TLR2-a Member of TLR Family Mediating Innate Immunity

Toll like receptors can recognize exogenous microbes and trigger a series of signal transduction, which will promote the release of inflammatory medium and initiate innate immunity. Currently, more than ten TLR family members have been found, the TLR2 is the most abundant one. It is widely distributed in monocytes, macrophages, neutrophils, dendritic cells, epithelial cells and tumor cells. The pathogenic microorganisms TLR2 recognizes are much more than other members in TLR family. As TLR2 plays a key role in innate immunity, it has great theoretical and practical significance for research of immunity. Consequently, the Cloud-Clone Corp. develops a series of testing tools, including the recombinant proteins, antibodies and ELISA kits for researchers.

 According to the structure analysis, TLR2 is a type I transmembrane protein and its sequence consists of extracellular section, transmembrane section, and intracellular section. As the extracellular section has 19 LRR repeat domains, the Pro47-Gly245 was chosen as the expressed recombinant protein after antigen epitopes analysis, protein structure prediction and the solubility analysis. The Pro47-Gly245 is an extracellular domain, which is convenient for researchers to study. The primers were designed according to the CDS of NM_003264.3 in NCBI. As there is an EcoRI restriction enzyme cutting site in the sequence, we add BamHI to the upstream primer and Xhol to the downstream primer for expressive plasmid construction,.

[PRIMER INFORMATION]

Upstream Primer:CGCggatccGAATCCTCCAATCAGGCTTCTCTG

Downsream Primer: CCGctcgagTCACAGTGCTGTCCTGTGACATTCCGAC

We obtained a 600bp sequences by PCR. After gene sequencing, the Cloud-Clone Corp. construted the expressive plasmid by the gene fragment and self-established E.coli expression vector pUSCNK-1 by traditional restriction enzymes digestion..The result of gene sequencing is shown in Fig 1.

1 CCCTCAGGGCTCACAGAAGCTGTAAAAAGCCTTGACCTGTCCAACAACAGGATCACCTAC

61 ATTAGCAACAGTGACCTACAGAGGTGTGTGAACCTCCAGGCTCTGGTGCTGACATCCAAT

121 GGAATTAACACAATAGAGGAAGATTCTTTTTCTTCCCTGGGCAGTCTTGAACATTTAGAC

181 TTATCCTATAATTACTTATCTAATTTATCGTCTTCCTGGTTCAAGCCCCTTTCTTCTTTA

241 ACATTCTTAAACTTACTGGGAAATCCTTACAAAACCCTAGGGGAAACATCTCTTTTTTCT

301 CATCTCACAAAATTGCAAATCCTGAGAGTGGGAAATATGGACACCTTCACTAAGATTCAA

361 AGAAAAGATTTTGCTGGACTTACCTTCCTTGAGGAACTTGAGATTGATGCTTCAGATCTA

421 CAGAGCTATGAGCCAAAAAGTTTGAAGTCAATTCAGAATGTAAGTCATCTGATCCTTCAT

481 ATGAAGCAGCATATTTTACTGCTGGAGATTTTTGTAGATGTTACAAGTTCCGTGGAATGT

541 TTGGAACTGCGAGATACTGATTTGGACACTTTCCATTTTTCAGAACTATCCACTGGTTGA

Figure 1 The result of gene sequencing

The homology of pUSCNK-1 hTLR2 and TLR2 gene from NCBI is 100%, which shows the accuracy of the sequence. The result of sequence alignment is shown in Fig 2.

Later, we got an approximately 29KD recombinant protein of TLR2 by IPTG induced expression. And it was purified by Nickel column as there’s a his-tag in the protein, and we obtained recombinant TLR2 protein with high purity (RPA663Hu01). The sequence of recombinant TLR2 protein is shown in Fig 3.

Figure 2 The result of sequence alignment

MGHHHHHHSGSEF- PSGL TEAVKSLDLS NNRITYISNS DLQRCVNLQA LVLTSNGINT IEEDSFSSLG SLEHLDLSYN YLSNLSSSWF KPLSSLTFLN LLGNPYKTLG ETSLFSHLTK LQILRVGNMD TFTKIQRKDF AGLTFLEELE IDASDLQSYE PKSLKSIQNV SHLILHMKQH ILLLEIFVDV TSSVECLELR DTDLDTFHFS ELSTG

Fig 3 The sequence of recombinant TLR2 protein

The SDS-PAGE result (Fig 4) shows the purity of the recombinant TLR2 exceeds 95%. Then we immunized mice by purified TLR2 and obtained anti-human TLR2 monoclonal antibody, and further purified the antibody by affinity chromatography to get antibodies with high specificity (MAA663Hu21). At the meanwhile, we also prepared anti-human TLR2 polyclonal antibodies (PAA663Hu01) by immunizing rabbits. Further, we tested the antibodies by western blot (Fig 5). Figure 4 SDS-PAGE Fig

At the same time, we did the IHC test (Fig 6) with human spleen tissue, and strong signal was shown in cytomembrane. All these data proved the antibody is highly specific. Further Cloud-Clone Corp. developed an Elisa kit (SEA663Hu ） by the recombinant TLR2 and anti-human TLR2 antibodies.

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