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Cloning, Expression and Preliminary Function Analysis of Chicken Toll-Like Receptor 1/2 Genes

Author: ChenHao
Tutor: WeiPing
School: Guangxi University
Course: Preventive Veterinary Medicine
Keywords: chicken TLR2 family stably transfect eukaryotic expression cell lines ligands recognition
CLC: S858.31
Type: Master's thesis
Year: 2011
Downloads: 38
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Abstract

Toll-like receptors are important pattern recognition receptors (PRR) in innate immune system. TLRs are type I integral transmembrane glycoproteins. This type of receptors can recognize the invading pathogens microbes, and cause inflammation, helping the body clear the pathogens in innate immune. To date, ten avian TLRs have been identified in chicken.The chTLR1LA,1LB,2t1 and 2t2 genes of chicken TLR family were amplified containing the full-length ORF (Opening reading frame) using four pair primers which series the Lumio tag sequence. The amplified genes products were cloned into eukaryotic expression vector pVITRO1-neo-mcs. We constructed four reconbinant expression plasmids pVITRO1-neo-mcs-chTLR1LA,1LA,2t1 and 2t2 identified by PCR and double DNA restriction enzymes digest. The expression plasmids were stably transfected single or double into vero cells and expressed in vitro and then was indentified by RT-PCR and protein page gel electrophoresis (Lumio fusion proteins). Conclusion the recombinant plasmids for fusion expression of chTLR1/2 genes were successfully constructed and stably expressed in vero cells. T1-T10 vero cell lines were successfully constructed. Then the detection plasmid pNiFty2-SEAP was stably transfected into T1-T10 Vero cells to construct T1’-T10’vero cell lines. They were stimulated by HEK-BlueTM Detection containing 10μg/mL LPS and Zymosan, with the non-stimulated control group, negative control group (vero cell transfected with the detection plasmid) and blank control group. After nine hours we could observe the color changes and determinate the culture medium OD (630nm) values. In a result, the expression of SEAP of T1’, T3’and T9’vero cells groups activated by LPS, T9’vero cells groups activated by zymosan and T4’, T8’vero cells control groups were very significantly higher than that of different from the negative control groups (P＜0.01).Finally, expression of chTLR1LA, chTLR2tl and co-expression of chTLR1LA and chTLR1LB of the chTLR2 subfamily result in NF-κB activation in response to LPS; Co-expression of chTLR1LA and chTLR1LB of the chTLR2 subfamily results in NF-κB activation in response to Zymosan; chTLR2t2 alone exhibits more ability to activate NF-κB than other members and the heterodimers of chTLR2 subfamily. It induces that chTLR2t2 alone may have the function of some unknown ligands recognition.

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