Construction Of Antibody Libraries Of Haemophilus Parasuis Based On Phage Display

Haemophilus parasuis is a opportunistic etiologic agent colonized commonly in upper respiratory tract of healthy conventional pigs, and causes the infection of Haemophilus parasuis in some stress conditions, also called as "Glasser’s disease" which is characterized by fibrinous polyserositis, polyarthritis, meningitis and pericarditis. In recent decade, infections caused by this bacterial pathogen have led to great economic losses in the pig industry worldwide. Vaccines used for preventing the infections are whole cell inactivated vaccine at home and abroad, including bivalent vaccine and polyvalent vaccine. Although these vaccines have played an important effect in preventing from the infections caused by homologous strains of Haemophilus parasuis, immune failures offen occur everywhere, perhaps due to serotype difference between the strains of vaccineis and infections. Pig farmers have used various antibiotics at large doses, so-called "health drug prevention" or pharmacotherapy to protect breakout of disease. Nevertheless, the etiologic agent is prone to inducing resistance of antibiotics, and even multiresitance, thus leading to failure of drug prevention or pharmacotherapy. In this study, the phage display library, which could generate a repertoire antibodies for H. parasuis, was established by virtue of the technology of phage display appeared in recent years. We attempted to select antibodies targeted to the protection and treatment of H. parasuis infections and to open a new avenue for preparation of antibody against H.parsuis.The main results in this study were as follows:1. The primary first-strand cDNA synthesis was progressed by countertranscription with Oligo (dT)15from total RNA extracted from the spleen tissue of slaughter pig immunized with vaccine against Haemophilus parasuis. Immunoglobulin G (IgG) heavy chain variable region (VH) and light chain (κ chain or λ chain) variable region (VL) genes were respectively amplified by PCR with a series of special primers of pig IgG.12genes, including5gens of VH,5genes of VH and2genes of Vλ, were obtained.2.12interesting gene fragments were respectively cloned into pMD19-T vector with TAKARA kit and seqenced. Each gene sequence was aligned with IgG V germlines in NCBI, the alignment results showed that these gene segments would belong to porcine VH, VKand Vλgene. According to V Quest Software of the IMGT database, the deduced amio acdio residues of porcine VH, VK and Vλ gene segments were analysed and separated into7regions which ranged from FR1, CDR1, FR2, CD2, FR3, CDR3and FR4. All CDR of the amio acdio residues was variable, especially CDR3.3. The individual rearranged Heavy-and Light-chain coding sequences are amplified separately and were linked through a series of overlap extension PCR steps which were fused with a linker DNA (Gly4Ser)3to give6scFv products.4.6scFv products were used for linking alone to phagemid pC3C, which was each transformed into XL1-Blue strain of E.coli. Each recombinant pC3C-scFv was analyzed using restriction digestion with sfil, and the results were consistent with expected results. After sequence, the deduced sequence of amio acdio residues was similar to above99%of expected results.The phage antibody library was established by helper phage VCSM13with postive transformant identifyed by sequence and sfil digestion.5. Antibody fragment, scFv with30kDa of the predicted molecular weight, was expressed on recombinant pC3C-H2.2κ1.1with lmM IPTG.6. The size of phage antibody library with recombinant pC3C-H2.2κ1.1was measured, showing1.7×1012capability.