重组日本血吸虫P14蛋白对血吸虫感染小鼠保护性免疫效果

Preliminary studies on protective immunity induced by recombinant protein of Schistosoma japonicum P14 gene and its mechanism against schistosome infection in mice

目的探讨重组日本血吸虫P14蛋白(SjP14)对血吸虫感染小鼠的免疫保护效果。方法构建SjP14原核表达质粒pET28a(+)SjP14,异丙基βD硫代半乳糖苷诱导表达重组蛋白rSjP14,纯化蛋白后,进行十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS PAGE)和Western blotting鉴定。将6周龄雌性BALB/c小鼠随机分为SjP14重组蛋白实验组(A组)、佐剂对照组(B组)、生理盐水对照组(C组),每组10只。每鼠每次注射抗原100μg,免疫3次,每次间隔2周,末次免疫后2周,血吸虫尾蚴攻击感染。分别于免疫前、免疫后6周和感染后6周经眶静脉采血,分离血清,采用酶联免疫吸附试验(ELISA)检测血清总IgG、IL 4和IFNγ。血吸虫尾蚴攻击感染后6周,剖杀小鼠,计算各组的减虫率和肝脏减卵率。结果SjP14原核表达产物相对分子质量(Mr)约为38 000;rSjP14能被SjP14多克隆抗体识别。血吸虫尾蚴攻击感染后6周,A组特异性IgG抗体水平升高,为(25.52±1.91)μg/ml,与C组[(18.65±3.16)μg/ml]和B组[(22.44±2.83)μg/ml]相比差异均有统计学意义(P均<0.05)。IFNγ浓度升高,为(171.30±70.12)ng/L,与C组[(136.89±37.62)ng/L]和B组[(153.64±43.44)ng/L]相比差异均有统计学意义(P均<0.05)。A组免疫后6周IL 4先略有升高,感染后6周下降,为(112.05±15.02)ng/L,与C组[(102.82±27.46)ng/L]相比差异有统计学意义(P<0.05)。A组小鼠减虫率和减卵率分别为29.2%和41.3%,与B、C组相比差异均有统计学意义(P均<0.05)。结论SjP14蛋白疫苗具有一定的抗血吸虫感染免疫保护作用。

Objective To explore the immune protective effect of the basic calponin-like protein （SjP14） in mice response to the infection of Schistosomajaponicum. Methods The recombinant plasmid pET28a（±）-SjP14 was built, and transferred into E. coli BL21 cell line. After induced, by isopropyl-13-D-thiogalactoside （IPTG）, the target protein was purified, and then analyzed by SDS-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting, respectively. Thirty female BALB/c mice （six- week age） were randomly divided into three groups （n = 10 for each）, including A group （rSjP14 experimental）, B group （adju- vant control）, and C group （saline control）. The mice in the A group were subcutaneously injected with 100 txg rSjP14 vaccine for three times at 2-week interval. After the final immunization, the mice were infected by cercariae of S. japonicum. The B or C groups experienced subcutaneous injections of adjuvant or saline, respectively, except for cercaria infection. The levels of sera IgG, IL-4 and IFN-~/were detected by ELISA before immunizing, 6 weeks after immunizing, and 6 weeks after the infection, respectively. The reduction rates of worms or eggs were also calculated at the 6th week after the infection. Results The SIP14 protein was about 38 kDa and was recognized successfully by polyclonal antibody of SjP14. The level of serum IgG in the A group at the 6th week after the infection of cercariae was significant increased [ （25.52±1.91） txg/mll compared with that in the B [ （ 18.65 ± 3.16）μg/ml] and C [ （22.44 ± 2.83） p,g,/ml] groups （P 〈 0.05） ; the level of serum IFN-γ [ （171.30 ± 70.12） ng/L] was also increased, compared with that in the B [ （136.89 ± 37.62） ng/L] and C [ （153.64 ±43.44） ng/L] groups （P 〈 0.05）. However, the level of serum IL-4 [ （112.05± 15.02） ng/L] in A group was significant decreased at the 6th week after the infection, compared with that in the C group [ （ 102.82 ± 27.46） ng/L] （P 〈 0.05）. The reduction rates of worms and eggs in the A group were 29.2% and 41.3%, respectively, after the infection, which had statistically significant differences compared with those of the C group （P 〈 0.05）. Conclusion SjP14 has a protective immunity against S. japonicum infection in mice.