Research Of Nano-gold To Enhance The Immunogenicity Of PRRSV GP5 Protein

Porcine reproductive and respiratory syndrome virus(PRRSV)is the causative agent of PRRS. It causes an important disease in pigs characterized by reproductive failure in sows , pneumonia and an increase in perinatal mortality, resulting in great economic losses to the swine industry. Since it was first described in 1987 in the United States and in Europe in 1990, PRRS has been one of the most economically important diseases of the global swine industry. Vaccine inoculation is the main method to prevent and control this disease. But the attenuated vaccines has the disadvantage of the transmitted virus,The inactivated vaccines and genetic-engineering vaccines are low immune efficacy.Nano-gold can improve the body humoral and cellular immunity level,it also can enhance the immune efficacy of vaccine as immunologic adjuvant.Glycoprotein 5(GP5) is immuno-proteins of PRRSV, it can induce the production of neutralization antibody. According to the gene of PRRSV publicized in GenBank, a pair of primers was designed. The full-length of ORF5 gene of PRRSV zhejiang strain was amplified by RT-PCR.In order to get more expressed protein, the gene segment g-ORF5 deleted signal peptide and three transmembrane region was amplified by overlap PCR with primers which was designed and synthesized according to the characteristic of flexible acid. Following insertion into pET-28a(+)and transformation into Escherichia coli BL21, the fragment g-ORF5 was expressed after induced with 1mM IPTG. The results of ELISA and Dot-ELISA showed that the protein specifically reacted with PRRSV-positive sera from pigs, not negative sera. The studies lay foundations for further study on the function and immunological characteristic of GP5 protein.The 15~20nm nano-gold which made from hydrochloro-auric acid was reduced by Sodium crtrate. After dialysed with double distilled water, the renaturation GP5 protein was peridiumed with nano-gold. The optimal peridium pH Value was 9.8, and the optimal GP5 protein peridium amount was 40μL. The concentration of GP5-NG protein is 0.4mg.GP5 protein and GP5-NG protein immuned 6-8 weeks ICR mice and Freund's adjuvant and Tris-HCl as the control group. The humoral and cellular immunological effect were detected by ELISA and lymphocytes stimulation test. The results of humoral immunological effect showed that protein NG-GP5 achieved higher levels of specific antibody than that of GP5 protein group, but lower than Freund's adjuvant group. showing a significant increment between the two group(P<0.01). Comparing the two way(subcutaneous injection and intramuscular injection)of inoculation, subcutaneous way proved a better immune effect; The results of cellular immunological effect indicated that GP5-NG can induce both T and B lymphocyte proliferation higher than that of GP5 protein group(P<0.01). Comparing the two way(subcutaneous injection and intramuscular injection)of innoculation, subcutaneous way induced higher both T and B lymphocyte proliferation.