pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB双价疫苗的构建及其免疫效果观察  

Construction of the Bivalent Vaccine pVAX1/SjRPS4·CB,pcDNA3.0/SjRPS4·CB against Female Fecundity of Schistosoma japonicum and Its Immunoprotection Effect

作　　者：罗四维[1] 秦永华[1] 汪希雅[1] 高冬梅[1] 何卓[1] 周帅锋[1] 陈秀春[1] 余路新[1] 汪世平[1] 

机构地区：[1]中南大学湘雅医学院寄生虫学系,长沙410078

出　　处：《热带医学杂志》2009年第9期980-984,F0002,共6页Journal of Tropical Medicine

基　　金：国家重点基础研究发展(973)计划(No.2007CB513108);国家科技支撑计划(No.2009BAI78B05);湖南省血吸虫病免疫与传播控制重点实验室;湖南省重点学科建设专项经费(No.07-985-2)

摘　　要：目的构建日本血吸虫天然分子疫苗相关靶基因的双价疫苗pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB,并观察其免疫保护效果。方法分别以pcDNA3.0/SjRPS4、pcDNA3.0/SjCB为模板,设计引物扩增SjRPS4、SjCB,重叠PCR法将SjRPS4、SjCB拼接,扩增,经双酶切后与真核质粒载体pVAX1、pcDNA3.0连接,转化DH5α细胞,筛选阳性克隆;提取双价重组质粒、空质粒及pVAX1/SjRPS4,经左腿股四头肌注射昆明小鼠,14d后取肌肉组织切片进行间接免疫荧光及酶免疫组织化学染色,证实双价重组质粒的表达;提取pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB、pcDNA3.0/SjRPS4、pcDNA3.0/SjCB及相应空载体,100μg/只或等量PBS经左腿股四头肌注射昆明小鼠,4周后以(20±1)条尾蚴贴腹感染,6周后检测减虫率、减卵率。结果PCR、EcoRI/XhoI双酶切及测序证实双价疫苗pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB构建成功;肌肉组织间接免疫荧光及酶免疫组织化学染色证实重组质粒能够在小鼠肌肉细胞内成功表达;与PBS组相比,pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB、pcDNA3.0/SjRPS4、pcDNA3.0/SjCB免疫小鼠各组间差异均有统计学意义(P<0.05),各组与相应的空质粒组相比各指标差异亦具有统计学意义(P<0.05);两种不同载体构建的双价疫苗之间比较差异则无统计学意义(P>0.05)。结论成功构建真核双价重组质粒pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB,二者免疫昆明小鼠均可以产生比单价疫苗更好的免疫保护效果。Objective To construct the bivalent vaccine pVAX1/SjRPS4. CB, pcDNA3.0/SjRPS4. CB and to study their immunoprotection effect. Methods SjRPS4 and SjCB were amplified with the templates of pcDNA3.0/ SjRPS4 and pcDNA3.0/SjCB and spliced to SjRPS4·CB by SOE-PCR （splicing by overlap extension）, then the SjRPS4·CB was cloned into the vector pVAX1 and pcDNA3.0 and transformed the competent E.coli DH5α cells to screen the positive clone. Recombinant plasmid DNA of pVAX1/SjRPS4, pVAX1 and pcDNA3.0 were injected to quadriceps femoris of Knnming mise to confirm their successful expression by indirect fluorescent assay （IFA） and immunohistochemistry assay. 7 groups of Kuming mice were immunized with either （A） pVAX1 （control）, （B） pVAX1/SjRP34-CB （bivalent）, （C） pcDNA3.0 （control）, （D） pcDNA3.0/SjRPS34-CB （bivalent）, （E） pcDNA3.0/ SjRP34, （F） pcDNA3.0/SjCB, or （G） PBS （control）. The immunized mice were challenged with cercariae of Schistosoma japonicum. Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden, intrauterine eggs and liver eggs. Results Restriction enzyme digestion, PCR and sequencing results confirmed that plasmids pVAX1/SjRPS4 .Cb and pcDNA3.0/SjRPSd·CB were successfully constructed. Results of IFA and immunohistochemistry demonstrated the expression of the recombinant plasmids pVAX1/SjRPS4 .CB, pcDNA3.0/ SjRPS4-CB were successful. The worm burden, uterus egg number and liver egg per gram （LEPG） in the pVAX1/ SjRP34-CB （bivalent）, pcDNA3.0/SjRPS34-CB （bivalent）, pcDNA3.0/SjRP34, and pcDNA3.0/SjCB groups were significantly reduced when compared with vector alone or PBS. LEPG of the pcDNA3.0/SjRPS34-CB （bivalent） group was significantly lower than that in pcDNA3.0/SjCB （monovalent） group. Conclusion The bivalent vaccine pVAX1/ SjRPS4.CB and pcDNA3.0/SjRPSd-CB are successfully constructed. Both vaccines can induce similar immunoprotection which is better than the monovalent vaccine on reducing

关 键 词：日本血吸虫病 疫苗 免疫保护 

分 类 号：R383.24[医药卫生—医学寄生虫学]