| Schistosomiasis is a serious threat to the public health. Although great achievements on prevention and treatment of Schistosomiasis have been obtained in our country, Schistosomiasis is still epidemic seriously in some areas. The effect of molluscicidal is difficult to protract and steady, but can bring about the ecosystem pollution of the environment. Moreover, some schistosome strains of praziquantel- resistant to the drug have been observed recently in several countries. As the complement to chemotherapy and other approaches, the development of Schistosomiasis vaccines is veiy necessary, as the use of sugar materials or molecules containing sugar (proteoglycans, glycoproteins and glycolipides) are the protective antigens of schistosoma, it is difficult to produce vaccines by the technique of genetic engineering. Anti-idiotypic antibody vaccines of Schistosomiasis have aroused great attention.Guan Xiaohong et al established a strain of monoclonal anti-idiotypic antibody NP30 of Schistosomia japonicum, which is an internal image anti-idiotypic antibody of gut associated antigen (GAA), its antibody isotype is IgM. NP30 has a partial cross reaction with soluble egg antigen (SEA) and membrane associated antigen (MAA). NP30 has been researched on many aspects. It has been confirmed that NP30 has sensitizing effect for hepatic egg granulomas of Schistosomia japonicum; immunization with NP30 in mice and goats also obtained good results. Anti-idiotypic antibody NP30 was confirmed with protective immunityof both anti-infection and anti- pathology and may act as a vaccine candidate for Schistosomiasis japonica. But NP30 is derived from murine hybridoma, as alloprotein, continued injection of human with NP30 eventually leads to the production of human anti mouse antibody (KAMA) or induces anaphylaxis. So it is necessary to humanized murine monoclonal anti-idiotypic antibody NP30 of Schistosoma Japonicum. Analyased by molecular biology computer programme, the 10 amino acids of VHCDR3 region are thought to be antigenic epitope of NP30, then NP30 6 VnCDR3 gene was artificially designed and constructed, on this bases, the aim of this study is to amplify NP30 6 VHCDR3 gene , then fused with hIL-2 gene and express fusion protein, and observe the protective immunity in mice induced by the fusion protein. Methods1. Based on the sequence of NP30 6 VHCDR3 gene in plasmid pFUW80 and the published nucleotide sequence of human IL-2, two pairs of primers were designed and synthesized. The plasmids of pFUW80 and pSSK-I were used as templates for polymerase chain reaction (PCR). Then the purified products of PCR were cloned into pUCm-T vector, and the recombinants were sequenced by TaKaRa.2. Having been amplified and sequenced, NP30 6 VHCDR3 gene and hIL-2 gene were inserted into corresponding sites of expression vector pET28a(+), so the fusion gene was constructed.3. The recombinant clones were tested by the methods of restrictional enzyme cut and PCR, and then were transfected into E.coli BL21. The fusion gene was induced to express by IPTG and detected with SDS-PAGE. Then the expressed product was purified.4. Both sexes of 6-8 week old (weight 18-20g) BALB/C mice were randomly divided into three groups of twelve mice each. The experimental group was immunized three times, separated by two weekintervals, by intraperitoneal injection of fusion protein (20 g/dose/mouse ). Fifteen days after final boosting, vaccinated and control mice were challenged precutaneously with S.japonicum cercariae (Chinese mainland strain) by the cover glass method. Mice were sacrificed by separating the cervical vertebra on the twenty-eighth day postchallenge. Thoracic and abdominal cavities were exposed and adult worms were recovered and counted by perfusion of the left ventricle-portal vein, then the percentage of resistance was calculated. Control group were injected with water or NP30, the other treatments were the same as the experimental group. Results1. Two ban... |
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