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目的:克隆、表达日本血吸虫酪蛋白激酶2α(SjCK2α)基因,并定位表达,观察其表达产物的免疫效果。方法:首先针对SjCK2α基因的开放阅读框设计特异性引物,以日本血吸虫成虫RNA逆转录后的cDNA为模板,扩增目的基因;并使其带上限制性内切酶酶切位点,获得SjCK2α的目的基因片段,回收后连入表达质粒pET28a(+)。重组质粒pET28a(+)-SjCK2α转化入BL21细胞,构建表达菌;并用IPTG诱导表达菌的表达;用Ni-NTA树脂纯化后透析,收集重组目的蛋白。用重组蛋白免疫家兔制备多克隆血清。免疫组化法对皮肤型童虫、肺型童虫和常规培养肺型童虫进行组织学定位。将重组蛋白免疫小鼠,再用日本血吸虫尾蚴攻击感染,6周后剖杀,灌注法收集日本血吸虫成虫,计算减虫率;摘取肝脏组织剪碎、KOH消化后收集虫卵,计算减卵率;摘取眼球取血,收集血清,ELISA检测细胞因子干扰素γ(IFN-γ)和白细胞介素4(IL-4)的含量。另设感染对照组和化工剂对照组进行比较分析。结果:扩增的目的基因SjCK2α基因片段长度为1047bp,构建的重组质粒pET28a(+)-SjCK2α转化入BL21细胞后,构建的表达菌在IPTG诱导下获得大量重组蛋白,纯化后免疫家兔获得多克隆血清。免疫组化染色发现,SjCK2α主要定位于童虫表面。将重组蛋白免疫小鼠后,攻击感染6周后获得15%的减虫率和17%的减卵率,与对照组的减虫率和减卵率差异无统计学意义(P>0.05);此时,感染对照组的IFN-γ和IL-4含量分别为42.77±12.11和17.31±9.13,佐剂对照组的IFN-γ和IL-4的含量分别为30.99±16.55和18.65±7.52,重组蛋白免疫组的IFN-γ和IL-4的含量分别为36.19±14.56和9.90±5.20,三组间两指标比较均无统计学差异(P>0.05)。结论:本实验成功克隆和表达SjCK2α基因,其主要表达于童虫表面;SjCK2α免疫小鼠后未能获得免疫性保护。
OBJECTIVE: To clone and express SjCK2α gene of Schistosoma japonicum and locate its expression, and observe its immunogenicity. Methods: Firstly, specific primers were designed according to the open reading frame of SjCK2α gene and the cDNA was reverse transcribed from Schistosoma japonicum as a template to amplify the target gene. Then, the target gene was digested with restriction endonuclease to obtain SjCK2α The target gene fragment was recovered and ligated into the expression plasmid pET28a (+). The recombinant plasmid pET28a (+) - SjCK2α was transformed into BL21 cells to construct the expression bacterium. The expression strain was induced by IPTG. The recombinant protein was purified by Ni-NTA resin and dialyzed to collect recombinant protein. Rabbit was immunized with recombinant protein to prepare polyclonal serum. Immunohistochemistry was used to histologically locate skin-type schistosomiasis, lung-type schistosomiasis and routine-cultured schistosomula. The recombinant protein was immunized in mice and then challenged with cercariae of Schistosoma japonicum. After 6 weeks, the mice were killed and the adult worms were collected by perfusion method to calculate the worm reduction rate. The liver tissues were excised and the eggs were harvested after digestion with KOH. The blood samples were taken from the eyes and the serum was collected. The levels of IFN-γ and IL-4 were detected by ELISA. Another infection control group and chemical control group for comparative analysis. Results: The amplified fragment of SjCK2α gene was 1047bp in length. The constructed recombinant plasmid pET28a (+) - SjCK2α was transformed into BL21 cells. The constructed recombinant bacterium obtained a large number of recombinant proteins induced by IPTG. Clone the serum. Immunohistochemical staining showed that SjCK2α mainly localized on the surface of the schistosomula. After the recombinant protein was immunized, the worm reduction rate of 15% and the egg reduction rate of 17% were obtained after 6 weeks of challenge. There was no significant difference between the control group and the control group (P> 0.05). At this point, the IFN-γ and IL-4 contents in the infected control group were 42.77 ± 12.11 and 17.31 ± 9.13, respectively. The levels of IFN-γ and IL-4 in the adjuvant control group were 30.99 ± 16.55 and 18.65 ± 7.52, The levels of IFN-γ and IL-4 in the immunized group were 36.19 ± 14.56 and 9.90 ± 5.20, respectively. There was no significant difference between the two groups in the three groups (P> 0.05). Conclusion: The SjCK2α gene was successfully cloned and expressed in this experiment, which is mainly expressed on the surface of Schistosoma japonicum. SjCK2α immunized mice failed to acquire immune protection.