Cloning And Expression Of Murine TLR-2 And Research Of Anti-mTLR-2 Antibodies

Cloning and Expression of murine TLR-2 and Research of Anti-mTLR-2 AntibodiesThe mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in mediating innate immunity and linking to acquired immunity. Nine members in murine TLR family and ten members in human TLR family have been found since the first TLR molecular was identified in 1997. TLRs participate in both infection and non-infection immunity, such as antigen presenting, immune tolerance, and cell apoptosis. TLRs play an important role in autoimmune diseases, allergic diseases, transplantation rejection and tumor. Toll-like receptor 2 (TLR-2) has been studied in substantial detail over the last years. TLR-2 can recognize a wide variety of pathogen associated molecular patterns (PAMPs) such as gram-positive bacteria, mycobacterium tuberculosis and their component, and mediate similar signal transduction and pro-inflammatory cytokine production. Therefore TLR-2 was considered as a central pattern recognition receptor in innate immunity. There have been a lot of research in vitro regarding the importance of TLR-2 and their ligands in inducing immune related events. But few work has been done in vivo regarding the exploration of TLR-2 in innate immune and induction of acquired immunity.Both mTLR-2 and hTLR-2 are Drosophila Toll homologues, and amino acid of mTLR-2 shared 83% homologous with hTLR-2. Based on high homologous structure and similar distribution and biological activitities, mouse is undoubtedly a good model for us to study the structure and functions of hTLR-2. In this research, complete gene of murine TLR-2 was cloned and expressed; antibody against B cell epitope on extracellular domain and a fragment of N terminal were prepared; the activities of antibody on anti-allergy and anti-tumor experiment were performed.-6-Part I Cloning and expression of murine TLR-2Based on the complete sequence of murine TLR-2 gene published in GenBank, four primers from P1 to P4 were designed and synthesized. The downstream gene and the upstream gene of murine TLR-2 was amplified using P2 and P4, P1 and P3 respectively, P1 and P4 were used to amplify the complete gene. One fragment of 1 500bp was amplified using cDNA from mouse liver as template, another fragment of 1 400bp was obtained by RT-PCR using the same template. Two amplified products were cloned into pUC-T vector respectively. The upstream clone U3 and the downstream clone D4 were obtained through blue/white color selection, PCR and sequence analysis. Then U3 and D4 as template, the fragment of over 2 500 bp was amplified and cloned into pUC-T vector using P1 and P4. The recombinant plasmid pUC-mTLR-2 was obtained, and identified by sequence analysis. It showed the complete gene of mTLR-2 was acquired. The gene had been accepted as accession number AY179346, which has 99.84% homology with the standard sequence of mTLR-2. Using BLAST2 online, there are substitutions of four bases between AY179346 and the standard sequence AF124741, among which t substitute c at nucleotide position 515, t substitute c at 963, a substitute g at 2476, and t substitute c at 2500. Compared the deduced sequence of amino acid from AY179346 with that from AF124741, there are three amino acid substitutions, F266L, Q770R and L778P.It is significant for us to obtain mTLR-2 protein, which is helpful to study the biological functions of mTLR-2 in vitro and in vivo. Now the expression system of mammalian cell, the methylotrophic yeast Pichia pastoris, Escherichia coli were chosen to express the complete or partial gene of mTLR-2. In briefly, (l)The mTLR-2 gene was cloned into pcDNA3.1 of mammalian expression vector, and the recombinant plasmid pcDNA-mTLR-2 was transfected into CHO cell by electroporation , the positive cell clones were obtained by screening with G418, and insertion of-7-exogenous gene was confirmed by PCR; One clear band was shown in relative molecular weight(Mr) of 95 000 using Western blot analysis, and mTLR-2 was identified to be expresse...