Observation On Immune Protective Effect Of PVAX1/SjRPS4·CB Enhanced ByIL-18Against Schistosomiasis Japonicum

BackgroundSchistosomiasis is a disease that could cause severe chronic lesion, and is prevalent in about1/3of the countries and regions in the world. It does great harm to the health of the people and livestock in the affected areas. After years of prevention and treatment, the infection rate and other epidemic indicators have reached the historical lowest level in our country. But the decline of Schistosomiasis is imbalance, and several province have eradicated the disease, such as Shanghai, Fujian, Guangdong, Zhejiang and Guangxi provinces(municipalities and autonomous regions), Yunnan, Sichuan and Jiangsu provinces has reached the spread and control standards. But in Hunan, Hubei, Jiangxi and Anhui four provinces, where were abundant with lakes remains endemic areas of schistosomiasis japonica. For current endemic trends infection source control serve as the main comprehensive prevention and control strategy put forward by our country to conquer this situation.For over a decade, a large number of experimental studies by our group confirmed:Schistosoma japonicum immature eggs soluble antigen of26-28kDa (SIEA26-28kDa) and adult worm antigen31-32kDa (SWA31-32kDa) molecular immunized animals induced strong ability in suppressing spawning through restraining eggs embryonic development and anti-fecundity. From which we seleceted two natural antigen SjRPS4and SjCB to build a pVAXl/SjRPS4·CB bivalent DNA vaccine, in animal experiments show that the bivalent vaccine can induce a better immune protection and disease resistance effect. However, we deem that the vaccine immune protection and disease resistance effect could be improved. In this subject, in order to increase the bivalent vaccine expression level, we intend to rebuild the pVAXl/SjRPS4·CB bivalent DNA vaccine. we also want to investigate the immune protection and disease resistance effect after cytokines immunoadjuvant was injected into animals with bivalent DNA vaccine.ObjectiveAfter building and efficient expression eukaryotic vector pVAX1/SjRPS4·CB and pVAXl/IL-18, injecting them into mice to investigate whether IL-18could enhance the immune protection and disease resistance effect of bivalent DNA vaccine SjRPS4·CB.Methods1Building and identification of efficient eukaryotic expression vector pVAX1/SjRPS4·CB and pVAXl/IL-18.Taking PET32a/SjRPS4·CB and pET28a/IL-18plasmid as templates, amplifying SjRPS4·CB and IL-18gene through polymerase chain reaction. After restriction enzyme digestion, the PCR products were linked to the same restriction enzyme digested pVAXl vector through their identical joint. Then the recombinant vector was transformed into E. coli DH5a, postive clones were selected to proceed bacterium fluid polymerase chain reaction, plasmid preparation, restriction enzyme digestion and sequencing. The right recombinant vector was transfected into293T cells, RT-PCR was used to detect the mRNA expression level of SjRPS4·CB and IL-18.2Investigations of the immune protection and disease resistance effect of SJRPS4·CB and IL-18in mice.A large number of DNA vaccines, pVAX1/SjRPS4·CB and pVAXl/IL-18were extracted from. Then they were injected into per mice100μg. Three weeks later, Schistosoma japonicums were released to infect the belly of each mice. ELISA was used to detect the expression level of IFN-y, IL-4and the induced specific antibody. Lymphocyte proliferation experiment was used to detect the spleen lymphocyte proliferation level of the mice. HE staining of mice liver tissue were used to detect the liver worm eggs granuloma area and masson staining was used to detect the collagen deposition around the eggs. Worm reduced rate was calculated according to the adult worm in mice. Intestine eggs reduced rate, liver eggs reduced rate and per female worm eggs reduced rate were calculated according to the number of Intestine eggs, liver eggs, and per female worm eggs.Results1Bacterium fluid polymerase chain reaction, restriction enzyme digestion and sequencing were all proved that the SjRPS4·CB and IL-18genes were identical to the canonical sequences. At the beginning of the insert fragment, the short sequence ANNATGG were found in both vectors. RT-PCR revealed that SjRPS4·CB and IL-18genes were dramaticlly expressed in293T cells, and the expression level were higher than the unmodified vectors. These proved that the recombinant vector pVAX1/SjRPS4·CB and pVAX1/IL-18were construct correctly.2After co-inject pVAX1/SjRPS4·CB and pVAX1/IL-18into mice muscle, we found that they can induce upregulation IFN-y express, and induce Thl type cells proliferation and enhance Thl type immunity, and they can increase worm burden reduction rate, liver egg reduction rate, intestine egg reduction and uterus egg reduction of per female worm, they can Reduce the area of liver egg granuloma and Collagen content. We also found that co-inject group’s IgG level is higher than other groups. According to those result, we can said that IL-18could intensify the immune protection and disease resistance effect of SjRPS4·CB vaccine through Thl type immunity., And it does not lower the Th2type immunity. Conclusion1. Recombinant vector pVAX1/SjRPS4·CB and pVAX1/IL-18were construct correctly.2. IL-18could intensify the immune protection and disease resistance effect of SjRPS4·CB vaccine through Thl type immunity., And it does not lower the Th2type immunity.