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Preliminary Study on Combination Immunization with Recombinant Proteins and DNA Nucleicacids of GST and CatL Genes of Fasciola Hepatica

Author: LiXiaoJuan
Tutor: PuFanZe
School: Heilongjiang Bayi Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Fasciola hepatica Glutathione S- transferase Cathepsin L Recombinant proteins DNA nucleic acid Combined immunodeficiency
CLC: S855.91
Type: Master's thesis
Year: 2009
Downloads: 19
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Abstract

Liver fascioliasis is parasitic in ruminants such as cattle, sheep liver bile ducts within the liver fasciolosis (Fasciola hepatica) caused a very serious harm to animals trematodes. Currently, the disease is mainly relying on medication, long-term medication, however, is not only the emergence of drug-resistant and will cause environmental pollution, vaccines have been developed, therefore, become the hotspot of disease prevention. Domestic and international study found, glutathione S-transferase (GST) and cathepsin L (CatL) against Fasciola hepatica infection are immune protective effect, and good prospects for application in of hepatic fascioliasis molecular vaccine developed showing . Therefore, this study intends Preparation of GST and CatL gene recombinant protein and DNA nucleic acid, immune rats analyze its immunogenicity. In this study, Fasciola hepatica total RNA as a template, the GST and CatL gene was amplified by RT-PCR. Were cloned into the pMD18-T vector and transformed into TG1 host strain, obtained positive recombinant plasmid pMD18-T-GST and pMD18-T-CatL known sequencing results with GenBank nucleotide sequence homology was 98.8% and 97.6%. Further subcloned into the prokaryotic expression vector pET-30a (), was transformed into E. coli BL21 (DE3) pLysS positive recombinant plasmid pET30a-GST and pET30a-CatL recombinant strains induced by IPTG, the fusion protein was highly expressed. Western blotting analysis of recombinant proteins can be immune serum of SD rats and goat serum to identify anti-Fasciola hepatica, confirmed that the the GST and CatL recombinant protein has good immunogenicity. Green fluorescence appeared reuse amplified by PCR for GST and CatL gene, respectively connected to the eukaryotic expression vector pEGFP-N1 construct recombinant plasmid pEGFP-GST and pEGFP-CatL Hela cells transfected with the recombinant plasmid and observed under a fluorescence microscope , Western blotting results further confirmed that the recombinant plasmid expression in vitro. 80 SD rats were randomly divided into eight groups, respectively GST and CatL recombinant protein immunohistochemistry GST, and CatL DNA nucleic acid immunohistochemistry GST, and CatL DNA nucleic acid-based immune then the corresponding recombinant protein boost immunization \Joint immunohistochemistry, pEGFP-N1 empty vector control group and PBS control group. Each rat immune three times, at intervals of three weeks. Before immunization (week 0) and 3,6,9,12,15 weeks after the first immunization from rat blood collection and application of indirect ELISA serum IgG antibody levels, results show that, compared with the control group, The immunohistochemical starting from the first three weeks of IgG antibody titers were improved to varying degrees, including the GST, and CatL recombinant protein immunohistochemical antibody level is always higher than that of other immune group, and reached to the highest (1:102 400 antibody levels in the first nine weeks ), 12 weeks began to decline; the GST and CatL DNA nucleic immunohistochemical antibody levels have been slowly rising, \Confirmed that the humoral immune response induced by the recombinant protein was significantly higher than the DNA nucleic acid, DNA nucleic acid in the rat in vivo role of time longer. MTT assay 11 weeks rat T lymphocyte transformation effects, results showed that the the various immunohistochemical average stimulation index was significantly higher than the empty vector control group and PBS control group (p lt; 0.05), including GST recombinant protein immunohistochemistry, CatL recombinant protein immunization groups and GST joint immunohistochemistry and CatL joint immunization group lymphocyte stimulating index difference is not significant with (p-gt; 0.05), but with the GST of DNA nucleic acid immunization group and CatL of DNA nucleic acid immunization group relative comparison differences are significant with (p lt ; 0.05). Description and DNA nucleic acid alone immune comparison, the recombinant protein and DNA nucleic acid / recombinant protein combined immunodeficiency is more effective to promote the growth and proliferation of rat spleen lymphocytes. Both vaccines combined immune not only enhanced cellular immune responses, but also triggered a humoral immune memory, has opened up new avenues for the in-depth study of liver fascioliasis genetically engineered vaccine.

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