Cloning Of TLR7 Gene Fragment And Semi-quantitation Of MRNA By RT-PCR In Chicken Vaccinated With Infections Bursa Disease Virus

Toll-like receptors (TLRs), a superfamily of pattern-recognition receptors (PRRs) play a pivotal role in host innate immunity against pathogen infection and bridge the innate and adaptive immune. It has been shown that in both human and other mammals TLR7 can be an important recognizing receptor of the single-stranded RNA viruses and activate the innate immune responses through a signal transduction pathway. To date only partial gene sequences of chicken TLRs (chTLRs) have been identified by using bioinformatics methods, while little is known about their biological functions and roles in against specific-pathogen infection. The purpose of this study is to investigate the role of chTLR7 against infectious Bursa disease virus infection by cloning chTLR7 gene fragment and developing a semi-quantitative RT-PCR assay.The sequence of the gene fragment encoding the chicken Toll-like receptor7(chTLR7) was cloned by RT-PCR using the specific primers designed according to the cDNA sequence by Eukaryota et al. (2005). The total RNA was extracted from the isolated lymphocytes of broiler chicken spleen,Bursal of Fabricius and used as the template for RT-PCR. The RT-PCR product was cloned into the pGEM-T-easy plasmid vector and then purified by agarose gel electrophoresis. The ligated product was transformed into E. coli DH 5α, and the positive transformants identified by PCR were transferred into LB medium for extracting its plamid. The cloned fragment was sequenced and the result showed the nucleotide sequence of the cloned gene fragment was 99.69% identifical to the homological fragment sequence in the target chTLR7 in GenBank. This cloned region is 846 bp in sequence length localized in the chTLR7 cDNA 2574-3419bp region.To investigate the change of chTLR7 gene expression in Bursa of Fabricius and spleen and its potential role in earlier period of immune response against infectious Bursa disease virus (IBDV), in vivo experiments was conducted in chicken. Ninety 11-day-old chicken were randomly divided into 3 group of 30 each: control group, low dose group (one dose each time), high dose group (three doses each time). Chickens were sacrificed at the designed time points and chTLR7 mRNA expression in Bursa of Fabricius and spleen lymphocytes was measured by Semi-quantitative RT-PCR at different time points. The low level of chTLR7 mRNA could be detected in both Bursa of Fabricius and spleen lymphocytes in control group. After vaccination the mRNA level in spleen lymphocytes for both low and high dose group increased rapidly, reaching the peak at 12 hours. Then it started decreasing gradually and approached to the level of before vaccination at 130h. Similarly, the mRNA level in Bursa of Fabricius started increasing 12 hours and reached the peak at 24 hours after vaccination, which gradually reduced to the level of before dosing at 72 hours. By comparison, chTLR7 mRNA in Bursa of Fabricius increased slowly, but decreased fast.Our data showed that the up-regulated expression of TLR7 mRNA can be induced by vaccine IBDV, suggesting that chTLR7 may play a defense role in the early infectious phase of Chicken Bursa virus.