、35.3%、41.4%;联合免疫组及rSj14-3-3和rSj26GST组,肝组织中每条雌虫平均产卵数显著低于感染对照组(P值均0.01)。联合疫苗组小鼠虫卵肉芽肿的直径为(173.9±35.0um),显著小于感染对照组的(267.7±28.6um)(P值0.01)。检测各组的透明质酸和层黏连蛋白水平,联合免疫疫苗组显著低于感染对照组(P值0.01),他们均高于正常对照组。 结论成功诱导表达rSj14-3-3和rSj26GST重组蛋白,并成功获得他们纯化后的重组蛋白。联合免疫组的保护作用优于rSj14-3-3和rSj26GST单独免疫组。联合免疫疫苗具有一定的抗虫卵肉芽肿及抗肝纤维化作用。
[Abstract]:Objective to construct a recombinant Schistosoma japonicum (Schistosoma japonicum) strain E.coli BL21/pET28a, which contains recombinant signal protein (rSj14-3-3) and recombinant glutathione S transferase (rSj26GST) gene of Schistosoma japonicum. The recombinant proteins rSj14-3-3 and rSj26GST were induced and immunized in mice. The protective effects of combined immunization with rSj14-3-3 vaccine and rSj26GST vaccine against Schistosoma japonicum were observed. At the same time, the effect of combined immunization with rSj14-3-3 and rSj26GST vaccine of Schistosoma japonicum on anti-disease immunity in mice was observed. Methods E.coli BL21 / pET28a, which contained rSj14-3-3 and rSj26GST coding genes, was revived at-80 鈩，

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