Detection Method Establishment And Eukaryotic Expression Of Swine Surfactant Proteins D, And Research On Its Gene Adjuvant Effect To T. Gondii DNA Vaccine

Surfactant Protein D（SP-D）, a member of the collectin family, is a pattern recognition protein,secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens.SP-D can be used as biological markers of a variety of diseases.In order to improve the efficiency of the detection of the expression levels of SP-D,this study established a RT-FQ-PCR method and gold nanoparticle PCR. Acording to swine SP-D mRNA gene sequence（NM₂14110）,we design and synthesis of primers and matching the TaqMan probe, and use swine SP-D gene CDS region sequence connection PMD-18 T vector to construct a new plasmid PMD-SP-D as a positive standard. We optimized RT-FQ-PCR reaction conditions, dilutions of standards and then established a standard curve, amplification equation: Y=-3.131 X +35.09 and amplification efficiency of 108%, R2 reached 0.999, Ct values batch coefficient of variation, and batch-to-batch variation The coefficients are less than 3%. Standard Ct value has a good linear relationship with the initial concentration of the template, the lowest concentration that can be detected up to3copies/μl. In this study we also establish the nanoparticle PCR method following primers that we used above, fumble optimal reaction conditions for the method and its sensitivity were measured. The results showed that the nanoparticle PCR method could also amplified specific bands consistent with the experimental design, its sensitivity was similar to RT-FQ-PCR method.This study for the detection of trace SP-D gene provides a set of efficient detection of double insurance,and made an exploration for the promotion and application of gold nanoparticle PCR.In this study,the SP-D gene was amplified by PCR using the PMD-SPD recombinant vector that contained the swine SP-D sequence as a template, and the specific primers with 2 restriction sites for Sal I and Hind III restriction enzymes. The PCR product was cloned in Sal I/Hind III restriction sites of the linearized pFast Bac-HTB vector and evaluated by using those restriction enzymes and sequencing. Then the recombinant pFastBac-HTB vector was transformed in DH10 Bac and the result was screened and confirmed by X-Gal discrimination and PCR.The results showed that the swine SP-D gene was successfully amplified and the PCR product was confirmed by using the related restriction enzymes and sequencing. Cloning of pFast Bac vector with the purified PCR product of SP-D gene was confirmed. Finally, the recombinant Bacmid wassuccessfully transformed in DH10 Bac. The recombinant Bac-SP-D expression vector is considered as an important tool to transfect the sf9 cell line and expression the SP-D protein.The recombinant SP-D was successfully purified and showed a good degree of purity. SP-D ELISA kit analysis showed SP-D protein was biologically active by the use of the baculovirus vector. Therefore, this study describes an efficient method to produce adequate amounts of biologically active swine SP-D, useful for immunomodulation studies in swine. The method described here can be used for further studies on the development of vaccines and medicaments with recombinant SP-D protein.SP-D play a critical role in the regulation of the homeostasis of naive and memory T cells.Co-administration the DNA vaccine with cytokines may improve its efficacy. In the study,mice were administrated with DNA vaccine（PcDNA3.1-AMA1） expressing T. gondii apical membrane antigen 1（AMA1）, in the presence or absence of SP-D plasmids（PcDNA3.1-SP-D）, immune responses were analyzed including lymphoproliferative assay and serum antibody measurements.Indirect immunofluorescence assay（IFA） and Immunofluorescence laser scanning confocal microscope（LSCFM） showed that SP-D was dispersed throughout the cytoplasm. Evaluation of proliferation of splenocytes using the MTT（3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide） assay indicated induction of cellular response. Measurement of antibody showed that the PcDNA3.1-SP-D+PcDNA3.1-AMA1 groups stimulated AMA1 antibody production in comparison with Pc DNA3.1-AMA1 groups（P<0.05）, the control groups（P<0.001）.Above all, the DNA vaccine and the genetic adjuvant Pc DNA3.1-SP-D revealed in this study might be new candidates for further vaccine development against T. gondii infection.