摘要
目的克隆表达日本血吸虫体表蛋白Sj29基因,分析其表达特性和免疫保护效果。方法根据日本血吸虫体表蛋白Sj29基因序列(GenBank登录号为AY814537)设计引物,PCR扩增目的基因,生物信息技术分析序列特性。将目的基因部分片段亚克隆到原核表达载体pET28c中,构建重组质粒pET28c-Sj29,将其转至大肠埃希菌后用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,组氨酸(His)柱亲和层析法纯化重组蛋白;蛋白质印迹(Western blotting)分析其免疫原性。30只雌性BALB/c小鼠均分3组,实验组每鼠皮下多点注射重组蛋白rSj29共100μl(0.1mg/ml,用206佐剂配制)、佐剂对照组和空白(PBS)对照组,分别免疫3次,每次间隔2周。末次免疫后2周用血吸虫尾蚴攻击感染小鼠,每鼠40±2条,感染后第53天剖杀,收集虫体计算减虫率;取肝脏和粪便计算减卵率。ELISA法检测实验鼠血清特异性IgG抗体水平,免疫组织化学法检测rSj29蛋白在各期虫体的定位,荧光定量PCR法分析各期虫体及攻击感染后42d虫体Sj29基因表达水平。结果获得576bp的Sj29基因。重组蛋白的相对分子质量(Mr)为22900,以包涵体形式存在。Western blotting表明,重组蛋白可被免疫小鼠血清和感染日本血吸虫的小鼠血清识别。攻击感染后42d,小鼠体内Sj29基因转录水平分别为佐剂对照组和空白对照组虫体的9.1倍和51.8倍。实验组小鼠成虫数(15.4±5.9)、肝组织虫卵数(40143.3±2995.9)和粪便虫卵数(3803.9±110.9)与空白对照组相比,差异均有统计学意义(P<0.05)。ELISA结果显示,免疫小鼠可产生高滴度(1∶32000)特异性IgG抗体。免疫组织化学结果表明,重组蛋白rSj29可在7、14d童虫,28、32d成虫和42d雄、雌虫的体表表达。荧光定量PCR结果表明,虫龄为32d的日本血吸虫成虫Sj29基因转录水平最高。结论重组蛋白rSj29有一定的免疫保护效果。
Objective To clone,express and characterize a tegument protein gene of Schistosoma japonicum（Sj29）,and investigate the immune protection of the recombinant protein against S.japonicum in mice.Methods The gene coding for Sj29 protein was amplified by PCR,and the sequence was analyzed by bioinformatics tools.Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c（＋）.The recombinant plasmid was transformed into E.coli BL21（DE3） and induced the recombinant with IPTG.The recombinant plasmid was transformed into E. coli BL21 （DE3） and induced the recombinant with IPTG. The recombinant protein （r S j29） was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times （two weeks interval） respectively with 100 Ixl recombinant rSj29 （0.1 mg/ml） , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40±2 cercariae of S. japonicum. At the 53th day after infection, the mice were sacrificed to obtain the number of adult worms,number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique, mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. Results A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22 900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms （15.4±5.9） , number of hepatic eggs （40 143.3±2 995.9） and number of fecal eggs （3 803.9±110.9） in recombinant protein group were significantly higher than those of PBS control group （20±3.4, 49 318.1±6 648.3, 5 238.1±303.5, respectively） （P〈0.05） . There was a high level of specific IgG against rSj29 （maximum dilution 1：32 000） in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum, mRNA level of Sj29 was the highest at the 32nd day post-infection. Conclusion The recombinant protein rSj29 induces certain degree of protective immunity in mice.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2009年第6期476-482,共7页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家科研业务费项目(2007-08)
国家科技支撑计划项目(No.2006BAD06A09)~~
关键词
日本血吸虫
体表蛋白
荧光定量PCR
免疫组化
Schistosoma japonicum
Tegument profein
Real-time quantitative PCR
Immunohistochemistry
