日本血吸虫P14基因重组蛋白诱导小鼠保护性免疫及其抗血吸虫感染机制的初步研究
[Preliminary studies on protective immunity induced by recombinant protein of Schistosoma japonicum P14 gene and its mechanism against schistosome infection in mice].
作者信息
Tang Xiao-niu, Ji Feng, Jiang Yu-xin, Zhan Xiao-dong, Li Chao-pin
机构信息
Department of Medical Parasitology, Wannan Medical College, Wuhu 241002, China.
出版信息
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2013 Aug;25(4):375-8, 410.
OBJECTIVE
To explore the immune protective effect of the basic calponin-like protein (SjP14) in mice response to the infection of Schistosoma japonicum.
METHODS
The recombinant plasmid pET28a(+)-SjP14 was built, and transferred into E. coli BL21 cell line. After induced, by isopropyl-beta-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 (adjuvant control), and C group (saline control). The mice in the A group were subcutaneously injected with 100 microg 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-gamma 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 SjP14 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) microg/ml] compared with that in the B [ (18.65 +/- 3.16) microg/ml] and C [(22.44 +/- 2.83) mcirog/ml] groups (P < 0.05); the level of serum IFN-gamma [(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.
目的
探讨碱性钙调蛋白样蛋白（SjP14）在小鼠对日本血吸虫感染反应中的免疫保护作用。
方法
构建重组质粒pET28a(+)-SjP14，并将其转入大肠杆菌BL21细胞系。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，纯化目的蛋白，然后分别通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法进行分析。30只六周龄雌性BALB/c小鼠随机分为三组（每组n = 10），包括A组（rSjP14实验组）、B组（佐剂对照组）和C组（生理盐水对照组）。A组小鼠每隔2周皮下注射100μg rSjP14疫苗，共三次。末次免疫后，小鼠感染日本血吸虫尾蚴。B组和C组除感染尾蚴外，分别皮下注射佐剂或生理盐水。分别在免疫前、免疫后6周和感染后6周通过酶联免疫吸附测定（ELISA）检测血清IgG、白细胞介素-4（IL-4）和γ-干扰素（IFN-γ）水平。感染后第6周还计算虫体或虫卵的减少率。
结果
SjP14蛋白约为38 kDa，能被SjP14多克隆抗体成功识别。感染尾蚴后第6周，A组血清IgG水平显著升高[（25.52±1.91）μg/ml]，高于B组[（18.65±3.16）μg/ml]和C组[（22.44±2.83）μg/ml]（P < 0.05）；血清IFN-γ水平[（171.30±70.12）ng/L]也升高，高于B组[（136.89±37.62）ng/L]和C组[（153.64±43.44）ng/L]（P < 0.05）。然而，感染后第6周A组血清IL-4水平[（112.05±15.02）ng/L]与C组[（102.82±27.46）ng/L]相比显著降低（P < 0.05）。感染后A组虫体和虫卵的减少率分别为29.2%和41.3%，与C组相比有统计学差异（P < 0.05）。
结论
SjP14对小鼠日本血吸虫感染具有保护性免疫作用。
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