Expression Of Human Toll-like Receptor-9 And Preparation Of Anti-hTLR-9 Antibody

Toll like receptors (TLR) are innate immune molecule in mammal. As pattern recognition receptor (PRR), they play an important role in recognizing and defencing varities of pathogen. TLR1—TLR13 are typical I-type transmembrane receptors which have been founded in mammal since 1997. Among them there are ten types of TLR existing in human (hTLR1—hTLR10) .TLR-9 is the representative of TLRs family. The CpG DNA contained in bacterium DNA is the only pathogen associated molecular patterns (PAMPs) that recognized by TLR-9. The CpG sequence is more frequently present in bacteria and virus genome than in vertebrate genome. Non-methylated cytosine and guanine dinucleotide (CpG) is the base of immunostimulatory oligonucleotide. The immune system of vertebrae discriminates bacterium DNA and primes protective immune response by identifying the characteristic structure of CpG. It has been an important topic in fundamental and clinical medicine study today, such as how CpG DNA function as an immunomodulator in allergy, tumor and autoimmune disease. TLR-9 as the only one receptor of CpG DNA has been brought to the notice of investigators.Considerable evidence has shown that TLR-9 can recognise CpG, then activate many kinds of immunocytes. However, little evidence is present concerning the proteinic characteristic of TLR-9, the binding with CpG and the subsequently induced immune response. The present study was designed to clone and express the human TLR-9 (hTLR-9) and prepare the anti-hTLR-9 antibodiy. Simultaneously we expressed hTLR-9 in eukaryotic expression system by HEK293 cells. The HEK293cell model transferred with hTLR9 gene stablely was constructed successfully. This cell model would play a part in further investigation of the biologic activitiy of hTLR-9 and its antibody.Part I Cloning and expression of human Toll-like receptor-9The foundation of anti-hTLR-9 Ab preparation and functional experiment require hTLR-9 protein. We expressed hTLR-9 in prokaryotic and eukaryotic expression systems.Escherichia coli were chosen to express the complete or partial gene of hTLR-9 as prokaryotic expression system. Based on the whole length gene of human TLR-9 gene published in GenBank, we designd and synthetized three couples primers by Primer premier 5.0 software. Using pTAdv-hT9 (plasmid containing complete hTLR-9 cDNA sequence) as template, we amplified 5'-terminal (707~l 167 bp)of hTLR9 based by PCR. Similar to this, the total length and extracellular domain of hTLR-9 gene were amplified. The three kins of PCR product were cloned into pET32a plasmid. PCR, enzymatic analysis and sequencing showed three different pET32a recombinant plasmid were constructed. After recombinat plasmids were transferred into Escherichia coly BL21(DE3), the protein expression was induced by IPTG. SDS-PAGE and Western blot analysis show that the hTLR-9 protein and it's fragment wer expressed correctly. Then we could prepare antibody respectively.We expressed hTLR-9 in eukaryotic expression system by HEK293 cells. HEK293 cell has been used as negative control in functional research of TLRs. We designed a couple of primer by Oligo6.0 software. So whole length hTLR-9 gene sequence was amplified with pTAdv-hT9 as template. The hTLR-9 gene was cloned into pcDNA3.1+ of mammalian expression vector, and the recombinant plasmid pcDNA-hTLR-9 was transfected into HEK293 cell by electroporation . Cell clones transferred with hTLR-9 gene permanently(HEK293-T9) were screeened with G418, and insertion of exogenous gene was confirmed by PCR product from genome DNA and mRNA; HTLR-9 was identified to be expressed on the surface of HEK293-T9 cells by immunohistochemical assay and flow cytometry analysis. HEK293-T9 can be used as vector carrying natural hTLR-9 protein to verify the specificity of antibodys prepared with such immunogen as polypeptide and protein expressed inprokaryotic system. Meanwhile it may provide a satisfactory cell model for hTLR-9function study.Part II Preparation and identification of antibody against N-terminal ofhTLR9Preparation and identification of polyclonal antibody against N-terminal of hTLR9By high-capacity expression of E. coli , We obtained N-terminal of hTLR9 fusion protein in form of inclusion bodies. The fusion protein was used as immunogen with sufficient quantility and purity by initial purification to prepare polyclonal antibody.Mice were immunized with the mixture of inclusion body in 8 M Urea and Freund's complete adjuvant. One week after the fouth immunization, mice seurm was isolated. The fact that this kind of antibody can bind hTLR9 expressed in eukaryotic system proved the specificity of antibody. The results showed that polyclonal antisera binding to natural hTLR9 can be prepared by the utilization of the inclusion bodys.We also proceed the large-scale prepareation of antibody against hTLR9 by immunizing two rabbits in the same way and got specific antisera 80ml. Then the serum was purified by SPG for subsequent experiment. Preparation and identification of monoclonal antibody against N-terminal of hTLR-9BALB/c mice were immunized with prepared HEK293T9 cells, and the hybridoma were generated from fusion cells of mouse spleen cells and myeloma cells NS-1. By screening all fusion cells with prokaryotic expressed N-terminal of hTLR-9, hybridoma cell lines which secrete monoclonal antibody against N-termal of hTLR9 were obtained after successively cloning. Two of hybridoma cell lines secreting anti-hTLR-9 mAb were established and named as 1F5 and 2F11. Both belong to IgGl isotype and can bind specifically to HEK293T9 cells, whereas they cann't react with nomal HEK293 cells.