Generation And Application Of Polyclonal And Monoclonal Antibodies Against Procine Beta-Defensin 2

Antimicrobial peptides (AMPs) are a class of micro proteins secreted by animals themselves to resist pathogenic microorganisms from the external world. AMPs are important for animal innate immunity. Because of their specific antibacterial mechanism, broad antimicrobial activity, and generating tolerance hardly, AMPs are a popular research area recently. A major family of short cationic AMPs found in mammals is the defensins, which can be divided into two classes, theα-andβ-defensins according to their structural features at the gene and peptide levels. Beta-defensins are AMPs of key interest because they operate in both innate and adaptive immune responses in mammals. Some AMP genes have been used to generate transgenic animals to improve the ability of disease resistance. This study aimed to generate polyclonal and monoclonal antibodies against porcineβ-defensin 2 (PBD2), and to establish the immunohistochemistry (IHC) and indirect immunofluorescence (IFA) methods to evaluate the expression of PBD2 in pigs and transgenic animals using the antibodies.An 11-mer peptide (between the third and fourth cysteines) and a 37-mer mature peptide were synthetized according to the known protein sequence of mature PBD2. The 11-mer peptide coupled with keyhole limpet hemocyanin (PBD2-KLH) was used as immunogen to immune Balb/C mice, and the synthetised 37-mer peptide (PBD2) was used as coating antigen to screen the PBD2 antibodies via indirect ELISA. After three times immune Balb/C mice, get their serum to detect the titers, all of their ELISA titers reached up to 1:12800. We get the Balb/C mice serum to prepared mouse anti-PBD2 polyclonal antibodies, an indirect immunofluorescence assay was also established using the PBD2 polyclonal antibodies.The spleen cells from the immunized mice with highly antibody levels were fused with the SP2/0 myeloma cells. The positive clones were selected by indirect ELISA method. After two times of subcloning by limiting dilution,5 strains of hybridoma,1C2,1F4,2H2, 3A2 and 4B1, were eventually obtained, which could secret high levels of monoclonal antibodies with good specificity. Their ELISA titers in the supernatants of cell cultures were 1:210, and those in the ascites were 1:106 respectively. The tissues from clinical healthy and Actinbacillus pleuropneumoniae 4074-infected pigs, such as liver, spleen, mesenteric lymph nodes and intestines, were selected to prepare histological sections. The SABC immunohistochemistrical straining was carried out on these sections using the PBD2 monoclonal antibodies. The results showed that the highest expression of PBD2 is in the liver, and the PBD2 is mainly distributed in the vascular endothelial cells and liver cells, both in cytoplasm and nucleus. Real-time RT-PCR and Sandwich ELISA may be needed to determine the quantitative difference of PBD2 expression in different tissues and animals.