IL-18增强pcDNA3.1/SjOST48抗血吸虫感染的免疫保护性研究  

IL-18 enhances the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection

作　　者：谭潇 肖楚丽 肖非 王硕 卿蕊 黄泽智 TAN Xiao;XIAO Chu-li;XIAO Fei;WANG Shuo;QING Rui;HUANG Ze-zhi(School of Medical iMboratory,Shaoyang University,Shaoyang 422000,China)

机构地区：[1]湖南省邵阳学院医学检验学院,邵阳422000

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第3期279-285,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：湖南省教育厅科学研究项目(No.15C1257);湖南省教育厅优秀青年项目(No.17B240)。

摘　　要：目的探讨白细胞介素18(IL-18)是否能增强DNA疫苗候选抗原pcDNA3.1/SjOST48对日本血吸虫病感染的免疫保护效果。方法构建pcDNA3.1/SjOST48和pcDNA3.1/IL-18真核载体,表达重组蛋白。Western blotting检测HeLa细胞内重组质粒pcDNA3.1/SjOST48和pcDNA3.1/IL-18表达情况,ELISA检测细胞培养基中IL-18的表达情况。100只雌性BALB/c小鼠随机均分为PBS组(空白对照组)、pcDNA3.1组(空质粒组)、pcDNA3.1/IL-18组、pcDNA3.1/SjOST48组和pcDNA3.1/SjOST48+pcDNA3.1/IL-18组等5组,各组小鼠左后腿股四头肌肌内注射相应的重组质粒(30μg),共免疫3次,每次间隔14 d。末次免疫后2周,各组取5只小鼠采集血样,ELISA检测各组小鼠血清IgG抗体及其亚类(IgG1和IgG2a)水平。末次免疫后3周,各组取5只小鼠脾组织,无菌分离小鼠脾淋巴细胞,ELISA检测小鼠脾淋巴细胞上清中TNF-α、INF-γ、IL-2、IL-4、IL-6和IL-17含量及脾淋巴细胞增殖水平。末次免疫后2周,各组取15只免疫小鼠感染血吸虫尾蚴(20±1)条/鼠。感染后6周处死小鼠,无菌分离肝,计算减卵率;门静脉灌注法收集成虫,计算减虫率;另取肝组织,切片后行HE染色,显微镜下观察肝虫卵肉芽肿数量及炎症浸润情况。结果末次免疫后2周,ELISA结果显示,pcDNA3.1/SjOST48+p cDNA3.1/IL-18组、pcDNA3.1/SjOST48组、pcDNA3.1/IL-18组小鼠的IgG抗体水平分别为0.82±0.07、0.41±0.06、0.16±0.05,均高于pcDNA3.1组的0.12±0.03(P<0.05);pcDNA3.1/SjOST48+pcDNA3.1/IL-18组较pcDNA3.1/SjOST48组和pcDNA3.1/IL-18组均增高(P<0.05);pcDNA3.1/SjOST48+pcDNA3.1/IL-18组和pcDNA3.1/SjOST48组的IgG2a/IgG1比值分别为4.02±0.01、2.51±0.01(P<0.05),均>1,其他3组均<1。末次免疫后3周,pcDNA3.1/SjOST48+pcDNA3.1/IL-18组小鼠脾淋巴细胞上清中的IL-2、TNF-α、INF-γ、IL-6和IL-17含量分别为(101.82±8.90)、(738.02±146.22)、(593.41±51.07)、(685.64±171.2)、(261.32±48.19)pg/ml,高于pcDNA3.1/SjOST48组[(55.82±9.69)、(538.21±85.26)、(393.41±51.07)、Objective To investigate if IL-18 can enhance the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection.Methods pcDNA3.1/SjOST48 and pcDNA3.1/IL-18 eukaryotic vectors were constructed to express recombinant proteins.Western blotting was performed to detect the expression of pcDNA3.1/SjOST48 and pcDNA3.1/IL-18 in HeLa cells,and ELISA was performed to examine IL-18 levels in culture medium.One hundred BALB/c female mice were randomly divided into 5 groups:PBS group(blank control),pcDNA3.1 group(empty vector),pcDNA3.1/IL-18 group,pcDNA3.1/SjOST48 group,and pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group,and were immunized with corresponding recombinant plasmid(30μg)for three times at intervals of 14 days by intramuscular injection(100μg/ml)at the quadriceps of the left hind leg femur.Two weeks after the last immunization,five mice in each group were selected for blood collection,and serum levels of IgG and its subclasses(IgG1 and IgG2 a)were detected by ELISA.Three weeks after the last immunization,5 mice of each group were selected for collection of spleen,to isolate spleen lymphocytes under sterile conditions;and the contents of TNF-α,INF-γ,IL-4,IL-6 and IL-8 in the lymphocytes culture supernatant and the proliferation of spleen lymphocytes were assessed by ELISA.Two weeks after the last immunization,15 mice of each group were infected with 20±1 cercariae of Schistosoma japonicum and sacrificed 6 weeks later.The liver tissue was obtained aseptically to calculate egg reduction rate;adult worms were collected by portal vein perfusion to calculate worm reduction rate.Another portion of liver tissue was used for hematoxylin&eosin(HE)staining to ob-serve the amount of S.japonicum egg granuloma and inflammatory infiltration.Results ELISA results showed that at two weeks after the last immunization,the IgG antibody levels in mice of the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group,the pcDNA3.1/SjOST48 group,and the pcDNA3.1/IL-18 group were 0.82±0.07,0.41±0.06,and 0.16±0.05,respectively,all significa

关 键 词：日本血吸虫 白细胞介素18 pcDNA3.1/SjOST48 候选抗原 免疫保护性 

分 类 号：R383.24[医药卫生—医学寄生虫学]