Construction Of CDNA Library For Adult Dirofilaria Immiti And Research On Immune-related Proteins

Dirofilaria immitis, belongs to the Spirurida Chitwood,Filariata Skrjabin,Onchocercidae,Dirofilariinae,and Diroflaria which caused Dirofilariasis. D. immitis resided in right ventricle and pulmonary arltery leading to circulatory disorder, dyspnoer, anemia, even suddenly death. It not only threatens dog breeding severely, but also is harmful to human health. Dogs, cats are natural hosts of this parasite, the animals such as wild carnivores, horses, beavers, apes, muskrats, and other primates are infected incidentally. D. immitis is commonly transmitted by mosquitoes that ingest blood containing the relatively long unsheathed microfilariae with tapered tails. More than 60 species of mosquitoes around the world have been shown to be susceptible to infection. So far there are no ideal diagnostic although many detection methods were reported, such as: microfilaria examination, blood clotting tests, agar diffusion test, complement fixation test, fluorescent antibody labeling, PCR method. To date there is no effective vaccines to control Dirofilariasis. In this study, cDNA library of adult D.immitis has been constructed and screened. Immune-related genes of D.immitis were identified and two novel genes were used as candidate genes for vaccines and diagnosis of D. immitis. Recombinant subunit vaccines and DNA vaccines encoding GSP1 and MTFP genes were developed and their protective immunity was identified, respectively. Also ELISA assay was developed using two recombinant proteins GSP1 and MTFP. They are very useful for the prevention and treatment of dirofilariasis.Construction of cDNA library for adult D.immitis In this study, cDNA library of adult D.immitis has been constructed using Clontech's SMART technology. After extracting total RNA of adult D.immitis, cDNA was synthesised by reverse transcription, then second strand was synthesised. At last, cDNA fragments were connected and packaged after digesting by proteinase K and separating by Sfi restriction. The results showed that cDNA library of adult D.immitis was constructed successfully. The titer of the unamplified library was 9×10⁵ pfu/mL. The results showed the library recombination rate was 98% by PCR identification on 100 randomly selected plaques. The library capacity was 1.0×10⁹ recons. The construction of the library laid the foundation for screening of immune-related genes.Screening of immune-related genes of D.immitis Immune-related genes of D.immitis had been screened in this study using SEREX technology. Firstly, theλphages of the library were cultured, and protein expression was induced with IPTG. Immune-related protein genes were screened from the library using immunized mouse with adult D.immitis antigens and naturally infected serum from dog. Eleven immune-related protein genes of D.immitis were identified: D.immitis paramyosin partial sequence, D. immitis partial sequence for a highly immunoreactive antigen, D. immitis cuticular antigen tandem repeats partial sequence, Brugia malayi Csnk2b protein partial sequence, D. immitis partial sequence for neurotrophil chemotactic factor precursor,, D. immitis 18S ribosomal RNA gene, partial sequence, D. immitis cuticular antigen tandem repeats partial sequence, B. malayi Fip1 motif family protein partial sequence, B. malayi Helicase conserved C-terminal domain containing protein partial sequence, B. malayi Myosin tail family protein partial sequence, B. malayi ATPase partial sequence, AAA family protein partial sequence. Two novel genes were selected as candidates for vaccines and diagnosis of D. immitis. GSP1 was 85% homology with B. malayi G protein pathway suppressor 1 gene; MTFP was 88% homology with B. malayi Myosin tail family protein gene.Recombinant subunit vaccines encoding GSP1 and MTFP genes After the amplification of GSP1 and MTFP genes, recombinant prokaryotic expression plasmid pGEX-4T-1-GSP1 and pGEX-4T-1-MTFP were constructed. The recombinant prokaryotic expression plasmids were transformed into competent E. coli DE3. The expression was induced by IPTG. SDS-PAGE and Western Blotting showed that the sizes of the recombinant proteins were about 34Ku and 36Ku and the proteins could be recognized by polyclonal antibody to adult D. immitis anitigens. Then the BALB/c mice were immunized with recombinant proteins. ELISA test showed that IgG titer of experimental group was gradually increased. After the third immunization, IgG titers showed significant increase in experimental groups immunization with recombination proteins compared with the control group (P<0.01). Detection of cellular immunity showed that the percentages of CD4⁺ T lymphocytes were significantly increase in experimental groups compared with the control group (P<0.01) and CD8⁺ T lymphocytes were not significantly increase (P>0.05). Also CD4⁺/ CD⁸⁺ T lymphocytes were significantly increase in experimental groups compared with the control group (P<0.01). GPS1 and MTFP genes are expected to be good candidate genes for subunit vaccines of D.immitis. This study laid a good foundation for the prevention of filariasis. DNA vaccines encoding GSP1 and MTFP genes GPS1 and MTFP genes were subcloned into the eukaryotic expression plasmid pVAX1 to form two recombinant eukaryotic expression plasmids pVAX1-GPS1 and pVAX1-MTFP, respectively. The recombinant plasmids were successfully transfected into HeLa cells and recombinant proteins expression was identified by Western Blotting. The recombinant proteins were recognized by polyclonal antibody to adult D. immitis anitigens and molecular weights were in accordance with expectation. Then BALB/c mice were immunized with two recombinant plasmids, respectively. Compared with the control groups, the level of humoral immunity in pVAX1- GPS1 and pVAX1- MTFP was significantly increasing (P<0.05). The percentage of CD4⁺ T cells or CD8⁺ T cells in pVAX1- GPS1 and pVAX1- MTFP was significantly higher than that of control group (P<0.01). The ratio of CD4⁺ and CD8⁺ T cells was significantly higher than that of control group (P<0.01). This study will be a theoretical basis of D.immitis prevention.Development of indirect ELISA assay for filariasis diagnosis Indirect ELISA assay for filariasis diagnosis was developed using the purified recombinant proteins GST-GSP1 and GST-MTFP. Square test was used to determine the optimum working concentration.The results showed that the opitimal working concentration of GST-GSP1 antigen was 5.5μg/100μL and that of GST-MTFP was 5μg/100μL. Serum dilution was 1:150. HRP-labeled goat anti-dog IgG was 1:2000. Furthmore, this ELISA assay was verified by blocking experiment, crossing response experiment, sensibitity experiment, replication experiment, coincidence rate and comparison of spot detection. The results showed that this antigen had high sensitivity (GSP1-1:1600, MTFP-1:3200), specificity and stability. Forty five dogs were detected by different assays, respectively. The positive rate detected by ELISA was the same as PCR examination (22.2%). It was higher than that of by Canine Heart Worm Ag Test Kit (20.0%). Also the coincidence rate with the PCR examination was 100%. Therfore, GSP1 and MTFP could be candidate genes of heartworm detection. The ELISA assay established in this study has laid a good foundation for filariasis diagnosis.