Preparation of Monoclonal Antibody to Porcine TNF-α and Expression of TNF-α in PAM Infected with PRRSV
|School||Nanjing Agricultural College|
|Course||Preventive Veterinary Medicine|
|Keywords||TNF-α McAb PRRSV PAM|
Tumor necrosis factor-alpha (TNF-a) is a kind of pleiotropic cytokine mainly producted by monocyte-macrophage cells and T cells. It involves in systemic inflammation and is a member of a group of cytokines that stimulates the acute phase reaction. The primary role of TNF-a is in the regulation of immune cells. TNF is able to induce apoptotic cell death and inflammation, and inhibit tumorigenesis and viral replication. Previous reports indicate that detection of TNF-a has an important significance for monitoring a variety of major diseases. Porcine reproductive and respiratory syndrome virus (PRRSV) can cause a highly contagious disease characterized with dysgenesia in pregnant pigs and dyspnea in piglet. It can be divided into two genotypes:European-type and Americas-type according to genomic and antigenic differences. And the pathogenicity was great different between the different isolates. The mechanism of pathogenicity has not been understood completely. In this study, porcine TNF-a was expressed in Ecoli and the monoclonal antibodies to TNF-a were preparaed. In order to clarify the role of TNF-a in the process of PRRSV infection, the levels of TNF-a in PAM infected with two PRRSV strains with different virulent were detected with Real-time quantitative PCR and ELISA. The content is as following:1. Expression of porcine TNF-a in Ecoli and preparatin of McAb to TNF-aBasing on the gene sequence of porcine TNF-a in GeneBank,two pairs of specific PCR primers were designed and the gene was amplified with PCR. The recombinant plasmid PET-28a-TNF-a was constructed and then transformed into E.coli BL21. And the recombinant bacteria was induced by IPTG and then confirmed by SDS-PAGE and Western blotting. To prepare monoclonal antibody specific to TNF-αprotein, Balb/c mice were immunized with purified TNF-αinclusion body. Two positive hybridoma cell strains,named 1E2 and 2F3,were screened by indirect ELISA. The McAb against TNF-αwill be useful for developing methods to detect this protein in the future.2. Comparsion of the expression levels of TNF-αin PAM infected with PRRSV with different virulentPorcine alveolar macrophage (PAM) were collected from 28-day-old health piglets and infected with high pathogenic PRRSV and MLV vaccine strain individually. Then the levels of PRRSV replication and TNF-αwere detected by using Real time quantitative PCR, using housekeeping geneβ-actin as internal control, and ELISA, respectively. Meanwhile, LPS (LPS) was used a positive control for stimulation of the pig alveolar macrophages. The results indicated that the two different PRRSV isolates could replicate in the cells. TNF-αmRNA could express in the cells 1-10h after infection with the PRRSV isolates, and then had a gradually declined trend until 24 h after inoculation. Meanwhile, TNF-αin the supernatant of the cultures were raised gradually by detected with ELISA. Moreover, the levels of TNF-αmRNA and protein in SY0608 group were significantly higher than those in MLV group. It indicated that TNF-αplay important role in the pathogenicity of PRRSV.