Neospora Caninum Major Surface Proteins, NcSRS2 And NcSAG1, And The Immunogenicity Of Recombinant Proteins

Neospora caninum, which is a protozoan parasite, is an obligate intracellular pathogen that appears to be capable of utilizing a fairly wide range of animals as intermediate hosts. N.caninum is considered to be a major cause of reproductive failure in cattle worldwide. Two surface proteins of N.caninum tachyzoite, NcSRS2(Nc-p43) and NcSAGl(Nc-p36), were involved in the adhesion and invasion process. They are considered to be important diagnostic candidates for the detection of antibodies to N.caninum in animals. In this study, NcSRS2 gene was cloned and expressed alone. And NcSRS2 gene and NcSAGlgene were cloned and inserted into the same expression vector to express a chimeric protein. At the same time, the immunogenicity of NcSRS2 protein and NcSRS2-NcSAGl protein were assessed.The molecular characters of amino acid sequences encoded by NcSRS2 and NcSAGl were analyzed using molecular softpackages. The truncated NcSRS2 (dNcSRS2) gene lacking a N-terminal hydrophobic signal peptide was amplified by PCR using oligonucleotide primers and then cloned into the bacterial expression vector, pGEX-6P-l. The resulting plasmid was designated as pGEX-dNcSRS2. The dNcSRS2 gene was highly expressed as a glutathione S-transferase (GST) fusion protein (GST-dNcSRS2) in E. coli (BL21 strain) induced by 1mM IPTG The recombinant GST-dNcSRS2 was expressed both as soluble protein and inclusion body. Of the total mass of bacterial proteins the GST-dNcSRS2 could amount to 32.3%. The molecular weight of the fusion protein is 62.6KD.The GST-dNcSRS2 was purified with Glutathione Sepharose 4B.The specific band was identified with GST antibody through western blot analysis.Two DNA fragments encoding respectively strong antigenic determinants of NcSRS2 and NcSAGl were cloned, they were inserted into the same expression vectors pGEX-6P-l. The resulting plasmid was designated as pGEX-tNcP43-P36. The expressed chimeric protein of NcSRS2 with NcSAGl in a form of inclusion body was about 14.9% of total proteins ofE.coli (BL21). The molecular weight of the chimeric protein is 64.5KD. Western blot analysis with sera against GST showed the specific band. The good renaturation was obtained using gradient dialysis with low protein concentration (0.042mg/ml).The Balb/C mice were immunized with purified recombinant proteins three times to assess the immunogenicity of the two proteins. Both GST-dNcSRS2 and GST-tNcP43-P36, which was mixed with Freunds adjuvant, were set up three doses groups which were high dose (100 μ g/mouse), middle dose (50 μ g/mouse) and low dose (25 μ g/mouse). At the same time, two groups of mice were inoculated with GST-dNcSRS2 or GST-tNcP43-P36 respectively, both in middle dose (50 μ g/mouse) without Freunds adjuvant, and the last group was the control group. Serum was collected before the first inoculation and every 10 days post inoculation to measure Ab levels by ELISA. The results showed that IgG levels of each inoculation groups were markedly increased, the GST-dNcSRS2 group in middle dose mixed with Freunds adjuvant was the highest among them. Lymphocyte proliferation assays (LPA) were performed every 10 days post inoculation. The results showed that GST-tNcP43-P36 in each dose could induce better lymphocyte amplification reactions than GST-dNcSRS2 in the same dose.