【作者】 门静涛；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2009， 博士

【摘要】 为了研制一种新型的血吸虫疫苗,本研究通过同源重组的方法成功的构建了表达日本血吸虫复合表位基因的重组伪狂犬病病毒和复合表位核酸疫苗,并对获得的重组病毒和表位核酸苗进行了小鼠和绵羊的免疫试验。本研究选取包括磷酸丙糖异构酶(TPI),脂肪酸结合蛋白(FABP),副肌球蛋白(Paramyosin),钙结合中性蛋白酶(Calpain)的几个抗原基因的主要抗原表位,人工合成一段多表位基因序列,命名为TPC,将此基因序列插入日本血吸虫26kDa GST基因,构建一个融合表达基因GST-TPC,将此复合表位基因克隆入pVAX1真核表达载体,构建复合表位核酸苗pVAX/GST-TPC,酶切获取复合表位基因表达盒,与PRV疫苗株Bartha-K61的中间转移载体(本室构建)连接,再与PRV/LacZ基因组共转染Vero细胞,蓝白斑筛选获得重组病毒PRV/GST-TPC,对重组病毒的PCR,RT-PCR,IFA鉴定表明融合基因GST-TPC整合入病毒基因组,并且能正常转录。同时为探讨白细胞介素18的免疫佐剂活性,本研究克隆了小鼠的IL-18基因,亚克隆入pVAX1真核表达载体,构建了真核表达质粒pVAX/IL-18,与多表位核酸苗和重组病毒联合进行了小鼠的攻虫实验。体液和细胞免疫检测结果表明,IL-18能有效增强复合表位核酸苗的抗血吸虫免疫应答,促进免疫反应向Th1型发展,有可能作为疫苗的候选佐剂之一。小鼠和绵羊的攻虫保护实验表明复合表位核酸苗和重组病毒均能对血吸虫的尾蚴攻击产生免疫保护作用。在小鼠中复合表位基因核酸苗和重组病毒产生32.1%和35%的成虫减虫率,肝脏减卵率分别为33.3%和36.2%。绵羊的减虫率分别为41.8%和48.6%,肝脏减卵率为26.3%和49.0%,为血吸虫疫苗的下一步研究打下了良好基础。更多还原

【Abstract】 The schistosomiasis japonica is a kind of parasitosis which threaten human’s health seriously,about 625 million people under it’s threat around the world,and as one of epidemic region,100 million people under it’s threaten in China.After more than 50 years work,the schistosomiasis japonica was under control at a tremendous degree,however,in recent years,the epidemic situation was bounced back obviously,and some new epidemic regions were found.A investigation of 2002 has demonstrated that about 820 thousand schistosomiasis patients in China,and the number was increasing year by year at a certain ratio.The schistosome does not proliferation in its host,so if we can develop a vaccine which can effectively reduce down in both schistosome burden and the egg quantity exportion,relieve pathology damage in the host,and decrease or control the transmission of the schistosomiasis.The early stage of developing vaccines against schistosomiasis were mainly in the exploration of attenuated cercarie or schistosomulum by the ray,in the lysate of different stages of life cycle and the purification of polypide antigen.The highest protection effect was in the cercarie or schistosomulum by the X-ray orγ-ray,as high as 60-80%,which demonstrated that the development of vaccines against schistosomiasis was feasible and prospective.However,due to the source,preparation, transportion and security of the ray,there were still many limitions for the application in large scale.So it has been important to develop vaccines using genetic engineering technology.So far,the vaccine candidates received by the WHO were GST, paramyosin(Sj97),triose phosphate isomerase(TPI),the membrane protein of 23kDa(Sj23),fatty acid-binding protein(SjFABP) and Gynecophoral canal protein(SjGCP).Yet,because of the complexity of the protective immunologic mechanism,which includes cellular and humoral immunity and some recombination proteins with single ingredient could only induce humoral immunity and Th lymphocyte response,could not induce cytotoxicity T lymphocyte response,more over, different candidate moleculars have different protective immunologic mechanisms, a single molecular could hardly generate complete protection to aim directly at different stages of the schistosome.These candidate moleculars could not induce the immunoprotection higher than 50% of the schistosome reduction rate stably,so,how to improve the immunoprotection of the vaccines has become a tough problem for the parasitologist to solve.It has become a new reseach field in studying vaccine using vaccine strain without pathogenicity or weakened viral pathogen as live vector,the major antigenic genes of pathogen was inserted into the genome of the vector, and it did not influence the proliferation of the vaccine strain to construct recombinant virus live vector vaccine.As one of the herpesvirus,the Pseudorabies virus(PRV)possesses enormous genome and nonessential region,it has potential to express exogenous genes,which has been regarded as ideal live vaccine vector.Recent years,few researches have been studied in expressing antigenic genes of Schistosoma japonicum by live vectors,so in this study, vaccine strain PRV Bartha-K61 was used as vector,and we constructed the recombinant Pseudorabies(rPRV/GST-TPC)which could express multiple epitope gene of Schistosoma japonicum and poly-epitope nucleic acid vaccine,and we carried animal immunity test on mice and sheep.Schistosoma japonicum 26 KDa GST gene were cloned from pGEX4T-1 vector by PCR method, and were subcloned into multiple clone site of PVAX1 vector to construct recombinant plasmid pVAX/GST.we selected several genic dominant antigen epitopes of Schistosoma japonicum,including Paramyosin,FABP,Calpain and TPI, named it TPC and synthesized by biology company.And then it was inserted into the downstream of GST gene, to construct multiple epitope genetic expression plasmid pVAX/GST-TPC.To acquire expression cassette of multiple epitope gene by enzyme digesting and it was cloned into Pseudorabies virus vector p8KD to construct intermedial transfer vector p8KD/GST-TPC,and we cotransfected p8KD/GST-TPC with Pseudorabies virus rPRV/LacZ,rPRV/LacZ on vero cells mediated by lipidosome, after blue and white patch screening,we obtained a multiple epitope genic recombinant Pseudorabies virus rPRV/GST-TPC.After extraction of the virus genome,we got 1000bp fragments use special primer of GST by PCR and RT-PCR technology,indirect immunofluorescence showed it could be recognized by polyclonal antibody of Schistosoma japonicum.So,we proved the multiple epitope gene can normal transcription in recombinant Pseudorabies virus PRV/GST-TPC.The stability test showed that after the recombinant virus passed down by 20 generations in vitro continuously,the expression of fusion gene could still be detected,which demonstrated that the rPRV/GST-TPC has good hereditary stability.To find an effective way to enhance the effect of this DNA vaccine,we cloned IL-18 gene of mouse,and subcloned it into pVAX1 vector to construct a eukaryotic expression plasmid pVAX/IL-18,we used this plasmid to immune mice with multiple epitope nucleic acid vaccine and recombinant virus,to justify if it could enhance the immune response of mice against Schistosoma japonicum.We carried animal tests on mice and sheep using recombinant virus PRV/GST-TPC and multiple epitope genic DNA vaccine pVAX/GST-TPC along with IL-18 respectively.The results showed the combinant virus PRV/GST-TPC and multiple epitope genic DNA vaccine pVAX/GST-TPC could both induce strong immune response against Schistosoma japonicum in mice and sheep.The groups of combined immunization showed higher immunoprotection rate than single one,but difference not significant amaong them.The detection results of humoral immunity showed the combined immunization groups could produce stronger IgG of soluble adult worm antigen than single ones,the IFN-γlevel were obvious higher in the combined groups than that of single ones in cytokine detection.The increasement of lymphocyte in vitro tests showed the transformation efficiency of combined groups was quite higher than single ones.So,we draw a conclusion that the recombinant virus PRV/GST-TPC and multiple epitope genic DNA vaccine pVAX/GST-TPC can induce mice and sheep against Schistosoma japonicum.and the IL-18 has the possibility to be an adjuvant to enhance the immune effect of vaccine.更多还原